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https://openalex.org/W4390705063	https://egusphere.copernicus.org/preprints/2023/egusphere-2023-2231/egusphere-2023-2231.pdf	English	null	Reply on RC2	null	2,024	cc-by	14,906	1 Introduction Estuaries are highly productive ecosystems. Their relative small area disproportionally contributes to the global carbon cy- cle, along with their role as a source of nutrients and hatching grounds for marine ecosystems (Cloern et al., 2014; Arevalo et al., 2023). While they are heavily inﬂuenced by anthropogenic stressors such as diking, dredging, and ﬁshing, they are of tremendous importance for anthropogenic usage (Jennerjahn and Mitchell, 2013; Brown et al., 2022; Wilson, 2002). Estuaries 15 present challenging dynamics to their smallest residents, due to strong salinity gradient and a net transport to the ocean. Here, l h h t l kt d ifti ll i d th t f th b i f t i f d b i t ithi Estuaries are highly productive ecosystems. Their relative small area disproportionally contributes to the global carbon cy- cle, along with their role as a source of nutrients and hatching grounds for marine ecosystems (Cloern et al., 2014; Arevalo et al., 2023). While they are heavily inﬂuenced by anthropogenic stressors such as diking, dredging, and ﬁshing, they are of cle, along with their role as a source of nutrients and hatching grounds for marine ecosystems (Cloern et al., 2014; Arevalo et al., 2023). While they are heavily inﬂuenced by anthropogenic stressors such as diking, dredging, and ﬁshing, they are of tremendous importance for anthropogenic usage (Jennerjahn and Mitchell, 2013; Brown et al., 2022; Wilson, 2002). Estuaries 15 present challenging dynamics to their smallest residents, due to strong salinity gradient and a net transport to the ocean. Here, we explore how phytoplankton, drifting small primary producers that form the basis of estuarine food webs, can persist within such dynamic environments. tremendous importance for anthropogenic usage (Jennerjahn and Mitchell, 2013; Brown et al., 2022; Wilson, 2002). Estuaries 15 present challenging dynamics to their smallest residents, due to strong salinity gradient and a net transport to the ocean. Here, we explore how phytoplankton, drifting small primary producers that form the basis of estuarine food webs, can persist within such dynamic environments. 15 Like most ecosystems - estuarine ecosystem dynamics are strongly controlled by primary producers, in particular phyto- plankton (Chen et al., 2023). Apart from bioﬁlm-forming phytoplankton which are attached to their substrate (Cheah and 20 Chan, 2022), the vast majority of phytoplankton organisms drifts passively in currents, though they may be able to inﬂuence their vertical movement. Phytoplankton Retention Mechanisms in Estuaries: A Case Study of the Elbe Estuary Laurin Steidle1 and Ross Vennell2 1Institute of Marine Ecosystem and Fishery Science, Universität Hamburg, Olbersweg 24, 22767 Hamburg, Germany 2Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand Correspondence: Laurin Steidle (laurin.steidle@uni-hamburg.de) We ﬁnd that vertical migration especially rising favors the retention, fast sinking does not. We further provide ﬁrst estimates on outwashing losses. Our simulations illustrate that riverbanks and tidal ﬂats are essential for the long-term survival of phytoplankton populations, providing refuges from strong downstream currents. These results contribute to the understanding needed to advance ecosystem-based management of estuaries. 10 5 10 Phytoplankton Retention Mechanisms in Estuaries: A Case Study of the Elbe Estuary Laurin Steidle1 and Ross Vennell2 1Institute of Marine Ecosystem and Fishery Science, Universität Hamburg, Olbersweg 24, 22767 Hamburg, Germany 2Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand Correspondence: Laurin Steidle (laurin.steidle@uni-hamburg.de) Abstract. Due to their role as primary producers, phytoplankton are essential to the productivity of estuarine ecosystems. However, it is important to understand how these nearly passive organisms are able to persist within estuaries, when river inﬂow results in a net outﬂow to the ocean. Estuaries are also representing challenging habitats due to a strong salinity gradient. So far, little is known about how phytoplankton are able to be retained within estuaries. We present a new individual-based Lagrangian model of the Elbe estuary which examines possible retention mechanisms for phytoplankton. Speciﬁcally, we 5 investigated how reproduction, sinking and rising, as well as diel vertical migration may allow for populations to persist within the estuary. We ﬁnd that vertical migration especially rising favors the retention, fast sinking does not. We further provide ﬁrst estimates on outwashing losses. Our simulations illustrate that riverbanks and tidal ﬂats are essential for the long-term survival of phytoplankton populations, providing refuges from strong downstream currents. These results contribute to the understanding needed to advance ecosystem-based management of estuaries. 0 Abstract. Due to their role as primary producers, phytoplankton are essential to the productivity of estuarine ecosystems. However, it is important to understand how these nearly passive organisms are able to persist within estuaries, when river inﬂow results in a net outﬂow to the ocean. Estuaries are also representing challenging habitats due to a strong salinity gradient. So far, little is known about how phytoplankton are able to be retained within estuaries. We present a new individual-based Abstract. Due to their role as primary producers, phytoplankton are essential to the productivity of estuarine ecosystems. However, it is important to understand how these nearly passive organisms are able to persist within estuaries, when river inﬂow results in a net outﬂow to the ocean. Estuaries are also representing challenging habitats due to a strong salinity gradient. So far, little is known about how phytoplankton are able to be retained within estuaries. We present a new individual-based Lagrangian model of the Elbe estuary which examines possible retention mechanisms for phytoplankton. Speciﬁcally, we 5 investigated how reproduction, sinking and rising, as well as diel vertical migration may allow for populations to persist within Lagrangian model of the Elbe estuary which examines possible retention mechanisms for phytoplankton. Speciﬁcally, we 5 investigated how reproduction, sinking and rising, as well as diel vertical migration may allow for populations to persist within the estuary. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Phytoplankton Retention Mechanisms in Estuaries: A Case Study the Elbe Estuary Laurin Steidle1 and Ross Vennell2 1Institute of Marine Ecosystem and Fishery Science, Universität Hamburg, Olbersweg 24, 22767 Hamburg, Germany 2Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand Correspondence: Laurin Steidle (laurin.steidle@uni-hamburg.de) Phytoplankton Retention Mechanisms in Estuaries: A Case Study of the Elbe Estuary Laurin Steidle1 and Ross Vennell2 1Institute of Marine Ecosystem and Fishery Science, Universität Hamburg, Olbersweg 24, 22767 Hamburg, Germany 2Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand Correspondence: Laurin Steidle (laurin.steidle@uni-hamburg.de) 1 Introduction With the estuary having a net outwards ﬂow, we would expect phytoplankton to be moving down- stream over time and to be washed out from limnic waters, via brackish into marine waters. Hence, the question arises how phytoplankton, as the drifting base of estuarine food webs, are able to maintain their population size without declining due 1 1 https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. to the net transport into the open ocean. If we assume that the population is not exclusively maintained by a self maintaining 25 source population upstream, that is washed into the estuary, then there must be some sort of retention mechanism that enables a phytoplankton population to persist within the estuary. So far, different theories exist on how estuarine phytoplankton populations are able to maintain their position. Previous to the net transport into the open ocean. If we assume that the population is not exclusively maintained by a self maintaining 25 source population upstream, that is washed into the estuary, then there must be some sort of retention mechanism that enables a phytoplankton population to persist within the estuary. So far, different theories exist on how estuarine phytoplankton populations are able to maintain their position. Previous observational studies suggested several possible mechanisms that could enable retention of phytoplankton populations within to the net transport into the open ocean. If we assume that the population is not exclusively maintained by a self maintaining 25 source population upstream, that is washed into the estuary, then there must be some sort of retention mechanism that enables a phytoplankton population to persist within the estuary. So far, different theories exist on how estuarine phytoplankton populations are able to maintain their position. Previous observational studies suggested several possible mechanisms that could enable retention of phytoplankton populations within estuarine systems - vertical migration in the form of sinking, rising or diel migration, stickiness. 30 Diel vertical migration is a process where organisms move up and down in the water column in response to the sun. This movement favors retention by allowing plankton to reduce the time in the faster downstream currents at the water surface. How- ever, this has only been demonstrated for larger motile estuarine organisms such as zooplankton (Hall et al., 2015; Kimmerer et al., 2002; Crawford and Purdie, 1991; Hall and Paerl, 2011). Yet, a study by Anderson and Stolzenbach (1985) showed that diel migrating dinoﬂagellates were able to out compete other non-motile phytoplankton in an embayment environment 35 and even compensate for outwashing losses through reproduction increasing their abundance. However, this also implies that the growing part of the population is somehow retaining their position. If the regrowing population is also continuously drift- ing downstream they will not able to sustain their population in that area and ultimately die out due to unfavorable salinity conditions in marine waters (Admiraal, 1976; von Alvensleben et al., 2016; Jiang et al., 2020). that diel migrating dinoﬂagellates were able to out compete other non-motile phytoplankton in an embayment environment 35 and even compensate for outwashing losses through reproduction increasing their abundance. However, this also implies that the growing part of the population is somehow retaining their position. If the regrowing population is also continuously drift- ing downstream they will not able to sustain their population in that area and ultimately die out due to unfavorable salinity conditions in marine waters (Admiraal, 1976; von Alvensleben et al., 2016; Jiang et al., 2020). that diel migrating dinoﬂagellates were able to out compete other non-motile phytoplankton in an embayment environment 35 and even compensate for outwashing losses through reproduction increasing their abundance. However, this also implies that the growing part of the population is somehow retaining their position. If the regrowing population is also continuously drift- ing downstream they will not able to sustain their population in that area and ultimately die out due to unfavorable salinity conditions in marine waters (Admiraal, 1976; von Alvensleben et al., 2016; Jiang et al., 2020). Estuaries are complex and strongly dynamic systems such that it is still difﬁcult to predict ecosystem dynamics or the ef- 40 fects of anthropogenic impacts due to their complex bathymetry (MacWilliams et al., 2016; Fringer et al., 2019). Nevertheless, previous modelling studies have investigated potential retention mechanisms of phytoplankton in estuaries. In Simons et al. (2006); Kimmerer et al. (2014) they used a Lagrangian model to study zooplankton retention. Simons et al. (2006) examined the dispersal and ﬂushing time of mussel larvae in the St. Lawrence Estuary while() (Kimmerer et al., 2014) examined zoo- plankton movement in the San Francisco Estuary. They were able to show that sinking and diel vertical migration slows the 45 outwashing process and might be a beneﬁcial retention strategy. However, they did so by ignoring key processes like reproduc- tion, mortality, and stranding or sedimentation processes. Moreover, both studies were based on low resolution structured grid models, which we suspect to under-represent the complex bathymetry of estuarine systems (Ye et al., 2018). Diatoms or benthic microalgae in particular have been observed to be strongly negatively buoyant, hence sinking to the plankton movement in the San Francisco Estuary. They were able to show that sinking and diel vertical migration slows the 45 outwashing process and might be a beneﬁcial retention strategy. However, they did so by ignoring key processes like reproduc- tion, mortality, and stranding or sedimentation processes. Moreover, both studies were based on low resolution structured grid models, which we suspect to under-represent the complex bathymetry of estuarine systems (Ye et al., 2018). Diatoms or benthic microalgae in particular have been observed to be strongly negatively buoyant, hence sinking to the Diatoms or benthic microalgae in particular have been observed to be strongly negatively buoyant, hence sinking to the riverbed and remaining there for a long time (Passow, 1991; Thomas Anderson, 1998). Studies also found sticky compounds 50 in phytoplankton aggregates that are suspected to allow them stick to suspended particles, enabling them to sink to the riverbed or sticking to their surroundings aiding retention (Kiørboe and Hansen, 1993; van der Lee, 2000). In summary, different retention mechanisms have been observed or examined in modeling studies. However, they did so either in isolation in the case of observational studies or with major simpliﬁcations in the modeling studies There is cur rently a lack of theoretical studies that allow for a more comprehensive overview into the interplay of vertical migration and 55 reproduction in combination with settling and stranding as retention mechanisms. Here, we explore possible retention mechanisms of phytoplankton using the Elbe estuary as a case study. It is located in the north of Germany and ﬂows into the North Sea. Like most alluvial estuaries, it is relatively shallow, with most of it averaging only a few meters in average depth. Similar to other European estuaries it experienced a strong anthropogenic pressure over the Here, we explore possible retention mechanisms of phytoplankton using the Elbe estuary as a case study. It is located in the north of Germany and ﬂows into the North Sea. Like most alluvial estuaries, it is relatively shallow, with most of it averaging only a few meters in average depth. Similar to other European estuaries it experienced a strong anthropogenic pressure over the 2 2 Figure 1. Bathymetry of the Elbe model around Hamburg. Note the bathymetric jumps from 5 m on the right, upstream side to a short 10 m step in the upper harbor area to the 20 m in the lower harbor area all the way to the North Sea. Also note that there exists no channel that does not pass through the 10 km of exclusively 20 m deep channel. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Fi 1 B th t f th Elb d l d H b N t th b th t i j f 5 th i ht t id t h t 10 https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 1. Bathymetry of the Elbe model around Hamburg. Note the bathymetric jumps from 5 m on the right, upstream side to a short 10 m step in the upper harbor area to the 20 m in the lower harbor area all the way to the North Sea. Also note that there exists no channel that does not pass through the 10 km of exclusively 20 m deep channel. last centuries. Most notably diking to restrain it to a narrow channel and dredging to improve access to the Hamburg harbor. 60 Unlike other major European harbors, the Hamburg harbor is located far inwards roughly 100 km away from the coast. To create port access the main channel experience a sudden jump in bathymetry from approximately 5 m at border of the city to up to 20 m in the harbor and downstream (see ﬁg. 1). This bathymetric jump is suspected to be the cause of a collapse of the phytoplankton, resulting in an increase in oxygen depletion and high ammonium remineralization downstream of the last centuries. Most notably diking to restrain it to a narrow channel and dredging to improve access to the Hamburg harbor. 60 Unlike other major European harbors, the Hamburg harbor is located far inwards roughly 100 km away from the coast. To create port access the main channel experience a sudden jump in bathymetry from approximately 5 m at border of the city to up to 20 m in the harbor and downstream (see ﬁg. 1). Blue represents ﬂoating, green particles stranded by the receding tide. The red area is the initial release location. The background map has been provided by © OpenStreetMap contributors 2023. Distributed under the Open Data Commons Open Database License (ODbL) v1.0. This bathymetric jump is suspected to be the cause of a collapse of the phytoplankton, resulting in an increase in oxygen depletion and high ammonium remineralization downstream of the bathymetric jump (Schroeder, 1997; Holzwarth and Wirtz, 2018; Sanders et al., 2018). Ongoing dredging is being carried out 65 to maintain the depth of the navigational channel causing high turbidity (Kappenberg and Grabemann, 2001). While important aspects of the along-channel biochemical dynamics have been studied, little is known about their vertical and shore-to-shore dynamics (Goosen et al., 1999; Dähnke et al., 2008; Sanders et al., 2018). For this purpose we further developed the individual-based Lagrangian model OceanTracker (Vennell et al 2021) and For this purpose, we further developed the individual-based Lagrangian model OceanTracker (Vennell et al., 2021) and applied it to the Elbe estuary using the hydrodynamics calculated by a recent model SCHISM (Pein et al., 2021). While 70 the Lagrangian model simulated the movement of the inanimate organism, we included key phytoplankton features such as reproduction and mortality, sinking and rising, as well as diel vertical migration. Using this model, we investigate under which conditions phytoplankton retention can be reproduced. applied it to the Elbe estuary using the hydrodynamics calculated by a recent model SCHISM (Pein et al., 2021). While 70 the Lagrangian model simulated the movement of the inanimate organism, we included key phytoplankton features such as reproduction and mortality, sinking and rising, as well as diel vertical migration. Using this model, we investigate under which conditions phytoplankton retention can be reproduced. applied it to the Elbe estuary using the hydrodynamics calculated by a recent model SCHISM (Pein et al., 2021). While 70 the Lagrangian model simulated the movement of the inanimate organism, we included key phytoplankton features such as reproduction and mortality, sinking and rising, as well as diel vertical migration. Using this model, we investigate under which conditions phytoplankton retention can be reproduced. 3 3 https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 2. Map of the full model domain, with Geesthacht being the upstream boarder on the right and the North-sea being the downstream border on the left. The black outline marks the edge of the model domain. Blue and green dots show an example snapshot of a fraction of the phytoplankton in the model. The location of the initial release is shown in red. 2.1 Model description 75 In our study we use a Lagrangian approach with the particle tracking model OceanTracker (Vennell et al., 2021). While off-line particle tracking on unstructured grids has been relatively computationally expensive until recently (Vennell et al., 2021), it offers several advantages. Firstly, it allows us to reuse computationally expensive hydrodynamic models to model tracer-like objects. This is overall much faster than recalculating the advection-diffusion-equation in an Eularian model. Secondly, because i l i i di id ll i l bl b h i k Thi k h i i f l we are simulating individually particles we are able to observe their tracks. This makes the interpretation of our results more 80 intuitive and allows us to include individual based properties and processes that can not or only indirectly be represented in Eulerian models. we are simulating individually particles we are able to observe their tracks. This makes the interpretation of our results more 80 intuitive and allows us to include individual based properties and processes that can not or only indirectly be represented in Eulerian models. three-dimensional unstructured grid to represent the full Elbe estuary from the weir at Geesthacht to the North Sea, including several side-channels and the harbor area (see ﬁg. 2). The model provides us with a node-based mesh containing a range of 85 information such as water velocity, salinity, water level and dispersion. The year represented in that dataset is 2012 with a temporal resolution of 1 hour and a dynamically varying spacial resolution with distance between nodes ranging from 5 to 1400 m with a median distance of approximately 75 m. several side-channels and the harbor area (see ﬁg. 2). The model provides us with a node-based mesh containing a range of 85 information such as water velocity, salinity, water level and dispersion. The year represented in that dataset is 2012 with a temporal resolution of 1 hour and a dynamically varying spacial resolution with distance between nodes ranging from 5 to 1400 m with a median distance of approximately 75 m. 4 4 Children of light-limited i h i h i i li h b d f h i parents inherit the remaining light budget of their parents. 110 We investigate the effect of different patterns of vertical motion. The ﬁrst is monodirectional upward or downward vertical motion, representing either positively or negatively buoyant phytoplankton. This buoyancy can be interpreted either as an active choice of buoyancy by the organism through adaptation, or as governed by the suspended matter aggregate on which they live. For monodirectional vertical motion, we assign each particle a vertical velocity, which remains constant throughout its lifetime. parents inherit the remaining light budget of their parents. 110 We investigate the effect of different patterns of vertical motion. The ﬁrst is monodirectional upward or downward vertical motion, representing either positively or negatively buoyant phytoplankton. This buoyancy can be interpreted either as an active choice of buoyancy by the organism through adaptation, or as governed by the suspended matter aggregate on which they live. For monodirectional vertical motion, we assign each particle a vertical velocity, which remains constant throughout its lifetime. The second mode of vertical motion is diel vertical migration. Here the particles change their direction of motion based on the 115 current phase of the sun, creating a motion pattern where they rise during the day and sink during the night within the same velocity range. We include a settling and resuspension model to represent tidal stranding and particles settling on the bed of the estuary. Particles become stranded when the current grid cell becomes dry. They are not allowed to move from wet cells to dry cells, by the random walk dispersion applied to all particles. A grid cell is considered dry based on the ﬂag given in the SCHISM 120 hydrodynamic model output. Once this cell is rewetted all stranded particles resuspend and are able to move again. Particles settle on the bed once they attempt to move below the bottom model boundary and are resuspended based on a critical sheer by the random walk dispersion applied to all particles. A grid cell is considered dry based on the ﬂag given in the SCHISM 120 hydrodynamic model output. Once this cell is rewetted all stranded particles resuspend and are able to move again. However, the main motivation for this choice is that most of the particles that die through this process have passed the isohaline for more than 12 hours, one tidal cycle, and are assumed not to return again through this isohaline. Anything outside the 20 PSU isohaline is not considered part of the estuary for the purposes of this study. Therefore, we are not tailoring our salinity tolerance is chosen based on a range of the salinity tolerances of estuarine phytoplankton species presented in (von Alvensleben et al., 100 2016). This is only an approximation and salinity tolerances many estuarine phytoplankton species deviate from this. However, the main motivation for this choice is that most of the particles that die through this process have passed the isohaline for more than 12 hours, one tidal cycle, and are assumed not to return again through this isohaline. Anything outside the 20 PSU isohaline is not considered part of the estuary for the purposes of this study. Therefore, we are not tailoring our salinity tolerance to a speciﬁc species, but rather testing whether they can retain themselves within this isohaline. We consider particles that are 105 stranded outside the water by the receding tide, and lie dry for more than 7 consecutive days to be dead and remove them. Particles also die if they are light-limited for 28 days (Walter et al., 2017). They are considered light-limited at a depth of 1m based on turbidity data presented in (Pein et al., 2021). A particle starts its life with a light budget of 28 days, and each minute below 1m reduces this budget by one minute, while the opposite applies when they are above 1m. Children of light-limited to a speciﬁc species, but rather testing whether they can retain themselves within this isohaline. We consider particles that are 105 stranded outside the water by the receding tide, and lie dry for more than 7 consecutive days to be dead and remove them. Particles also die if they are light-limited for 28 days (Walter et al., 2017). They are considered light-limited at a depth of 1m based on turbidity data presented in (Pein et al., 2021). A particle starts its life with a light budget of 28 days, and each minute below 1m reduces this budget by one minute, while the opposite applies when they are above 1m. OceanTrackers recent advances in computational efﬁciency (Vennell et al., 2021) and buffer handling make it possible to simulate a large number of particles over a long period of time for the ﬁrst time on unstructured grids. We perform several simulations for a range of reproduction rates that are constant over the lifetime of the particle and the chance to reproduce is evaluated every minute. While a ﬁxed reproduction rate 95 is a simpliﬁcation that does not allow for more realistic simulation of the population dynamics of a particular species, it does allow us to investigate the general mechanisms that enable plankton retention. g p g p constant over the lifetime of the particle and the chance to reproduce is evaluated every minute. While a ﬁxed reproduction rate 95 is a simpliﬁcation that does not allow for more realistic simulation of the population dynamics of a particular species, it does allow us to investigate the general mechanisms that enable plankton retention. Mortality is induced either by high salinity, when they dry-out while stranded, or due to long term light limitation. When particles are exposed to high salinity water above 20PSU, a mortality probability of 0.5% per minute is imposed. This threshold particles are exposed to high salinity water above 20PSU, a mortality probability of 0.5% per minute is imposed. This threshold is chosen based on a range of the salinity tolerances of estuarine phytoplankton species presented in (von Alvensleben et al., 100 2016). This is only an approximation and salinity tolerances many estuarine phytoplankton species deviate from this. However, the main motivation for this choice is that most of the particles that die through this process have passed the isohaline for more than 12 hours, one tidal cycle, and are assumed not to return again through this isohaline. Anything outside the 20 PSU isohaline is not considered part of the estuary for the purposes of this study. Therefore, we are not tailoring our salinity tolerance is chosen based on a range of the salinity tolerances of estuarine phytoplankton species presented in (von Alvensleben et al., 100 2016). This is only an approximation and salinity tolerances many estuarine phytoplankton species deviate from this. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. We add a set of biological features on top of the otherwise inanimate organism. These features include reproduction and mortality, vertical movement in form of sinking, rising or diel vertical migration, stranding, and settling on the riverbed. 90 mortality, vertical movement in form of sinking, rising or diel vertical migration, stranding, and settling on the riverbed. 90 Reproduction is represented as a chance of creating copies of themselves. This is a novel feature applied in OceanTracker that has not been included in any previous Lagrangian model of this kind. OceanTrackers recent advances in computational efﬁciency (Vennell et al., 2021) and buffer handling make it possible to simulate a large number of particles over a long period of time for the ﬁrst time on unstructured grids. We perform several simulations for a range of reproduction rates that are constant over the lifetime of the particle and the chance to reproduce is evaluated every minute. While a ﬁxed reproduction rate 95 is a simpliﬁcation that does not allow for more realistic simulation of the population dynamics of a particular species, it does allow us to investigate the general mechanisms that enable plankton retention. Mortality is induced either by high salinity, when they dry-out while stranded, or due to long term light limitation. When particles are exposed to high salinity water above 20PSU, a mortality probability of 0.5% per minute is imposed. This threshold Reproduction is represented as a chance of creating copies of themselves. This is a novel feature applied in OceanTracker that has not been included in any previous Lagrangian model of this kind. OceanTrackers recent advances in computational efﬁciency (Vennell et al., 2021) and buffer handling make it possible to simulate a large number of particles over a long period of time for the ﬁrst time on unstructured grids. We perform several simulations for a range of reproduction rates that are constant over the lifetime of the particle and the chance to reproduce is evaluated every minute. While a ﬁxed reproduction rate 95 Reproduction is represented as a chance of creating copies of themselves. This is a novel feature applied in OceanTracker that has not been included in any previous Lagrangian model of this kind. This allows us for example to compare successfully retained particles (older than three months) unsuccessfully retained particles (dead after less than three months). These observables are recorded every 12 hours starting at midnight. Model simulations and visualizations were performed in Python making heavy use of Numba, a LLVM-based Python JIT compiler (Lam et al., 2015) to signiﬁcantly speed up the simulations (Vennell et al., 2021). Trajectories were calculated using 140 a second order Runge-Kutta scheme with a ﬁxed time step of 60 s. Flow velocities, like any other hydrodynamic data, were interpolated linearly in time and space using barycentric coordinates, with the exception of water velocity in the bottom model cell, where logarithmic vertical interpolation is used. compiler (Lam et al., 2015) to signiﬁcantly speed up the simulations (Vennell et al., 2021). Trajectories were calculated using 140 a second order Runge-Kutta scheme with a ﬁxed time step of 60 s. Flow velocities, like any other hydrodynamic data, were interpolated linearly in time and space using barycentric coordinates, with the exception of water velocity in the bottom model cell, where logarithmic vertical interpolation is used. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. velocity of 0.009 ms−1 (see appendix for details). The velocity proﬁle in the bottom layer, or log layer, is calculated by U(z) = u∗ κ ln z z0 , ( velocity of 0.009 ms−1 (see appendix for details). The velocity proﬁle in the bottom layer, or log layer, is calculated by U(z) = u∗ κ ln z z0 , (1 U(z) = u∗ κ ln z z0 , (1) where U is the friction velocity representing the drag at height z above the seabed, κ is the van Karman constant, z0 is a length 125 scale reﬂecting the bottom roughness, and u∗is the critical friction velocity. If the friction velocity is above the critical friction velocity the particle is resuspended. Particles that are stranded or settled on the bed are allowed to reproduce. Particles are not only advected but also dispersed based on eddy diffusivity. This allows us implement a dynamic dispersion that is crucial to represent tidal-pumping processes. Dispersion was modeled using a random walk using a random number generator with a normal distribution. Horizontally the standard distribution of the random walk was set to 0.1 ms−1. The displacement by 130 vertical dispersion ∂z of particle i is calculated by a normal distribution. Horizontally the standard distribution of the random walk was set to 0.1 ms . The displacement by 130 vertical dispersion ∂z of particle i is calculated by ∂zi = K ′ v(zi(n))∂t + N(0,2Kv(zi)) (2) based on Yamazaki et al. (2014) where zi is the vertical position of the particle, K ′ v is the vertical eddy diffusivity gradient, Kv is the vertical eddy diffusivity and N is the normal distribution. The term based K ′ v is needed to avoid particle accumulation (2) ∂zi = Kv(zi(n))∂t + N(0,2Kv(zi)) (2) based on Yamazaki et al. (2014) where zi is the vertical position of the particle, K ′ v is the vertical eddy diffusivity gradient, Kv is the vertical eddy diffusivity and N is the normal distribution. The term based K ′ v is needed to avoid particle accumulation on the top and bottom of the water column from the hydrodynamic model output. 135 For each particle we log their distance traveled, age, water depth, and status (whether they are drifting or settled on the river bank or bottom). Particles settle on the bed once they attempt to move below the bottom model boundary and are resuspended based on a critical sheer by the random walk dispersion applied to all particles. A grid cell is considered dry based on the ﬂag given in the SCHISM 120 hydrodynamic model output. Once this cell is rewetted all stranded particles resuspend and are able to move again. Particles settle on the bed once they attempt to move below the bottom model boundary and are resuspended based on a critical sheer 5 3 Results 3 Results 3.1 Retentions success in different scenarios The results of the retention experiments are visualized as heatmap in ﬁg. 3. Fig. 3a shows the results for the monodirectional vertical migration scenarios i.e. constant sinking or rising. Fig. 3b shows the results for the diel vertical migration scenarios. Each pixel in the heatmap represents a simulation with a speciﬁc combination of vertical velocity and reproduction rate. The 70 coloring indicates the relative population change after one year. White pixels and the boundary between green and brown pixels represent net zero growth rate simulations In this case the losses are equal to the growth Therefore we can use the The results of the retention experiments are visualized as heatmap in ﬁg. 3. Fig. 3a shows the results for the monodirectional vertical migration scenarios i.e. constant sinking or rising. Fig. 3b shows the results for the diel vertical migration scenarios. vertical migration scenarios i.e. constant sinking or rising. Fig. 3b shows the results for the diel vertical migration scenarios. Each pixel in the heatmap represents a simulation with a speciﬁc combination of vertical velocity and reproduction rate. The 170 coloring indicates the relative population change after one year. White pixels and the boundary between green and brown pixels represent net-zero growth rate simulations. In this case, the losses are equal to the growth. Therefore, we can use the reproduction rate as an estimate for the total relative losses due to downstream transport, drying out while being stranded, and light starvation. Our simulations show that the population is able to successfully retains itself under certain conditions. Passively drifting 175 particles are able to sustain themselves in the estuary if they have a reproduction rate that doubles their population size within approximately 3 months. Note that the growth rates realized in nature may vary from this value due to e.g. nutrient or temper- ature limitations. The reproduction thresholds should be interpreted as an upper bound rather than an accurate estimate of the growth rate. For the case of the monodirectional movement we see that a higher positive velocity (representing buoyancy) and higher 180 reproduction rates are more beneﬁcial for retention success than a downward oriented velocity (sinking) and lower reproduction rates. As expected, simulations in which the reproduction is set to zero do not show any retention success. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. active simultaneously counted over all cases over 500,000 time steps. The initial population is homogeneously distributed in a volume covering the full water column at the weir in Geesthacht (see ﬁg. 2) and examine how the population distributes itself over the estuary and whether it is able to maintain its population size over time. Conceptually, we consider a population to be successfully retained if it is showing long term growth. Practically, this is evaluated by comparing the population size at the end of the year to the size after release The ﬁrst three months of the simulations are considered as initial model spin up time 60 active simultaneously counted over all cases over 500,000 time steps. The initial population is homogeneously distributed in a volume covering the full water column at the weir in Geesthacht (see ﬁg. 2) and examine how the population distributes itself over the estuary and whether it is able to maintain its population size over time. Conceptually, we consider a population to be successfully retained if it is showing long term growth. Practically, this is evaluated by comparing the population size at the end of the year to the size after release. The ﬁrst three months of the simulations are considered as initial model spin-up time 160 during which the initial population is dispersed downstream throughout the estuary. Population size changes are measured at the end of the year relative to the population size after this initial spin-up time. active simultaneously counted over all cases over 500,000 time steps. The initial population is homogeneously distributed in a volume covering the full water column at the weir in Geesthacht (see ﬁg. 2) and examine how the population distributes itself over the estuary and whether it is able to maintain its population size over time. Conceptually, we consider a population to be successfully retained if it is showing long term growth. Practically, this is evaluated by comparing the population size at the 160 end of the year to the size after release. The ﬁrst three months of the simulations are considered as initial model spin-up time 160 during which the initial population is dispersed downstream throughout the estuary. Population size changes are measured at the end of the year relative to the population size after this initial spin-up time. 2.2 Experimental conﬁgurations We perform two sets of experiments to test the inﬂuence of different vertical movements on the retention success of phyto- 145 plankton in the Elbe estuary. In the ﬁrst experiment, we examine a range of different monodirectional upward or downward particle velocities from −10 to +10 mm s−1 in 2 mm s−1 steps representing sinking or rising phytoplankton organisms (Fennessy and Dyer, 1996). Each vertical velocity is examined for a range of different reproduction rates, expressed as population doubling times in idealized We perform two sets of experiments to test the inﬂuence of different vertical movements on the retention success of phyto- 145 plankton in the Elbe estuary. In the ﬁrst experiment, we examine a range of different monodirectional upward or downward particle velocities from −10 to +10 mm s−1 in 2 mm s−1 steps representing sinking or rising phytoplankton organisms (Fennessy and Dyer, 1996). Each vertical velocity is examined for a range of different reproduction rates, expressed as population doubling times in idealized conditions ranging from 40 to 404 days with a logarithmic scaling. In the following, we will use reproduction rate to refer to 150 the prescribed population growth rate under ideal conditions and use growth rate whenever we describe population growth in nature. Hence, a total of 187 different scenarios are tested. In the second set of model experiments, we study the inﬂuence of possible diel vertical migration patterns for the same vertical velocities and reproduction rates. In both sets of experiments, we release 10,000 individuals representing the studied phytoplankton population at the beginning of the year. This results in over 1 billion individual particles simulated for each case with approximately 1 million particles 155 In both sets of experiments, we release 10,000 individuals representing the studied phytoplankton population at the beginning of the year. This results in over 1 billion individual particles simulated for each case with approximately 1 million particles 155 6 Computations were performed on the supercomputer Mistral at the German Climate Computing Center (DKRZ) in Hamburg, Germany. The simulations were performed on a compute node with two Intel Xeon E5-2680 v3 12-core processor (Haswell) and 128 GB of RAM with a total run time of approximately 4.5 hours. 165 3.1 Retentions success in different scenarios While it is easy to understand that high reproduction rates aid retention, we were surprised that buoyant phytoplankton particles are more successful in maintaining their growth in an estuary than sinking ones. For the case of the diel vertical migration in the velocity range of 4 to 10 mm s−1 we see an equal or higher retention 185 success compared to the case with no vertical migration. A diel velocity of 2 mm s−1 is less successful than no migration. Most importantly, none of the diel migration scenarios improve the retention success, when compared to passively drifting organisms. For the case of the diel vertical migration in the velocity range of 4 to 10 mm s−1 we see an equal or higher retention 185 success compared to the case with no vertical migration. A diel velocity of 2 mm s−1 is less successful than no migration. Most importantly, none of the diel migration scenarios improve the retention success, when compared to passively drifting organisms. 7 7 3.2 Spatial factors We are now taking a closer look at spacial factors that allow phytoplankton particles to maintain net growth in the estuary. For 190 this analysis we used data from both sets of experiments. Fig. 4 compares two box plots showing the average water depth at the location of each particle between particles that remained alive for less than three months (short-living) and for more than three months (long-living). Depth is measured relative to the current water surface. Hence, a value above zero indicate that the particle is stranded on the river bank. These analyses demonstrate that long-living particles predominantly live close to the river banks in shallower waters or on tidal ﬂats. 195 We moreover analyzed the horizontal spacial distribution of long and short-living particles in ﬁg. 5. To do this, we divide the model domain into equally sized hexagons. The color of each hexagon indicates the average age of the particles within it. Hexagons with a yellow color indicate an average age of over three months. These yellow areas are mainly found along the river banks in shallow waters or tidal ﬂats. To further investigate the reasons for the positive effect of buoyancy and the importance of shallow waters and tidal ﬂats, 200 we repeated the ﬁrst set of simulations and disabled the reproduction of settled and stranded particles. Under these conditions, populations were unable to retain themselves in the estuary, regardless of their vertical velocity and reproduction rate indicating that tidal ﬂats are essential for the survival of the population. To further investigate the reasons for the positive effect of buoyancy and the importance of shallow waters and tidal ﬂats, 200 we repeated the ﬁrst set of simulations and disabled the reproduction of settled and stranded particles. Under these conditions, populations were unable to retain themselves in the estuary, regardless of their vertical velocity and reproduction rate indicating that tidal ﬂats are essential for the survival of the population. To further investigate the reasons for the positive effect of buoyancy and the importance of shallow waters and tidal ﬂats, 200 we repeated the ﬁrst set of simulations and disabled the reproduction of settled and stranded particles. Under these conditions, populations were unable to retain themselves in the estuary, regardless of their vertical velocity and reproduction rate indicating that tidal ﬂats are essential for the survival of the population. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 3. Relative population changes for the monodirectional movement (a) and diel migration (b) scenarios. Positive vertical velocities indicate an upwards drift. Positive population changes represent a retention success (green) while negative population changes represent a loss of the population (brown). The vertical black lines indicate the boundary between successfully and unsuccessfully retained scenarios. Figure 3. Relative population changes for the monodirectional movement (a) and diel migration (b) scenarios. Positive vertical velocities indicate an upwards drift. Positive population changes represent a retention success (green) while negative population changes represent a loss of the population (brown). The vertical black lines indicate the boundary between successfully and unsuccessfully retained scenarios. Figure 3. Relative population changes for the monodirectional movement (a) and diel migration (b) scenarios. Positive vertical velocities indicate an upwards drift. Positive population changes represent a retention success (green) while negative population changes represent a loss of the population (brown). The vertical black lines indicate the boundary between successfully and unsuccessfully retained scenarios. 8 8 Figure 4. Box and violin plot showing the vertical distribution of particles that are passively drifting. Short-living are those younger than 3 months and long-living all those older than that. Depth is measured relative to the current water surface with positive numbers being above the water surface i.e. stranded on shore. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. Box and violin plot showing the vertical distribution of particles that are passively drifting. Short-living are those younger than 3 months and long-living all those older than that. Depth is measured relative to the current water surface with positive numbers being above the water surface i.e. stranded on shore. 3.3 Interpretation and contextualization of Results In this study, we investigated different strategies to explain how phytoplankton populations are able to maintain their population 205 growth in estuaries while constantly being at risk to be transported into the open ocean. 9 Figure 5. Hex-bin heatmap of the Elbe with Hamburg in the bottom right showing average particle age per bin. Colors indicate the age of the particles, with yellowish colors indicating an average age of over three months. Yellow areas are mainly found along the river banks in shallow waters or tidal ﬂats. The important areas are Mühlenberger Loch (a), Wedeler Marsch (b) Haseldorfer Binnenelbe (c), Asseler- and Schwarztonnensand (d), at the mouth of Wischhafener Süderelbe (f), , and Stör (e), and at Nordkedding (g) and Neufelder Marsch (h). https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 5. Hex-bin heatmap of the Elbe with Hamburg in the bottom right showing average particle age per bin. Colors indicate the age of the particles, with yellowish colors indicating an average age of over three months. Yellow areas are mainly found along the river banks in shallow waters or tidal ﬂats. The important areas are Mühlenberger Loch (a), Wedeler Marsch (b) Haseldorfer Binnenelbe (c), Asseler- and Schwarztonnensand (d), at the mouth of Wischhafener Süderelbe (f), , and Stör (e), and at Nordkedding (g) and Neufelder Marsch (h). Figure 5. Hex-bin heatmap of the Elbe with Hamburg in the bottom right showing average particle age per bin. Colors indicate the age of the particles, with yellowish colors indicating an average age of over three months. Yellow areas are mainly found along the river banks in shallow waters or tidal ﬂats. The important areas are Mühlenberger Loch (a), Wedeler Marsch (b) Haseldorfer Binnenelbe (c), Asseler- and Schwarztonnensand (d), at the mouth of Wischhafener Süderelbe (f), , and Stör (e), and at Nordkedding (g) and Neufelder Marsch (h). The limit for population doubling times that we found to be necessary for survival for passively drifting plankton is of the order of around 4 months. Doubling times typically realized in natures are of the order of a few days which is two magnitudes small then those that we found necessary in our model (Koch et al., 2004; Wirtz, 2011). 3.3 Interpretation and contextualization of Results The low reproduction rates required for successful retention demonstrate that our model is also meaningful under more realistic environmental conditions, for example 0 if maximum growth rates cannot be reached due to nutrient or temperature limitations. The limit for population doubling times that we found to be necessary for survival for passively drifting plankton is of the order of around 4 months. Doubling times typically realized in natures are of the order of a few days which is two magnitudes small then those that we found necessary in our model (Koch et al., 2004; Wirtz, 2011). The low reproduction rates required for successful retention demonstrate that our model is also meaningful under more realistic environmental conditions, for example if maximum growth rates cannot be reached due to nutrient or temperature limitations. The limit for population doubling times that we found to be necessary for survival for passively drifting plankton is of the order of around 4 months. Doubling times typically realized in natures are of the order of a few days which is two magnitudes small then those that we found necessary in our model (Koch et al., 2004; Wirtz, 2011). The low reproduction rates required for successful retention demonstrate that our model is also meaningful under more realistic environmental conditions, for example 0 if maximum growth rates cannot be reached due to nutrient or temperature limitations. Our results suggest that shallow areas are very important for maintaining the estuary phytoplankton population. Plankton that consistently ﬁnds itself in areas that are dry due to the tides will regularly become stranded and therefore not move for much of the tidal cycle. We further see that positively buoyant plankton are more successful in retaining themselves. This is probably because they are more likely to be transported high up on the river bank where the water is less likely to reach them. 215 This effect is emphasized in ﬂatter regions as the distance between the wash margin and constantly ﬂooded areas is larger, increasing the chance of settlement or them stranding again. Initially, we expected sinking particles to have a higher retention success than buoyant ones. However, we found that faster sinking particles are less successful in retaining themselves. Sinking velocities of less than 2mm s−1 are common for diatoms (Passow, 1991) while larger velocities have been observed for aggregates in the Elbe estuary (Fennessy and Dyer, 1996). https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. We suspect that the reason for the increased retention success of diel migrating organisms is similar to the monodirectional 230 case. When the upwards diel migration coincides with high tide, particles are more likely to be stranded far out on the shore, reducing their risk of being washed out quickly. The higher the upward velocities, the greater the chance of being at the waterline during high tide. However, because they are sinking for half of the day they also tend to be light limited more frequent than positively buoyant particles. It seems like these favorable and unfavorable processes balance each other out, resulting in a similar retention success as for the monodirectional case. 235 We suspect that the reason for the increased retention success of diel migrating organisms is similar to the monodirectional 230 case. When the upwards diel migration coincides with high tide, particles are more likely to be stranded far out on the shore, reducing their risk of being washed out quickly. The higher the upward velocities, the greater the chance of being at the waterline during high tide. However, because they are sinking for half of the day they also tend to be light limited more frequent than positively buoyant particles. It seems like these favorable and unfavorable processes balance each other out, resulting in a similar retention success as for the monodirectional case. 235 3.3 Interpretation and contextualization of Results 220 Sinking particles have a reduced downstream velocity because ﬁnd themselves either settled on the riverbed not moving at all or close to the bed where the average downstream velocity is lower. In addition, the deeper water layers of the Elbe have on average a lower downstream velocity than the upper water column or move upstream due to temperature-induced density stratiﬁcation (Pein et al., 2021). Nevertheless, buoyant particles were more successful in their retention in our simulations. (Passow, 1991) while larger velocities have been observed for aggregates in the Elbe estuary (Fennessy and Dyer, 1996). 220 Sinking particles have a reduced downstream velocity because ﬁnd themselves either settled on the riverbed not moving at all or close to the bed where the average downstream velocity is lower. In addition, the deeper water layers of the Elbe have on average a lower downstream velocity than the upper water column or move upstream due to temperature-induced density stratiﬁcation (Pein et al., 2021). Nevertheless, buoyant particles were more successful in their retention in our simulations. The low chance of survival in the estuary for sinking particles might be explained by their light limitation in deeper waters. 225 We expected particles to die if they are exposed to dark conditions for more than two weeks. Thus, sinking particles have a disadvantage to buoyant particles since they are more likely to become light limited and eventually die. This suggests that dredging has a negative impact on sinking plankton because it increases both depth and turbidity (de Jonge et al., 2014), which increases the aphotic depth and therefore the volume of dark water relative to the amount of light water. The low chance of survival in the estuary for sinking particles might be explained by their light limitation in deeper waters. 225 We expected particles to die if they are exposed to dark conditions for more than two weeks. Thus, sinking particles have a disadvantage to buoyant particles since they are more likely to become light limited and eventually die. This suggests that dredging has a negative impact on sinking plankton because it increases both depth and turbidity (de Jonge et al., 2014), which increases the aphotic depth and therefore the volume of dark water relative to the amount of light water. 10 3.4 Model limitations & future perspectives In this study, we aimed to thoroughly investigate different possible retention mechanisms in a complex Lagrangian model system with a highly resolved bathymetry. Due to this computational and spatial complexity, the complexity of the biological particle properties needed to remain simple to keep computational cost manageable and due to a lack of high resolution validation data. 240 Our model design does not resolve more complex ecosystem dynamics such as nutrient limitation and grazing by higher trophic levels. The Lagrangian model is performed ofﬂine, meaning it is not coupled to the Eulerian model that calculates the hydrodynamics and is performed after the fact. Therefore, modeling the advection and dispersal of changes in concentration ﬁelds e.g. nutrients due to growth or remineralization was not easily possible. Future modeling efforts could couple the La- grangian model to a Eulerian model that disperses changes in concentrations ﬁelds by biotic activity throughout the model 245 domain. However, this would have drastically increased both, developing and computational time to a point where it would have been infeasible in our time frame and due to the lack of appropriate validation data. The key draw back of this is that growth rates could only be modeled as a constant rate in the current model description, similar to "ad libitum" experiments. This can lead to systematic errors in estimating population growth. In reality, it can happen that nutrient limitation, which slows grangian model to a Eulerian model that disperses changes in concentrations ﬁelds by biotic activity throughout the model 245 domain. However, this would have drastically increased both, developing and computational time to a point where it would have been infeasible in our time frame and due to the lack of appropriate validation data. The key draw back of this is that growth rates could only be modeled as a constant rate in the current model description, similar to "ad libitum" experiments. This can lead to systematic errors in estimating population growth. In reality, it can happen that nutrient limitation, which slows down the growth of the population, can occur, especially in the most light-saturated areas near the shore. For this reason, we 250 may overestimate the role of shallow areas in our model. To be consistent with the complexity of the representation of biotic mechanisms, we use a simplistic light limitation. 3.4 Model limitations & future perspectives Particles are expected to be light limited below a water depth of 1 m and not light limited above this threshold. We have not included a more complex light limitation model that takes into account current light availability and attenuation. A more realistic down the growth of the population, can occur, especially in the most light-saturated areas near the shore. For this reason, we 250 may overestimate the role of shallow areas in our model. To be consistent with the complexity of the representation of biotic mechanisms, we use a simplistic light limitation. Particles are expected to be light limited below a water depth of 1 m and not light limited above this threshold. We have not included a more complex light limitation model that takes into account current light availability and attenuation. A more realistic formulation of light limitation could particularly favor particles that exhibit diel vertical migration. A process we mostly ignore 255 in our study is dormancy. Our organisms can survive for 28 days in light limited waters. However, phytoplankton species have life stages in which they can remain dormant for a longer period of time and germinate again when they ﬁnd themselves in more favorable waters (Thomas Anderson, 1998). In the process of choosing the light limitation threshold, we conducted sensitivity studies testing the effect of higher light budgets. We found that light budgets over 3 months begin to signiﬁcantly increase the survivability of sinking organisms when we crudely assume that they could still reproduce under these conditions Whether 260 formulation of light limitation could particularly favor particles that exhibit diel vertical migration. A process we mostly ignore 255 in our study is dormancy. Our organisms can survive for 28 days in light limited waters. However, phytoplankton species have life stages in which they can remain dormant for a longer period of time and germinate again when they ﬁnd themselves in more favorable waters (Thomas Anderson, 1998). In the process of choosing the light limitation threshold, we conducted sensitivity studies testing the effect of higher light budgets. We found that light budgets over 3 months begin to signiﬁcantly increase the survivability of sinking organisms, when we crudely assume that they could still reproduce under these conditions. Whether 260 dormancy plays a signiﬁcant role in an environment where the river bed is continuously dredged is unknown. 3.4 Model limitations & future perspectives Another limitation in our modelling efforts is the lack of sub-grid-resolution structure on the shores. In our representation we assume perfectly ﬂat surfaces with a median distance between nodes of approximately 60 m. This ’polished’ model rep- survivability of sinking organisms, when we crudely assume that they could still reproduce under these conditions. Whether 260 dormancy plays a signiﬁcant role in an environment where the river bed is continuously dredged is unknown. Another limitation in our modelling efforts is the lack of sub-grid-resolution structure on the shores. In our representation Another limitation in our modelling efforts is the lack of sub-grid-resolution structure on the shores. In our representation we assume perfectly ﬂat surfaces with a median distance between nodes of approximately 60 m. This ’polished’ model rep- 11 Future monitoring 275 efforts should therefore also include data along the river shores on tidal ﬂats or shore-to-shore to quantify the effect of potential future changes by dredging, diking or restoration attempts. Frequently stranded plankton have been shown to be essential to the survival of populations in our model. However, data on their ability to survive under these conditions are scarce. Our results suggest that these conditions may be as important as their Chlorophyll data with a sufﬁcient temporal and spacial resolution is only gathered in the center of the river. Future monitoring 275 efforts should therefore also include data along the river shores on tidal ﬂats or shore-to-shore to quantify the effect of potential future changes by dredging, diking or restoration attempts. Frequently stranded plankton have been shown to be essential to the survival of populations in our model. However, data on their ability to survive under these conditions are scarce. Our results suggest that these conditions may be as important as their ability to quickly regrow under more favorable conditions, and we suggest further research on plankton survivability when 280 stranded. For several decades, the annual average chlorophyll concentration in the Elbe estuary has been decreasing (data available at www.fgg-elbe.de/elbe-datenportal.html or see (Hardenbicker et al., 2014; Schöl et al., 2014)), while upstream concentrations do not show this effect. The reasons for this are not fully understood, but one possible reason is the increase in dredge activity. ability to quickly regrow under more favorable conditions, and we suggest further research on plankton survivability when 280 stranded. For several decades, the annual average chlorophyll concentration in the Elbe estuary has been decreasing (data available at www.fgg-elbe.de/elbe-datenportal.html or see (Hardenbicker et al., 2014; Schöl et al., 2014)), while upstream concentrations do not show this effect. The reasons for this are not fully understood, but one possible reason is the increase in dredge activity. ability to quickly regrow under more favorable conditions, and we suggest further research on plankton survivability when 280 stranded. For several decades, the annual average chlorophyll concentration in the Elbe estuary has been decreasing (data available at www.fgg-elbe.de/elbe-datenportal.html or see (Hardenbicker et al., 2014; Schöl et al., 2014)), while upstream concentrations do not show this effect. The reasons for this are not fully understood, but one possible reason is the increase in dredge activity. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. resentation can lead to an underestimation of the retention success, since the surface area on which phytoplankton organisms can settle is underestimated. In reality, vegetation, rocks or other surface irregularities cause a larger surface area on which the 265 phytoplankton organisms can settle. Our hydrodynamics data set was limited to the year 2012. Therefore, we were not able to study different release times with the same methodology. While we do not expect the general dynamics to change, future research could examine the effect of varying discharge throughout the seasons on retention. , p y p g can settle is underestimated. In reality, vegetation, rocks or other surface irregularities cause a larger surface area on which the 265 phytoplankton organisms can settle. Our hydrodynamics data set was limited to the year 2012. Therefore, we were not able to study different release times with the same methodology. While we do not expect the general dynamics to change, future research could examine the effect of varying discharge throughout the seasons on retention. While our model does have a settling and resuspension mechanic based on critical sheer velocities we still assume a static 270 bathymetry with sediments not able to move or bury particles. This masks potential losses due to particles being buried but also decreases resuspension times. Our results clearly suggest the importance of tidal ﬂats and shallow areas along the river banks for the successful maintenance of primary production in the Elbe estuary. However, their effect can currently not be quantiﬁed due to the lack of validation data. While our model does have a settling and resuspension mechanic based on critical sheer velocities we still assume a static 270 bathymetry with sediments not able to move or bury particles. This masks potential losses due to particles being buried but also decreases resuspension times. also decreases resuspension times. Our results clearly suggest the importance of tidal ﬂats and shallow areas along the river banks for the successful maintenance of primary production in the Elbe estuary. However, their effect can currently not be quantiﬁed due to the lack of validation data. Chlorophyll data with a sufﬁcient temporal and spacial resolution is only gathered in the center of the river. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. This increases the average turbidity and thus the aphotic depth, reducing the volume of water in which phytoplankton can 285 grow. A large fraction of the phytoplankton measured upstream of Hamburg harbor consists of diatoms (Muylaert and Sabbe, 1999), which typically have negative buoyancy (Passow, 1991), making them particularly susceptible to sinking in light-limited waters. Our ﬁnding that sinking particles have a harder time surviving in the estuary supports this theory. Another mechanism that might in part explain the drop in phytoplankton concentration at the bathymetric jump, which is not yet explored in our model, is the phytoplankton stickiness. Phytoplankton, especially blooming one, has been shown 290 to be sticky due to exudates (Kiørboe and Hansen, 1993; van der Lee, 2000; Dutz et al., 2005). Some phytoplankton also produce transparent exopolymer particles, which increase their stickiness to other particles (Windler et al., 2015; De Brouwer et al., 2005). We suspect that this in combination with higher turbidity induced by dredging results in losses due to plankton aggregates sticking to negatively buoyant suspended matter and subsequently sinking to the ground where they are starved of light. A future model study could create estimates on chlorophyll concentration losses caused by this effect. 295 12 4 Conclusions In this study, we investigated the role of different possible retention strategies for drifting phytoplankton organisms to remain in an estuarine environment. Our model simulations suggest that realistically high growth rates are sufﬁcient for populations to prevent decline of estuarine populations. Moreover, buoyancy and strong diel vertical migration enhance retention within the estuary. The reason for this is that phytoplankton organisms with strong buoyancy or the potential to move upward in the 300 water column are more likely to be transported to shallow areas near the river banks or tidal ﬂats, where currents are slower and the chances of settlement under sufﬁciently light conditions to allow reproduction are higher than in deeper waters. These results illustrate the importance of shallow nearshore areas to allowing the productivity of estuarine ecosystems to persist. Our results also highlight the need for informed ecosystem-based management to avoid the degradation of estuarine ecosystems by dredging and diking activities. 305 in an estuarine environment. Our model simulations suggest that realistically high growth rates are sufﬁcient for populations to prevent decline of estuarine populations. Moreover, buoyancy and strong diel vertical migration enhance retention within the estuary. The reason for this is that phytoplankton organisms with strong buoyancy or the potential to move upward in the 300 water column are more likely to be transported to shallow areas near the river banks or tidal ﬂats, where currents are slower and the chances of settlement under sufﬁciently light conditions to allow reproduction are higher than in deeper waters. These results illustrate the importance of shallow nearshore areas to allowing the productivity of estuarine ecosystems to persist. Our results also highlight the need for informed ecosystem-based management to avoid the degradation of estuarine ecosystems by dredging and diking activities. 305 Code and data availability. Input data can be requested from Johannes Pein (johannes.pein@hereon.de). OceanTracker’s source code is available atgithub.com/oceantracker/oceantracker. Model conﬁguration and output is available at doi.org/10.25592/uhhfdm.13235 Code and data availability. Input data can be requested from Johannes Pein (johannes.pein@hereon.de). OceanTracker’s source code is available atgithub.com/oceantracker/oceantracker. Model conﬁguration and output is available at doi.org/10.25592/uhhfdm.13235 Author contributions. LS and RV contributed to conception of the study. LS designed the studies details and organized the hydrodynamic data. RV provided the source code for OceanTracker. RS,LS improved on the original physical model and LS developed the biological model. LS performed the model simulations, post-processing, and visualization. LS wrote the draft of the manuscript. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2231 Preprint. Discussion started: 5 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. 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An assessment by means of stable nitrate isotopes in the Elbe estuary, 335 Limnology and Oceanography, 53, 1504–1511, https://doi.org/10.4319/lo.2008.53.4.1504, 2008. De Brouwer, J. F., Wolfstein, K., Ruddy, G. K., Jones, T. E., and Stal, L. 4 Conclusions All authors contributed to 310 manuscript revision, read, and approved the submitted version. Author contributions. LS and RV contributed to conception of the study. LS designed the studies details and organized the hydrodynamic data. RV provided the source code for OceanTracker. RS,LS improved on the original physical model and LS developed the biological model. LS performed the model simulations, post-processing, and visualization. LS wrote the draft of the manuscript. All authors contributed to 310 manuscript revision, read, and approved the submitted version. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. We thank Johannes Pein for providing and supporting the implementation of the hydrodynamic data, Jana Hinners for her guidance through the project, and Hans Burchard for his input on dispersion. Further, we thank, Sina Remmers and Philipp Porada for providing helpful comments on the manuscript. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research 315 Foundation) within the Research Training Group 2530: “Biota-mediated effects on Carbon cycling in Estuaries” (project number 407270017), contribution to Universität Hamburg and Leibniz-Institut für Gewässerökologie und Binnenﬁscherei (IGB) im Forschungsverbund Berlin e.V. Acknowledgements. We thank Johannes Pein for providing and supporting the implementation of the hydrodynamic data, Jana Hinners for her guidance through the project, and Hans Burchard for his input on dispersion. Further, we thank, Sina Remmers and Philipp Porada for providing helpful comments on the manuscript. 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https://openalex.org/W3036304317	https://sajbm.org/index.php/sajbm/article/download/1817/1572	English	null	Can involvement increase trust in a confusing online setting? Implications for marketing strategy	South African journal of business management	2,020	cc-by	9,005	South African Journal of Business Management ISSN: (Online) 2078-5976, (Print) 2078-5585 South African Journal of Business Management ISSN: (Online) 2078-5976, (Print) 2078-5585 Page 1 of 10 Original Research Page 1 of 10 Original Research Page 1 of 10 Original Research Page 1 of 10 Dates: Received: 20 Oct. 2019 Accepted: 19 Mar. 2020 Published: 22 June 2020 Practical implications: To increase customer trust, marketers should invite regular customers to recommend their products and services, become associated with e-service providers and brands and design distinct logos, slogans and advertising styles. How to cite this article: Tzeng, S.-Y., & Shiu, J.Y. (2020). Can involvement increase trust in a confusing online setting? Implications for marketing strategy. South African Journal of Business Management, 51(1), a1817. https://doi.org/10.4102/ sajbm.v51i1.1817 Originality/value: This paper explores the direct effect of involvement on trust in multiple online contextual situations (e.g., platforms, brands and e-vendors), as well as the moderating effect of confusion on the involvement–trust relationship. Keywords: online transactions; confusion; involvement; trust; China. Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Can involvement increase trust in a confusing online setting? Implications for marketing strategy Authors: Shian-Yang Tzeng1 Jerry Y. Shiu2 Affiliations: 1Cultural Creativity and Tourism School, Guangdong University of Finance and Economics, Guangzhou, China 2School of Business, Macau University of Science and Technology, Macau, China Corresponding author: Jerry Shiu, ywshiu@must.edu.mo Dates: Received: 20 Oct. 2019 Accepted: 19 Mar. 2020 Published: 22 June 2020 Authors: Shian-Yang Tzeng1 Jerry Y. Shiu2 Affiliations: 1Cultural Creativity and Tourism School, Guangdong University of Finance and Economics, Guangzhou, China 2School of Business, Macau University of Science and Technology, Macau, China Corresponding author: Jerry Shiu, ywshiu@must.edu.mo Dates: Received: 20 Oct. 2019 Accepted: 19 Mar. 2020 Published: 22 June 2020 Authors: Shian-Yang Tzeng1 Jerry Y. Shiu2 Affiliations: 1Cultural Creativity and Tourism School, Guangdong University of Finance and Economics, Guangzhou, China 2School of Business, Macau University of Science and Technology, Macau, China Corresponding author: Jerry Shiu, ywshiu@must.edu.mo Dates: Received: 20 Oct. 2019 Accepted: 19 Mar. 2020 Published: 22 June 2020 Purpose: Customer trust toward e-commerce has been unsettled by recent unethical events. Involvement, the level of personal relevance of an object or event, has been proved to enhance trust. Nevertheless, in a complicated online shopping environment, the relationship between involvement and trust might undergo changes. Therefore, this study aims to investigate how consumer involvement can be translated into trust that is crucial to the success of online transactions in such a confusing setting. Design/methodology/approach: This study explores the relationships between involvement (i.e., purchase and product involvement) and trust (i.e., trust in e-vendors, retail websites and brands). This study also tests the moderating effects of confusion on the purchase involvement– trust relationship. Using 570 effective samples randomly collected in Guangdong, China, this study employed structural equation modelling to test a proposed hypotheses. Findings: The results show that purchase involvement has a negative impact on trust in e-vendors, retail websites and brands, whereas product involvement demonstrates a positive effect. Moreover, confusion reinforces the negative relationship between purchase involvement and trust in e-vendors. Introduction Because of the characteristics of Internet transactions, consumer trust is the foundation of e-commerce (Kim, Ferrin, & Rao, 2008). However, the emergence of negative news, such as Facebook’s privacy breach (Vogelstein, 2018), has perturbed consumer–supplier trust. For example, according to a recent survey of 25 countries (CIGI-Ipsos, 2018), 57% of the respondents expressed their increasing concerns about online privacy and 12% of the participants claimed that they would make fewer online purchases in the future. Moreover, in China, 21.5% of cross-border e-commerce users have confronted quality problems; and 39.4% of interviewed shoppers reported that they will switch to other platforms after learning about the quality problems of online commodities (iiMedia Report, 2018). The aforementioned situations might decrease customer trust toward web-based retailing. Therefore, determining how to regain customer trust in online transactions is essential for both practitioners and scholars. Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Copyright: Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Involvement has been proven to enhance trust beliefs (Chen, Wu, & Chien, 2016; Sanchez-Franco, 2009). Customers with a high level of knowledge about a service tend to make better purchase decisions than those with a low level of knowledge (Yang, Hung, Kai, & Farn, 2006). When consumers invest long-term, considerable effort in searching for and processing information or expressing ongoing concerns about products or services, the perceived risks associated with the products or services can be reduced (Bloch & Richins, 1983). Therefore, enduring involvement (i.e., product involvement) can enable customers to effectively assess retailers’ capabilities and benevolence, which in turn helps to build customers’ trust (Chen et al., 2016; Sanchez-Franco, 2009). By contrast, other consumers spend time and effort seeking or processing information to evaluate alternatives only during the purchase process because of the high perceived risks. This type of situational involvement (i.e., purchase involvement) is temporarily established, which can Involvement has been proven to enhance trust beliefs (Chen, Wu, & Chien, 2016; Sanchez-Franco, 2009). Customers with a high level of knowledge about a service tend to make better purchase decisions than those with a low level of knowledge (Yang, Hung, Kai, & Farn, 2006). When consumers invest long-term, considerable effort in searching for and processing information or expressing ongoing concerns about products or services, the perceived risks associated with the products or services can be reduced (Bloch & Richins, 1983). Therefore, enduring involvement (i.e., product involvement) can enable customers to effectively assess retailers’ capabilities and benevolence, which in turn helps to build customers’ trust (Chen et al., 2016; Sanchez-Franco, 2009). By contrast, other consumers spend time and effort seeking or processing information to evaluate alternatives only during the purchase process because of the high perceived risks. This type of situational involvement (i.e., purchase involvement) is temporarily established, which can Open Access http://www.sajbm.org Page 2 of 10 Original Research Page 2 of 10 involvement have been widely acknowledged as its two main categories (Belanche, Flavián, & Pérez-Rueda, 2017; Bloch & Richins, 1983; Lockshin, Spawton, & Macintosh, 1997; Vanwesenbeeck, Walrave, & Ponnet, 2016). Trust in online shopping Consumer trust in products or service providers is a major factor that fuels the rapid development of e-commerce (Gefen, 2000). Given their inability to confirm the quality of a product in person, consumers have to trust the entire e-commerce mechanism to determine the transactions that follow (Lee & Turban, 2001). Customer trust is a psychological state that allows consumers to accept their own vulnerability and take risks based on their positive expectations regarding the intentions and behaviours of the other party (Singh & Sirdeshmukh, 2000). Grabner–Kräuter and Kaluscha (2003) performed an integrative review of 11 empirical studies on trust in e-commerce and found that most of these studies have explored the consumers’ trust in online retailers or retail websites. Online retailers are stores which sell products or services via the Internet, while retail websites are online portals that allow online businesses to manage their website, marketing, sales and operations. Many authors highlighted brand trust as another important research object that can help customers reduce their uncertainty and simplify their choices of online shopping (Plassmann, Kenning, Deppe, Kugel, & Schwindt, 2008). Even though many e-merchants are unknown to consumers, they still engage in business online because e-service providers play an intermediary role in guaranteeing customers’ equity. Moreover, brands, public images conceived of as something to be marketed, can shape some consumers’ perceptions toward products. When product information is accessed from unfamiliar retailers, the brand name can increase the customer’s confidence in claims (Delgado–Ballester & Munuera-Alemán, 2001). Trust in online shopping must be conceptualised as a separate construct that functions differently in the e-commerce mechanism with an aim to reduce uncertainty. To fully consider the online shopping environment, this paper contributes by exploring the direct effect of purchase involvement and product involvement on trust in all online contextual situations (e.g., platforms, brands and e-venders) and the moderating effect of confusion on the involvement– trust relationship. Copyright: Product involvement (or enduring involvement) is a feeling of interest, enthusiasm and excitement about specific product categories that consumers build over the long term (Houston & Rothschild, 1978), while purchase involvement (or situational involvement) is stimulated by a particular situation, such as a purchase occasion, and prompts pre- purchase internal or external information searches to reduce the perceived risks associated with the selection of products or services (Bloch & Richins, 1983; Houston & Rothschild, 1978). also increase their trust toward retailing (Sanchez-Franco, 2009). Although several scholars have argued that trust considerably influences consumer involvement in trusted partners, such as financial companies (Chen et al., 2016; Hansen, 2017) and trading partners (Li, Li, & Feng, 2015), the reverse direction (i.e., the effect of involvement on trust) deserves more attention to a lack of initial trust. However, determining whether involvement can be successfully translated into online trust remains a challenge. Over the past few years, many global brands on e-commerce platforms have grown at a relatively rapid rate (Ecommerce Foundation, 2017). Through their rapid expansion efforts, many brands or e-retailers have adopted remarkably similar logos, product designs and displays, and marketing tactics, particularly in China (Bao, 2013). In such a confusing online shopping environment, consumers with limited product knowledge can easily become overwhelmed and struggle when making purchase decisions (Shiu & Tzeng, 2018; Walsh, Hennig-Thurau, & Mitchell, 2007). Massive amounts of unfamiliar and uncertain information raise the threshold of involvement that consumers must cross if they are to trust an e-seller (Kim et al., 2008). The continued increase of uncertainty in e-markets necessitates further examination of two questions: whether both types of involvement still positively influence trust; and whether consumer confusion affects the involvement–trust relationship. Consumer involvement others based on what they know (Riquelme, Román, Cuestas, & Iacobucci, 2019), but in an uncertain situation, people confirm their beliefs through experiential evidence (Ouyang, Gursoy, & Chen, 2019), such as their perceptions toward a website. After identifying their needs, those customers with a high purchase involvement engage in an information search to avoid uncertainty. They feel vulnerable in the unfamiliar online world because of their lack of confidence in their ability to purchase their desired product online (Sanchez-Franco, 2009). Therefore, those purchase-involved consumers who temporarily increase the relevance or their interest toward a product or service tend to seek information from formal or informal sources to evaluate their alternatives and ponder over their purchase decisions. These temporal risk-reduction activities may reinforce the customers’ trust in any trading party in e-commerce (Sanchez-Franco, 2009). As their degree of purchase involvement increases, these customers’ level of trust in e-retailers, brands being sold online and e-service providers increases. The following hypotheses are proposed: Consumer confusion has long been considered an important topic in the consumer behaviour literature (Mitchell et al., 2005; Walsh & Mitchell, 2010; Walsh et al., 2007). Understanding consumer confusion can help businesses improve their marketing strategies and establish sustainable and profitable relationships with their target consumers (Turnbull et al., 2000). Most papers on consumer confusion have investigated its antecedents and consequences (Cheng, Lu, & Gursoy, 2015; Tjiptono, Arli, & Bucic, 2014; Walsh & Mitchell, 2010; Walsh et al., 2007; Wobker, Eberhardt, & Kenning, 2015). Among those consequences, marketplace trust has been verified to be significantly decreased by consumer’s confusion (Cheng et al., 2015; Walsh & Mitchell, 2010; Walsh et al., 2007). H1a: A consumer’s purchase involvement is positively related to his or her trust in e-retailers. H1a: A consumer’s purchase involvement is positively related to his or her trust in e-retailers. H1b: A consumer’s purchase involvement is positively related to his or her trust in brands being sold online. H1c: A consumer’s purchase involvement is positively related to his or her trust in e-service providers. On the other hand, familiarity with products can make consumers feel secure when shopping online because they know how to identify useful information and where to find bargains (Kim et al., 2008). Product involvement allows customers to develop expectations regarding the trading parties (Sanchez-Franco, 2009). Consumer involvement The customers’ degree of involvement affects their purchase decisions (Laurent & Kapferer, 1985; Pieniak, Verbeke, Scholderer, Brunsø, & Olsen, 2008). To further understand the behaviour of consumers when making decisions, scholars have studied their involvement in various industries, such as banking (Sanchez-Franco, 2009), food (Pieniak et al., 2008), wine (Rahman & Reynolds, 2015) and tourism (Ferns & Walls, 2012). From the perspectives of different industries, these studies draw a full-scale map of the relationships between involvement and several factors, such as satisfaction, trust, commitment and positive mood. Their findings offer managerial implications for marketers who attempt to incorporate involvement into their marketing strategies. The inherent nature of perceived risks somehow bridges the relationship between involvement and trust in the e-commerce context. Sanchez-Franco (2009) investigated the moderating effect of involvement on the relationship between trust and commitment and argued that when product involvement exists, the importance of trust is reduced. By contrast, high purchase involvement results from a high perceived risk, thereby enhancing the relevance of trust. People tend to make trust-related assumptions about Involvement, which exhibits relevance to personal values (Barki & Hartwick, 1994; Bloch & Richins, 1983), is an individual, internal interest of intensity, direction and persistence toward specific products (Andrews, Durvasula, & Akhter, 1990). Although many papers have classified involvement into various types, product and purchase http://www.sajbm.org Open Access Page 3 of 10 Original Research Original Research Page 3 of 10 et al., 2000; Walsh et al., 2007). Similarity, confusion refers to the consumers’ propensity to think that different products in a product category are similar with the brand names, attributes or quality (Walsh et al., 2007). Overload confusion takes place when individuals are bombarded with an abundance of information (Mitchell et al., 2005). Ambiguity confusion occurs when individuals are forced to re-evaluate and revise current beliefs or assumptions about the product or purchasing environment (Mitchell et al., 2005). These three types of confusion not only take place when shopping in physical stores but also when shopping online (Mitchell et al., 2005). Browsing an excessive number of similar websites or reading excessively similar product information from multiple sources may engender similarity confusion; introducing newly updated online payment technologies or assorted product combinations at varying prices can trigger ambiguity confusion; and providing a vast amount of information may cause overload confusion (Walsh et al., 2007). Consumer involvement Therefore, given their rich experience or knowledge about e-commerce, those consumers with a high product involvement build their faith in others’ benevolence, competence and integrity, which consequently develops their trust in online businesses (McKnight, Choudhury, & Kacmar, 2002). The following hypotheses are proposed: However, only a few papers have discussed the moderating effects of consumer confusion. In the complex online shopping environment, this study examines whether consumer confusion can alter the strength or direction of the relationships between distinct involvement types and specific trust objects (Baron & Kenny, 1986). Product-involved consumers who adopt a long-term uncertainty-reduction strategy are less susceptible to the influence of a confusing context. Conversely, purchase- involved consumers often spend time and effort in seeking or processing information to evaluate their alternatives during the purchase process (Sanchez-Franco, 2009); this type of situational involvement is easily influenced by product attributes (e.g., product complexity and similarity) and situational variables (e.g., asymmetry in the availability of information related to decision-making) (Houston & Rothschild, 1978). Therefore, a complex and unfamiliar shopping environment places the consumers’ purchase decisions in a highly confusing situation. If these consumers feel confused when shopping online, then the effect of their purchase involvement on their trust may be reduced. On the basis of the above discussion, we formed the following hypotheses: H2a: A consumer’s product involvement is positively related to his or her trust in e-retailers. H2b: A consumer’s product involvement is positively related to his or her trust in brands being sold online. H2c: A consumer’s product involvement is positively related to his or her trust in e-service providers. H2c: A consumer’s product involvement is positively related to his or her trust in e-service providers. Confirmatory factor analysis Maximum likelihood estimation was conducted to estimate the confirmatory and structural equation models. The multi- dimensional factor of consumer confusion was tested before evaluating the fitness of the entire structural model. Confirmatory factor analysis was performed first to test the model fit of three second-order components (i.e., similarity, overload and ambiguity confusion) and one first-order construct (i.e., consumer confusion). As shown in Tables 1 and 2, all factor loadings (i.e., standardised coefficients) are above 0.6. The Cronbach’s α and CR of the second-order components all exceed 0.7, while their average variance extracted (AVE) meet the suggested level of 0.5, thereby confirming the convergent validity and internal consistency of the measurement scales (Fornell & Larcker, 1981; Hair, Black, Babin, & Anderson, 2010). Discriminant validity was also achieved because the inter-factor correlations in the corresponding rows and columns were less than the square roots of AVE on the main diagonal (Fornell & Larcker, 1981). The fit indices showed a favourable overall fit for the second- order model (Hair et al., 2010). Consequently, the items concerning the three types of consumer confusion were averaged dimensionally to obtain three indicators of the main construct. The English measurement items were translated into Chinese and then translated back into English by a bilingual expert to ensure translation accuracy. The survey instrument was then pilot-tested among 35 randomly selected college students to ensure its validity and reliability. In China, most college students are active Internet shoppers (Huang, Zhou, Liao, Mo, & Wang, 2017), which justifies the pilot-test sampling. Based on the results of reliability analysis and item analysis, some measure items were deleted from the original scales to reach better scale consistency. Questionnaire development This article followed all ethical standards for carrying out research without direct contact with human or animal subjects. This study developed a two-section questionnaire based on the literature review. The first section consists of three constructs and eight dimensions: consumer confusion (i.e., similarity, overload and ambiguity), product involvement (i.e., product and purchase) and trust (i.e., in e-vendor, retail website and brand). The consumer confusion scale was adapted from Walsh et al. (2007), while product involvement and purchase involvement were measured by using scales from Sanchez-Franco (2009) and Lockshin et al. (1997). The respondents’ trust was measured by using three dimensions, namely, trust in e-vendors, trust in retail websites and trust in brands, with scales adapted from Bhattacherjee (2002), Gefen (2000), Delgado–Ballester and Munuera-Alemán (2001) and Jarvenpaa, Tractinsky and Vitale (2000) (see Table 1-A1, Appendix 1 for details). The second section gathers the demographic information of the respondents, including their gender, age, education, marital status, years of online shopping and monthly personal income. All items in the constructs were measured by using a seven-point Likert-type scale. Results Structural equation modelling (SEM) was performed to analyse the collected data because of its ability to measure and test the causal relationships among latent variables. The second-order structure of consumer confusion must be analysed before estimating the entire proposed model because of its multi-dimensional character. Confirmatory factor analysis (CFA) was initially performed to test the convergent and discriminant validities of the second-order model of consumer confusion and the overall model before evaluating their fitness. Afterward, the proposed hypotheses were tested in the conceptual model. Online consumer confusion Consumer confusion is a mental state with conscious and unconscious elements in which a customer struggles when making purchase decisions (Moon, Costello, & Koo, 2016). Consumer confusion can occur before or after the purchase (Mitchell, Walsh, & Yami, 2005) and can be viewed as the failure to develop an accurate interpretation of the various facets of a product or service during the information processing procedure (Turnbull, Leek, & Ying, 2000). Three types of confusion, namely, similarity, overload and ambiguity have been identified from the literature (Turnbull H3a: A consumer’s confusion negatively moderates the relationship between his or her purchase involvement and trust in e-retailers. http://www.sajbm.org Open Access Original Research Page 4 of 10 H3b: A consumer’s confusion negatively moderates the relationship between his or her purchase involvement and trust in brands being sold online. 307 women (53.9%). Respondents aged below 33 years and with a bachelor’s degree or above accounted for 82.4% and 67.5% of the sample respectively. Almost all respondents (92.8%) had more than 2 years of online shopping experience, and most of them (64.9%) were earning below 5000 RMB every month. 307 women (53.9%). Respondents aged below 33 years and with a bachelor’s degree or above accounted for 82.4% and 67.5% of the sample respectively. Almost all respondents (92.8%) had more than 2 years of online shopping experience, and most of them (64.9%) were earning below 5000 RMB every month. H3c: A consumer’s confusion negatively moderates the relationship between his or her purchase involvement and trust in e-service providers. Sampling and data collection CR, composite reliability; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; NFI, normed fit index; CFI, comparative fit index; GFI, goodness of fit index; TLI, Tucker–Lewis index; RMSEA, root mean square error of approximation. reliable, α > 0.8 very reliable, α > 0.9 extremely reliable. CR, composite reliability; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; NFI, normed fit index; CFI, comparative fit index; GFI, goodness of fit index; TLI, Tucker–Lewis index; RMSEA, root mean square error of approximation. y rity confusion; OC, overload confusion; AC, ambiguity confusion; NFI, normed fit index; CFI, comparative fit index; GFI, goodness of fit index; TLI, Tucker–Lewis ror of approximation. TABLE 2: Second-order factor means, standard deviations, Pearson correlations and validities (n = 527). Construct Dimensions Mean SD AVE SC OC AC Consumer confusion (CC) Similarity confusion (SC) 4.941 1.229 0.528 0.727 - - Overload confusion (OC) 4.591 1.349 0.621 0.569 0.788 - Ambiguity confusion (AC) 5.037 1.153 0.610 0.527 0.569** 0.781 Note: The square roots of AVE are located along the diagonal and the inter-construct correlations are shown in the lower left off-diagonal elements in the matrix. SD, standard deviations; AVE, average variance extracted; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion. , p < 0.05, , p < 0.01. e: The square roots of AVE are located along the diagonal and the inter-construct correlations are shown in the lower left off-diagonal elements in the matrix. standard deviations; AVE, average variance extracted; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion. < 0.05, , p < 0.01. TABLE 3: First-order confirmatory factor analysis, reliability and model fit (n = 527). Sampling and data collection Since the population this study focused on the Internet shoppers, we applied the online survey as our sampling method. The online survey, which was run by a professional opinion pollster, randomly selected participants from online users in Guangdong Province for 3 months. According to the Alibaba Group (China Business Intelligence Network, 2018), e-retailing sales in Guangdong Province accounted for 25% of national sales and exceeded those in other parts of China in 2017, thereby enhancing the representativeness of the collected data. Those respondents who had never shopped online were excluded from the interviews. A total of 598 participants completed the questionnaire, among which 28 were excluded because they were inconsistent or had too many missing data, thereby leaving 570 effective samples for the analysis. The sample included 263 men (46.1%) and The proposed model was tested after performing second- order CFA on consumer confusion. An item-parcelling strategy was applied across the entire model to obtain highly reliable indicators, to estimate fewer parameters and to simplify the model interpretation (Hau & Marsh, 2004). Confirmatory factor analysis was also conducted to examine Open Access http://www.sajbm.org Page 5 of 10 Original Research TABLE 1: Second-order confirmatory factor analysis, reliability and model fit (n = 527). Construct Dimensions Items Factor loadings Cronbach’s α CR χ2(24) p χ2/df NFI CFI GFI TLI RMSEA Consumer confusion (CC) Similarity confusion (SC) SC1 0.81 0.763 0.769 - - - - - - - - SC2 0.71 - - - - - - - - - - SC3 0.65 - - - - - - - - - - Overload confusion (OC) OC1 0.80 0.825 0.829 - - - - - - - - OC2 0.88 - - - - - - - - - - OC3 0.67 - - - - - - - - - - Ambiguity confusion (AC) AC1 0.81 0.822 0.824 - - - - - - - - AC2 0.81 - - - - - - - - - - AC3 0.72 - - - - - - - - - - Goodness of fit indices - - - - - 52.988 0.000 2.208 0.973 0.985 0.979 0.977 0.048 Note: Kaiser-Meyer-Olkin (KMO) test = 0.839; Bartlett’s test of sphericity = 1913.627, p = 0.000; varimax with Kaiser normalisation, 3 factors extracted; standardised 9-item alpha = 0.721; α > 0.7 reliable, α > 0.8 very reliable, α > 0.9 extremely reliable. Sampling and data collection Note: Kaiser-Meyer-Olkin (KMO) test = 0.893; Bartlett’s test of sphericity = 4408.95, p = 0.000; varimax with Kaiser normalisation, 6 factors extracted; standardised 23-item alpha = 0.885; α > 0.7 reliable, α > 0.8 very reliable, α > 0.9 extremely reliable; CR, composite reliability; AVE, average variance extracted; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; NFI, normed fit index; CFI, comparative fit index; GFI, goodness of fit index; TLI, Tucker–Lewis index; RMSEA, root mean square error of approximation. Note: Kaiser-Meyer-Olkin (KMO) test = 0.893; Bartlett’s test of sphericity = 4408.95, p = 0.000; varimax with Kaiser normalisation, 6 factors extracted; standardised 23-item alpha = 0.885; α > 0.7 reliable, α > 0.8 very reliable, α > 0.9 extremely reliable; CR, composite reliability; AVE, average variance extracted; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; NFI, normed fit index; CFI, comparative fit index; GFI, goodness of fit index; TLI, Tucker–Lewis index; RMSEA, root mean square error of approximation. Note: Kaiser-Meyer-Olkin (KMO) test = 0.893; Bartlett’s test of sphericity = 4408.95, p = 0.000; varimax with Kaiser normalisation, 6 factors extracted; standardised 23-item alpha = 0.885; α > 0.7 reliable, α > 0.8 very reliable, α > 0.9 extremely reliable; whether the collected data fit the hypothesised measurement model (i.e., consumer confusion, product involvement, purchase involvement, trust in e-vendor, trust in retail website and trust in brand). As shown in Tables 3 and 4, a favourable model fit is achieved because the χ2/d.f. ratio is less than 3, the values of NFI, GFI, CFI and TLI are all above 0.90 and the RMSEA is below 0.08 (Hair et al., 2010). The convergent validity and internal consistency of the measurement scales were also confirmed because almost all values for the factor loadings, Cronbach’s α, and CR exceeded 0.7 while their AVEs met the suggested level of 0.5. Similarly, the test for discriminant validity presented a satisfactory outcome because the square root AVE estimates of a single construct were greater than the inter-construct correlations. whether the collected data fit the hypothesised measurement model (i.e., consumer confusion, product involvement, purchase involvement, trust in e-vendor, trust in retail website and trust in brand). As shown in Tables 3 and 4, a favourable model fit is achieved because the χ2/d.f. Sampling and data collection ratio is less than 3, the values of NFI, GFI, CFI and TLI are all above 0.90 and the RMSEA is below 0.08 (Hair et al., 2010). The convergent validity and internal consistency of the measurement scales were also confirmed because almost all values for the factor loadings, Cronbach’s α, and CR exceeded 0.7 while their AVEs Sampling and data collection Constructs Items Factor loadings Cronbach’s α CR χ2(307) p χ2/df NFI CFI GFI TLI RMSEA Consumer confusion (CC) SC 0.73 0.716 0.791 - - - - - - - - OC 0.78 - - - - - - - - - - AC 0.78 - - - - - - - - - - Product involvement (PD) PD1 0.70 0.846 0.850 - - - - - - - - PD2 0.88 - - - - - - - - - - PD3 0.84 - - - - - - - - - - Purchase involvement (PC) PC1 0.80 0.831 0.863 - - - - - - - - PC2 0.89 - - - - - - - - - - PC3 0.76 - - - - - - - - - - PC4 0.57 - - - - - - - - - - Trust in e-vendor (TV) TV1 0.72 0.832 0.834 - - - - - - - - TV2 0.77 - - - - - - - - - - TV3 0.88 - - - - - - - - - - Trust in retail website (TW) TW1 0.83 0.845 0.846 - - - - - - - - TW2 0.74 - - - - - - - - - - TW3 0.74 - - - - - - - - - - TW4 0.73 - - - - - - - - - - Trust in brand (TB) TB1 0.72 0.811 0.815 - - - - - - - - TB2 0.79 - - - - - - - - - - TB3 0.77 - - - - - - - - - - TB4 0.61 - - - - - - - - - - Goodness of fit indices - - - - 572.296 0.000 1.864 0.905 0.953 0.923 0.947 0.041 CR, composite reliability; AVE, average variance extracted; SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; NFI, normed fit index; CFI, comparative fit index; GFI, goodness of fit index; TLI, Tucker–Lewis index; RMSEA, root mean square error of approximation. Simultaneous regression paths SEM regression paths were constructed to test the proposed hypotheses in the conceptual model as shown in Figure 1. Purchase involvement is negatively related to trust in http://www.sajbm.org http://www.sajbm.org Open Access Page 6 of 10 Original Research TABLE 4: First-order factor means, standard deviations, Pearson correlations and validities (n = 527). Constructs Mean SD AVE CC PD PC TV TW TB Consumer confusion (CC) 4.848 0.995 0.558 0.747 - - - - - Product involvement (PD) 5.550 1.041 0.657 0.402* 0.811 - - - - Purchase involvement (PC) 5.162 1.096 0.584 0.353 0.459 0.764 - - - Trust in e-vendor (TV) 4.420 0.999 0.629 -0.148 0.437 -0.418 0.793 - - Trust in retail website (TW) 4.800 1.073 0.579 -0.258 0.269 -0.269 0.537 0.761 - Trust in brand (TB) 4.734 0.955 0.527 -0.207 0.352 -0.300 0.526 0.519 0.726 Note: The square roots of AVE are located along the diagonal and the inter-construct correlations are shown in the lower left off-diagonal elements in the matrix. CC, consumer confusion; PD, product involvement; PC, purchase involvement; TV, trust in e-vendor; TW, trust in retail website; TB, trust in brand; SD, standard deviations; AVE, average variance extracted. , p < 0.05, , p < 0.01. Note: The square roots of AVE are located along the diagonal and the inter-construct correlations are shown in the lower left off-diagonal elements in the matrix. CC, consumer confusion; PD, product involvement; PC, purchase involvement; TV, trust in e-vendor; TW, trust in retail website; TB, trust in brand; SD, standard deviations; AVE, average variance extracted. , p < 0.05, , p < 0.01. Note: The square roots of AVE are located along the diagonal and the inter-construct correlations are shown in the lower left off-diagonal elements in the matrix. CC, consumer confusion; PD, product involvement; PC, purchase involvement; TV, trust in e-vendor; TW, trust in retail website; TB, trust in brand; SD, standard deviations; AVE, average variance extracted. 2.0 2.5 3.0 4.0 5.0 3.5 4.5 Low PI Purchase involvement Trust in e-vendor High PI High confusion Low confusion FIGURE 2: Interaction of purchase involvement and confusion on trust in e-vendors. Purchase involvement Consumer confusion Product involvement Trust in brands Trust in retail websites Trust in e-vendors 0.795 P < 0.05, * P < 0.01,* P < 0.001 ns signiﬁcant –0.805* –0.823 3.109* 2.920* 3.034* –1.332* –1.238* –0.032, ns –0.047, ns –0.153 –0.187 ns, not significant. Simultaneous regression paths , p < 0.05; , p < 0.01; , p < 0.001. FIGURE 1: Estimation results of structural model. Purchase involvement Consumer confusion Product involvement Trust in brands Trust in retail websites Trust in e-vendors 0.795 P < 0.05, * P < 0.01,* P < 0.001 ns signiﬁcant –0.805* –0.823 3.109* 2.920* 3.034* –1.332* –1.238* –0.032, ns –0.047, ns –0.153 –0.187 FIGURE 2: Interaction of purchase involvement and confusion on trust in e-vendors. ns, not significant. , p < 0.05; , p < 0.01; **, p < 0.001. FIGURE 1: Estimation results of structural model. FIGURE 1: Estimation results of structural model. FIGURE 1: Estimation results of structural model. e-vendors (β = -0.153, p < 0.01). By contrast, the moderating effects of consumer confusion on the relationships between purchase involvement and trust in retail websites (β = -0.047, ns) and trust in brands (β = -0.032, ns) fail to reach a significant level. Therefore, H3a is supported, while H3b and H3c are rejected. The interacting effect of purchase involvement and confusion on trust in e-vendors is illustrated in Figure 2. The plot of interaction shows that trust in e-vendors decreases along with purchase involvement; however, this slope descends more sharply for higher confusion. This finding indicates that high purchase- involved and confused consumers tend to have lower trust in e-vendors. e-vendors (β = -1.187, p < 0.001), retail websites (β = -1.238, p < 0.001) and brands (β = -1.332, p < 0.001), thereby not supporting H1a, H1b and H1c. Meanwhile, consumers with a high purchase involvement exhibit low trust in e-commerce. Hypothesis 2, which predicts a positive relationship between product involvement and trust in e-business, was also verified by the results concerning the e-vendors (β = 3.034, p < 0.001), trust in retail websites (β = 2.920, p < 0.05) and trust in brands (β = 3.109, p < 0.001). Therefore, H2a, H2b and H2c are all supported. In other words, as the consumers’ product involvement increases, they tend to trust retailers, retail websites and brands while shopping online. Discussion A quadratic latent variable with a single indicant was applied to test the moderating effect of consumer confusion. Although all possible pairwise products of the main effect were recommended as indicators of the latent product to examine the interaction (Kenny & Judd, 1984), unfortunately, the product terms violate the multivariate normality of the maximum likelihood assumption (Cortina, Chen, & Dunlap, 2001; Ping, 1995). Given this problem, many authors have proposed a single indicator of the latent product (Jöreskog & Yang, 1996; Mathieu, Tannenbaum, & Salas, 1992; Ping, 1995). The moderating effects of consumer confusion was tested by using a single indicant specification, and the results are also shown in Figure 1. Consumer confusion demonstrates a significant and negative moderating effect on the negative relationship between purchase involvement and trust in Shoppers struggle when making decisions in the confusing, low-trust e-commerce environment. This study investigated the involvement–trust relationship and the moderating effect of consumer confusion. The results show that purchase involvement has a negative impact on online trust in e-vendors, retail websites and brands, whereas product involvement demonstrates a positive impact. In a confusing shopping environment, involvement is not always positively related to trust, which demonstrates a new relationship between involvement and trust. This finding supplements the theory proposed in Chen et al. (2016) and Sanchez-Franco (2009) in a low-trust context. This study also confirms that consumer confusion negatively moderates the relationship between purchase involvement and trust in e-vendors. http://www.sajbm.org Open Access Original Research Page 7 of 10 Page 7 of 10 Given their poor knowledge in online shopping, purchase- involved consumers have low trust in e-vendors, retail websites and brands. Purchase involvement is situational and can be easily affected by one’s surroundings (Houston & Rothschild, 1978). Consumers with a high purchase involvement are greatly affected by the negative image of online shopping and readily show low levels of trust belief toward e-commerce. In the worst-case scenario, these consumers may no longer trust online shopping and refrain from engaging in e-commerce transactions, thereby leading to a trade reduction. By contrast, product-involved consumers constantly focus on certain products or services before starting the purchase process. They exhibit great familiarity from enthusiasm and excitement, are capable of distinguishing good products and services from bad ones and can adopt effective strategies to reduce their perceived risks. Discussion Therefore, consumers with a high product involvement can easily identify trustworthy e-vendors, brands and retail websites with which they can transact online. Meanwhile, consumer confusion has a significant and negative moderating effect on the negative relationship between purchase involvement and trust in e-vendors. Given that consumer confusion is negatively related to trust in e-vendors (see Figure 1; β = -0.795, p < 0.01), consumer confusion reinforces the negative relationship. Conclusion In the face of these situational involvements, consumers take certain precautions against risks, such as by seeking additional information. When searching for information before conducting transactions, purchase-involved shoppers must browse numerous unfamiliar choices. Online retailers greatly outnumber the e-merchant marketplaces or brands that operate online. For instance, two top-ranked US e-commerce platforms, namely Amazon and eBay, held over half of e-commerce sales in 2017, while Tmall.com and JD.com, the two most well-known business-to-consumer (B2C) retail markets in China, already dominated more than three-quarters of the entire online market share (China Internet Watch, 2016). Conversely, the number of online retailers has increased dramatically. In 2017, the number of e-commerce companies in the US amounted to 300 000 (Murthy, 2017) and, surprisingly, the number of customer-to- customer (C2C) stores in China had reached 12 million (E-commerce statistics, 2017). These small stores grow faster than large ones because of their minimal start-up costs, yet enjoy a lower reputational advantage (Gao, Chan, Chi, & Deng, 2016). Therefore, in such a complex information environment created by e-retailers, purchase-involved consumers struggle when making purchase decisions because of their insufficient product and service knowledge and the lack of trustworthy stores. As a result, consumer confusion can eventually lead to a low trust in e-vendors. By contrast, given the small number of e-service providers and brands being sold online, consumers are less likely to become confused in platform and brand shopping, thereby explaining the insignificant moderating effects of consumer confusion on the relationships between purchase involvement and trust in the other two objects (i.e., retail websites and brands). Few studies have recognised the effects of involvement on trust in the full-scale context of online shopping. The moderating effect of confusion has also received little academic attention. Chinese shoppers struggle in the confusing, low-trust environment of the largest retail e-commerce market. This study, therefore, explored the relationships between consumer involvement and trust in the e-commerce setting. Moreover, the moderating effect of confusion was also assessed regarding these associations. The results show that purchase involvement has a negative impact on online trust in e-vendors, retail websites and brands, whereas product involvement has a positive impact. This study also confirms that confusion moderates the relationship between purchase involvement and trust in e-vendors. Managerial implications For practitioners, the findings provide important implications that consumer trust is an essential factor in e-commerce transactions. To enhance consumer trust in online settings, marketers must learn how to decrease uncertainty and confusion in the purchase process. Moreover, the involvement in establishing a trust-based relationship between consumers and suppliers must be emphasised. Some recommendations for practitioners are as follows: (1) To enhance the trust beliefs of purchase-involved consumers, marketers should invite regular customers (product-involved customers) to recommend their products and services during online transactions. Current customers, serving as a trusted third party, can be rewarded by allowing them to invite their in- group members to be involved in transactions. (2) To lessen the moderating effect of confusion, online retailers should set up shops on well-known e-commerce platforms or sell famous brands. Through being associated with e-service providers and brands, e-merchants can increase purchase- involved consumers’ trust beliefs toward e-retailers. (3) Finally, to reduce consumer confusion, e-retailers should differentiate themselves from others in the same merchandise category. 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Computers in Human Behavior, 75, 58–69. https://doi.org/10.1016/j.chb.2017.04.050 The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. iiMedia Report. (2018). China imports e-Commerce authentic research report. Retrieved from http://www.iimedia.cn/63004.html Jarvenpaa, S., Tractinsky, N., & Vitale, M. (2000). Consumer trust in an Internet store. Information Technology and Management, 1(1–2), 45–71. https://doi.org/ 10.1023/A:1019104520776 Authors’ contributions Gao, B., Chan, W.K.V., Chi, L., & Deng, X.N. (2016). Size and growth dynamics of online stores: A case of China’s Taobao.com. Electronic Commerce Research and Applications, 17, 161–172. https://doi.org/10.1016/j.elerap.2016.04.005 All authors contributed equally to this work. Gefen, D. (2000). E-commerce: The role of familiarity and trust. Omega-International Journal of Management Science, 28(6), 725–737. https://doi.org/10.1016/S0305- 0483(00)00021-9 Competing interests The authors have declared that no competing interest exists. Fornell, C., & Larcker, D.F. (1981). Evaluating structural equation models with unobservable variables and measurement error. Journal of Marketing Research, 18(1), 39–50. https://doi.org/10.1177/002224378101800104 Funding information Grabner-Kräuter, S., & Kaluscha, E.A. (2003). Empirical research in on-line trust: A review and critical assessment. International Journal of Human-Computer Studies, 58(6), 783–812. https://doi.org/10.1016/S1071-5819(03)00043-0 This research is supported by the Institute of Guangdong & Taiwan (KFJJ201802), Philosophy and Social Science Planning Project of Guangdong Province (GD18CGL05) and Guangdong Science and Technology Project (180917114960500). Hair, J.F., Black, W.C., Babin, B.J., & Anderson, R.E. (2010). Multivariate data analysis: A global perspective. 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SC3 Sometimes I want to buy a product seen in an advertisement but cannot identify it clearly between scores of similar products. Overload confusion (OC) OC1 I do not always know exactly which products meet my needs best. OC2 There are so many brands to choose from that I sometimes feel confused. OC3 Most brands are very similar and are therefore hard to distinguish. Ambiguity confusion (AC) AC1 Products often have so many features that a comparison of different brands is barely possible. References AC2 The information I get from advertising often are so vague that it is hard to know what a product can actually perform. AC3 When purchasing certain products, I need the help of sales personnel to understand differences between products. Product involvement (PD) PD1 I like to use the products I buy personally in my life. PD2 I often use the products I buy. PD3 The product I use says a lot about who I am. PD4 It is important for me to choose a product that ‘feels’ right. Purchase involvement (PC) PC1 In order to buy more cost-effective goods, I would like to spend extra time to do comparison shopping. PC2 Being a smart shopper is worth the extra time it takes. PC3 Because of my personal values, I feel smart shopping ought to be important to me. PC4 I usually spend a lot of time and effort making an expensive purchase decision. Trust in e-vendor (TV) TV1 Based on my online shopping experience, the store is trustworthy. TV2 I trust that the store keeps my best interests, such as providing needed information, in mind. TV3 The store wants to be known as one who keeps promises and commitments. TV4 The company will always be honest with me. Trust in retail website (TW) TW1 Even if not monitored, I’d trust the retail website to do the job right. TW2 I trust the retail website I often visit. TW3 I am quite certain what to expect from the retail website. TW4 It performs the work according to the entries on the registration protocol, such as the legitimate use of personal information in accordance with the privacy policy. Trust in brand (TB) TB1 The brand offers me a product with a constant quality level. TB2 The brand helps me to solve any problem I could have with the product. TB3 The brand is interested in my satisfaction. TB4 The brand offers me recommendations and advice on how to make the most of this product. SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; PD, product involvement; PC, purchase involvement; TV, trust in e-vendor; TW, trust in retail website; TB, trust in brand. Page 10 of 10 Original Research SC, similarity confusion; OC, overload confusion; AC, ambiguity confusion; PD, product involvement; PC, purchase involvement; TV, trust in e-vendor; TW, trust Appendix 1 Appendix 1 TABLE 1-A1: Questionnaire items. http://www.sajbm.org Open Access
https://openalex.org/W2029611175	https://europepmc.org/articles/pmc3238352?pdf=render	English	null	Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART	Advances in urology	2,012	cc-by	9,282	Correspondence should be addressed to Marlea Di Santo, disanto@tecnobiosprocreazione.it Received 5 August 2011; Revised 22 September 2011; Accepted 27 September 2011 Copyright © 2012 Marlea Di Santo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cryopreservation of human spermatozoa—introduced in the 1960’s—has been recognized as an eﬃcient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the eﬀects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation aﬀects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to aﬀect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates. Hindawi Publishing Corporation Advances in Urology Volume 2012, Article ID 854837, 12 pages doi:10.1155/2012/854837 Hindawi Publishing Corporation Advances in Urology Volume 2012, Article ID 854837, 12 pages doi:10.1155/2012/854837 Hindawi Publishing Corporation Advances in Urology Volume 2012, Article ID 854837, 12 pages doi:10.1155/2012/854837 There are two main conventional freezing techniques used in sperm cryopreservation: slow freezing and rapid freezing 2.1. Slow Freezing. The slow freezing technique proposed by Behrman and Sawada [8] consists of progressive sperm cooling over a period of 2–4 h in two or three steps, either manually or automatically using a semiprogrammable freezer. The manual method is performed by simultaneously decreasing the temperature of the semen while adding a cryoprotectant in a stepwise manner and after plunging the samples into liquid nitrogen [9]. It has been shown that the optimal initial cooling rate of the specimen from room temperature to 5◦C is 0.5–1◦C/min [10]. The sample is then frozen from 5◦C to −80◦C at a rate of 1–10◦C/min. The specimen is then plunged into liquid nitrogen at −196◦C [11]. In spite of reports showing successful sperm freezing with manual techniques, the reproducibility of this procedure could pose some problems. For this reason, programmable freezers have been investigated [12]. The freezers use a plate to hold the straws; these are cooled by liquid nitrogen held in a storage tank under the plate. Liquid nitrogen is poured into the tank, and the machine, once programmed, uses the software data logging to obtain cooling from 20◦C to −80◦C at rate of 1.5◦C/min and then at 6◦C/min; at completion of the freezing the straws are removed and stored into liquid nitrogen at −196◦C. This takes about 40 min [12]. Programmes are simple to use and allow for a cooling combination which does not require continuous operator intervention and have been used to increase the reproducibility of the freezing operations [12]. 2.4. Cryoprotectants. Cryoprotectants are low-molecular- weight and highly permeable chemicals used to protect spermatozoa from freeze damage by ice crystallization. There are four main well-known cryoprotectants: glycerol, ethylene glycol, dimethyl sulphoxide, and 1,2-propanediol. Cryopro- tectants act by decreasing the freezing point of a substance, reducing the amount of salts and solutes present in the liquid phase of the sample and by decreasing ice formation within the spermatozoa [16]. Usually the cryoprotectants are added in an equal volume of semen in a dropwise manner, gently mixed at room temperature, and then placed at 37◦C for 10–15 minutes to allow for proper equilibration between the cells and the medium [7]. It is necessary that the medium interacts with the cells. Indeed, the eﬀectiveness of cryoprotecting substances is also a function of the time of interaction between the cryoprotectants and the cells [7]. 1. Introduction low reproducibility, indeed, the temperature drop curve cannot be controlled, and the freezing temperatures may vary from −70, −80, and −99◦C [7]. recommended to preserve fertility in those subjects who— for one reason or another—are exposed to potentially toxic agents which may interfere with gametogenesis [7]. low reproducibility, indeed, the temperature drop curve cannot be controlled, and the freezing temperatures may vary from −70, −80, and −99◦C [7]. 1. Introduction germ cells caused by adjuvant therapy. In particular, the risk associated to therapy depends on several factors: the age of the patient at the time of treatment, the dose, site, and type of treatment [3]. Also some nonmalignant diseases, such as diabetes and autoimmune disorders, may lead to testicular damage. Cryopreservation is advisable also in these conditions [4]. In countries in which heterologous fertilization is allowed by law and in donor insemination programmes cryopreservation is necessary to have enough time to screen donors for infectious agents, such as the human immunodeﬁciency (HIV) and hepatitis B viruses, before the cryopreserved semen is used for clinical purposes [5]. In azoospermic patients, who have undergone testic- ular sperm extraction or percutaneous epididymal sperm aspiration, sperm cryostorage is also used to avoid repeated biopsies or aspirations [6]. Furthermore, cryopreservation is routinely performed in patients who—having to start an assisted reproduction treatment—decide to preemptively freeze the semen sample to avoid inconveniences due to failed ejaculation often associated with “semen collection stress,” certain emotional states, or other commitments at the time of oocyte retrieval [7]. Finally, male gamete freezing is largely The procedure that makes it possible to stabilize the cells at cryogenic temperatures is called cryopreservation, also known as an applied aspect of cryobiology or the study of life at low temperatures. Many advances in the cryopreservation technology have led to the development of methods that allow for low-temperature maintenance of a variety of cell types including male and female gametes, small multicel- lular organisms, and even more complex organisms such as embryos. Cryopreservation of human spermatozoa— introduced in the 1960’s [1]—has overcome many space and time limitations and now forms integral part of assisted reproduction technologies (ARTs). This technique becomes particularly important in cases of preservation of male fertility before radiotherapy or chemotherapy [2] which may lead to testicular failure or ejaculatory dysfunction. In fact, semen cryostorage seems to be the only proven method that may oﬀer these couples a chance of having children in the future: cancer therapy could in fact lead to damage, resulting in subfertility or sterility due to gonad removal or permanent damage to 2 2 Advances in Urology low reproducibility, indeed, the temperature drop curve cannot be controlled, and the freezing temperatures may vary from −70, −80, and −99◦C [7]. 2. Techniques for Cryopreservation 2.3. Cryopreservation of Small Numbers of Spermatozoa. The conventional methods of sperm cryopreservation described above are not ideal to cryopreserve small numbers of cells, such as epididymal and testicular spermatozoa. Eﬃcient cry- opreservation of surgically retrieved spermatozoa, as men- tioned earlier in this chapter, reduces the number of surgical interventions and avoids the logistic problem associated with coordinating the women’s oocyte retrieval and also the risk of no sperm being found on the day of oocyte retrieval [15]. Thus, novel cryopreservation approaches have been designed to cryopreserve limited numbers of motile sperms in a very small volume (Table 1). Both biological and nonbiological carriers have been tried for the cryopreservation of low numbers of spermatozoa, although, to date, no prospective randomised trials have been conducted to demonstrate that any single carrier is superior to the others [15]. Furthermore, to date there is a limited use of these technologies in the majority of IVF programs. This suggests that novel cryopreservation technology, designed to handle small sperm numbers and needs to be further explored [15]. Whatever the method used, to obtain good results it is essential to correctly perform all the steps before and after cryopreservation: the choice of more suitable cryoprotectants and the thawing procedure are particularly important. There are two main conventional freezing techniques used in sperm cryopreservation: slow freezing and rapid freezing There are two main conventional freezing techniques used in sperm cryopreservation: slow freezing and rapid freezing Glycerol is the permeating cryoprotectant most widely used for human sperm acting on: the membrane structure, permeability and stability of the lipid bilayer, the association of surface proteins and the cellular metabolism. Its employment gives an unfavorable outcome on membrane and acrosome structure, although allowing the freezing of poor quality sperm [7]. Sherman’s [14] studies showed that the use of glycerol may cause few alterations such as: presence of an undulating membrane, alteration in acrosomal internal membrane, nucleus inhomogeneity and disorganization in mitochondrial crests. Following this observations other protective substances were proposed such as the dimethyl sulfoxide (DMSO), which has deleterious eﬀects on human Some authors argue that conventional slow freezing, either manual or automated, causes extensive chemical-phys- ical damage to the sperm probably because of ice crystalliza- tion [13]. 2.2. Rapid Freezing. Rapid freezing was ﬁrst proposed by Sherman [14]. This technique requires direct contact between the straws and the nitrogen vapours for 8–10 min and immersion in liquid nitrogen at −196◦C. Inside nitrogen vapours there is a thermal gradient, as a function of the distance and the volume of the liquid below. The sample is initially mixed in dropwise manner with equal volume of cold cryoprotectant; the mixture is loaded into the straws and left to incubate at 4◦C for 10 minutes. The straws are then placed at a distance of 15–20 cm above the level of liquid nitrogen (−80◦C) for 15 min; after this stage, the straws are immersed in liquid nitrogen. During cooling it is preferable to place the straws in horizontal position to minimize the heat diﬀerence between the two ends. This technique has some drawbacks among which; for example, 3 3 Advances in Urology Table 1: Approaches to cryopreserve limited number of spermatozoa. Cryopreservation techniques Authors Principle Main advantages Main disadvantages Empty zona pellucida Borini et al. [69] Storage of individual spermatozoa in animal or human empty zona pellucida. Avoid waste of time in screening to locate motile sperm; cryoprotectants can be added and removed without loss of spermatozoa sequestered in the zona Risk of biological contamination Cohen et al. [70] Walmsley et al. [71] Montag et al. [72] Hsieh et al. [73] Liu et al. [74] Levi-Setti et al. [75] Cesana et al. [76] Hassa et al. [77] Microdroplets Gil-Salom et al. (iii) thawing at room temperature for 15 min. Once the semen is thawed, it is separated from the cryo- preservation medium by washing in culture medium and centrifuging [7]. 2.5. Thawing Procedure. The thawing procedure is an equally important step: the cell must be allowed to recover its normal biological activities trying to avoid abrupt thermal changes as far as possible. Generally speaking, the cryopreservation protocols use a thawing temperature of 37◦C; even if higher thawing temperatures allow for more rapid heating, they are not used because of the risks associated to cell damage. There are two main conventional freezing techniques used in sperm cryopreservation: slow freezing and rapid freezing [78] Storage of droplets of sperm/cryoprotectants mixture on the surface of dry ice and directly plunged into liquid nitrogen Avoid sperm loss through adherence to the vessel Risk of cross-contamination; shape and size of dishes make diﬃcult to handle and store in conventional freezers and liquid nitrogen tanks Sereni et al. [79] Quintans et al. [80] Bouamama et al. [81] ICSI pipette Gvakharia et al. [82] Storage of spermatozoa in ICSI pipettes Sterile, simple, and convenient system Not practical for long-term storage; fragility of ICSI pipettes; risk of cross-contamination Sohn et al. [83] Volvox globator spheres Just et al. [84] Storage of sperm into spheres of Volvox globator Signiﬁcant postthaw recovery of motile sperm Exposure to genetic material from the algae; constant source of algae Alginate beads Herrler et al. [85] Microencapsulation in alginate beads Inert nature of alginate beads Decrease sperm motility with encapsulation Cryoloop Nawroth et al. [86] Individual spermatozoa deposited directly on cryoprotectant ﬁlm covering the nylon loop and immersed in liquid nitrogen Excellent vessel for vitriﬁcation; no additional preparation Open system: risk of cross-contamination Schuster et al. [87] Isachenko et al. [42] Isachenko et al. [42] Desai et al. [88] Desai et al. [89] Agarose microspheres Isaev et al. [90] Storage of sperm loaded in agarose microspheres Nonbiological carrier Clinical value of this approach not evaluated Straws Desai et al. [91] Sperm/cryoprotectants loaded into the ministraw Sterile, simple, and convenient system Not ideal for severely impaired specimens; sperm loss due to adherence to the vessel Isachenko et al. [92] Koscinski et al. [93] Table 1: Approaches to cryopreserve limited number of spermatozoa. (iii) thawing at room temperature for 15 min. sperm when used at 4◦C, and the 1,2-propanediol slightly used in sperm cryopreservation [7]. sperm when used at 4◦C, and the 1,2-propanediol slightly used in sperm cryopreservation [7]. 3. Detrimental Effects of Cryopreservation on Sperm Integrity A rapid cooling rate causes severe intracellular ice formation, since the eﬄux of water across the membrane is impaired, thus, inducing supercooling. Ice crystals formed breach the membranes and aﬀect the organelle function. This condition leads to impaired cell survival. On the other hand, a too slow cooling rate deter- mines the eﬄux of water from the internal to the external environment, increasing the concentration of solutes and the osmotic pressure. This condition leads to cell volume changes associated with the movement of water, dehydration, and toxicity damage due to high solute concentration [9]. Cryoinjury is not limited to the freezing process but may also occur during the thawing process as the ice melts or recrystallizes [9]. The phenomenon of recrystallization of both intracellular and extracellular ice, in frozen samples, occurs as smaller ice crystals with a rate of recrystallization that increases with increasing temperature [13]. It has largely been reported that chilling injury can modify the structure and integrity of plasma membranes [19, 20] mainly composed by phospholipids and cholesterol [21]. Even though high concentrations of cholesterol and polyunsaturated fatty acids give more ﬂuidity to the mem- brane at lower temperature [22], during cryopreservation the cooling process causes phase transition of membrane lipids and impairs membrane protein function. In particular, the outer layer of the spermatozoal plasma membrane consists of a glycocalyx, a carbohydrate-rich zone that mainly contains oligosaccharide chains that bind to the integral protein of the plasma membrane (glycoproteins) or lipids (glycolipids) [23]. Generally, cryopreservation may have a detrimental eﬀect changing the carbohydrate composition of the glyco- calyx, thus impairing the function of membrane proteins which are responsible for ion transport and metabolism and aﬀecting the fertilizing ability [24]: the glycocalyx is involved in some physiological functions such as immune protection of the female genital tract [25], acrosomal reaction [26], and early gamete interaction. 3.1. Cryopreservation and DNA Damage. While the eﬀects of cryopreservation on the fertilization capacity, motility, mor- phology, and viability of spermatozoa are well documented, still open is the question of the possible alteration of sperm DNA integrity after freezing-thawing procedures. There is no agreement in the literature neither on whether cryopreserva- tion induces DNA damage nor on the amount of damage. In some studies, authors have reported signiﬁcant alterations of sperm DNA integrity after cryopreservation/thawing [6, 40], whereas other studies have expressed a diﬀerent opinion [41, 42]. 3. Detrimental Effects of Cryopreservation on Sperm Integrity Compared with other cell types, spermatozoa seem to be less sensitive to cryopreservation damage because of the high ﬂuidity of the membrane and the low water content (about 50%). Despite this, cryopreservation may lead to deleterious changes of sperm structure and function [17]. It was largely reported that several damaging processes could occur during freezing-thawing of human spermatozoa, such as thermal shock with formation of intracellular and extracellular ice crystals, cellular dehydration, and osmotic shock [18]. At the present time, several thawing techniques are used, among which are (i) thawing at room temperature for 10 min and subse- quent thermostat pass at 37◦C for another 10 min, (ii) thawing in a thermostat and water-bath at 37◦C for 10 min, 4 Advances in Urology potential and release of ROS [9]. The peroxidative damage induced by increased concentration of ROS is associated with damage to the sperm plasma membrane and impairment of the axonemal structure [30]. In addition, cryopreservation has been shown to diminish the antioxidant activity of the spermatozoa making them more susceptible to ROS damage [31]. High concentration of ROS and fall of antioxidant enzymes lead to cell apoptosis [32]. In this context the apoptosis cascade is mediated by activation of the BCL2 family proteins: there is a permeabilization of the outer mito- chondrial membrane through the Bax and BAK proteins and the release of cytochrome [33]. In turn, caspase 9 is activated along with APAF-1 to form an apoptosome [34]. The release of apoptosis-inducing factors from the mitochondria leads to DNA fragmentation [35]. Several studies examined the role of in vitro antioxidant supplementation in protecting the sperm DNA from oxidative damage. For example, when added to the seminal ﬂuid during cryopreservation, genistein [36], resveratrol [37], and ascorbic acid [37] seem to reduce DNA damage; on the contrary, vitamin E [38], ascorbate, and catalase [39] seem to improve motility and reduce ROS levels, though they do not improve spermatozoal viability and do not reduce DNA damage. In any case, the number of these studies and the number of patients they include is still too limited to draw any conclusions on the eﬃcacy of antioxidant supplementation in protecting DNA from freezing-induced damage. The primary cause of cellular damage during cryopreser- vation is the formation of intracellular or extracellular ice crystals. During the freezing process, the cooling rate plays an important role in determining the extent of cryoinjury to the spermatozoa [9]. 3. Detrimental Effects of Cryopreservation on Sperm Integrity This controversy between one study and the other could ﬁrst of all be explained by the fact that the ﬁndings do not refer to a considerable number of samples and is also due to the use of (1) diﬀerent freezing procedures, (2) diﬀerent tests to evaluate the DNA integrity, and (3) diﬀerent semen preparation techniques before cryopreser- vation (i.e., swimup or density gradient centrifugation). For example, Donnelly and colleagues [6] investigated pre- cryopreservation and postcryopreservation DNA integrity of both semen and prepared sperm samples (density gradient centrifugation or direct swimup) in 50 men. They reported that freezing sperm in seminal plasma improves postthaw DNA integrity: sperm-frozen unprepared in seminal ﬂuid seems to be more resistant to freezing damage than frozen prepared sperm; further improvement can be achieved by preparing sperm and freezing after readdition of seminal plasma. This may be due to the presence of abundant antioxidants in seminal plasma. Concerning the variability linked to diﬀerent freezing procedures, in the study of Petym and colleagues [43], the authors evaluated cryodamage on sperm chromatin comparing two diﬀerent procedures: The plasma and mitochondrial membranes have the same susceptibility to cryopreservation [27]. Mitochondria are placed along the midpiece between the plasma mem- brane and the nine ﬁbrous columns, to form a coating that provides energy necessary for sperm motility [27]. The greatest amount of energy is provided by molecules of ATP synthesized either by glycolysis in the cytoplasm [28] or through oxidative phosphorylation (oxphos) in the mitochondria [10]. The ATP generated by oxphos in the inner mitochondrial membrane is transferred to the microtubules, to drive motility [29]. Therefore, an impairment of mitochondrial activity may explain the reduction in motility [27]. An alteration in mitochondrial membrane ﬂuidity may also lead to an alteration in mitochondrial membrane 5 Advances in Urology liquid nitrogen vapour versus computerized program freezer. They analyzed 50 semen samples using acridine orange and concluded that DNA damage was signiﬁcantly higher following freezing with liquid nitrogen. since spermatogenesis results in the discarding of cytoplasm, leaving the spermatozoa incapable of undertaking DNA repair. According to this hypothesis, Kalthur and colleagues [49], evaluating sperm morphology and sperm DNA damage before and after cryopreservation, reported that the suscep- tibility of morphologically abnormal sperm to DNA damage during the freezing process is signiﬁcantly higher than that of sperm with normal morphology. 3. Detrimental Effects of Cryopreservation on Sperm Integrity They hypothesised that sperm with head abnormalities may have altered membrane physical properties and thereby have altered tolerance to cold stress. However, there are no studies conducted to assess whether or not a morphologically abnormal sperm can retain its chromatin integrity during cryopreservation. g g q g From a detailed analysis of the references currently available in the literature, it was found that there are basically three diﬀerent lines of thought about the question: “Does the freezing-thawing procedure induce sperm DNA damage?”. f g g p p g According to several authors the answer is “YES” (Table 2(a)). Spano and his group [44] reported that overall sperm quality deteriorates after freezing-thawing, including sperm DNA integrity assessed by SCSA in 19 samples. These ﬁndings have been conﬁrmed in a study by De Paula and colleagues [40] on 77 patients, where the authors have eval- uated the degree of sperm DNA fragmentation by TUNEL assay before and after cryopreservation: the authors stressed that the freezing-thawing procedure negatively aﬀects DNA integrity. Furthermore, from the data published in the literature, it is also clear that among the authors who argue that cryopreservation induces sperm DNA damage there is sometimes no agreement on the mechanism which actually induces that damage. For example, in spite of the fact that ROS was widely reported to play an important role in the pathophysiology of damage to human spermatozoa, includ- ing DNA fragmentation, Zribi and his group [45] stated that there is no relationship between DNA fragmentation and DNA oxidation. They suggested that cryopreservation induces sperm DNA fragmentation through other pathways beside oxidative stress, such as defects in DNA repairing enzymes or enhancement of defects already present in sperm cells. This hypothesis is controverted by Thomson and colleagues [46]: despite the use of the same technique to assess DNA oxidation, ﬂuorescent assay for the detection of 8 oxoguanine, they reported that human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspase and apoptosis. g y g y In opposition to these two answers to the above question, there is a third line of thought: some authors say: “NO, the freezing-thawing procedure does not compromise sperm DNA integrity” (Table 2(c)). For example, Duru and his group [41] evaluated the eﬀects of cryopreservation on DNA fragmentation and membrane integrity in 21 patients using the TUNEL assay and annexin V. 3. Detrimental Effects of Cryopreservation on Sperm Integrity Their results indicated that cryopreservation altered plasma membrane symmetry and was associated with translocation of phosphatidylserine, while DNA integrity was maintained. In addition, Isachenko and colleagues [42], comparing the eﬀects of slow freezing and vitriﬁcation on sperm DNA integrity in the absence of cryoprotectant, found that the integrity of DNA is unaﬀected by cryopreservation. The lack of eﬀects of cryopreservation on sperm DNA has also been conﬁrmed by data of Paasch and colleagues [50]. They demonstrated that cryopreser- vation was signiﬁcantly associated with disruption of the mitochondrial membrane potential, as well as activation of caspase 3, 8, and 9, but no signiﬁcant changes were observed in DNA fragmentation, as assessed by the TUNEL assay in 84 samples. Even if the opinions on the issue of “cryopreservation and DNA damage” are still highly controversial, the evaluation of the impact of cryopreservation on sperm chromatin is of extreme importance. Likewise, sperm DNA integrity is an important factor for the success of ART [51–53]. p p p About the question “Does the freezing-thawing proce- dure induce sperm DNA damage?” some authors follow another line of thought and answer “YES, but with some conditions” (Table 2(b)). They support the hypothesis of less susceptibility to freezing damage in the spermatozoa of fertile men, classiﬁed using WHO criteria, than those of infertile men. In the study of Donnelly and colleagues [6], semen samples were obtained from 17 fertile and 40 infertile men, and sperm integrity was assessed before and after cryopreservation using the Comet assay. The authors showed that semen from fertile men appears to be more resistant to freezing damage than sample from infertile men; moreover, in fertile man, there was no signiﬁcant decrease in DNA integrity after cryopreservation. These results support the observation that spermatozoa from infertile men have a greater incidence of irregular chromatin organization and show signiﬁcantly decreased resistance to thermal denatu- ration compared with spermatozoa from fertile men [47, 48]. In fact, as a consequence of reduced protamination, poor-quality spermatozoa often contain partially decon- densed chromatin that generates functional immaturity [6]. Chromatin condensation is fundamental for spermatozoa 4. Cryopreservation and Reproductive Outcome Cryopreservation is widely known to raise impaired sperm motility and decrease fertilization rate through detrimental eﬀects on membranes, acrosomal structure, and acrosin activity [54]. The freezing-thawing procedure of human spermatozoa may also be detrimental to the chromatin structure [55], leading to a potential risk of decondensation of the sperm nucleus after injection into the oocyte, thus, reducing fertilization rate [56]. However, a cumulative eﬀect of cryopreservation on sperm fertilization capacity is not deﬁnitely established. Considering the decrease in sperm fertilization power induced by cryopreservation, it can be easily understood that intrauterine insemination and conventional in vitro fertilization (IVF) with frozen-thawed spermatozoa result in lower pregnancy rates compared with insemination with fresh sperm [1]: this is the reason why cryopreservation of semen samples before intrauterine insemination or conventional IVF is not recommended. 6 Advances in Urology Table 2: (a)–(c) Evaluation of DNA integrity after cryopreservation: description of the experimental design and conclusions. (a) Authors Test to evaluate DNA integrity Number of samples Cryopreservation method “Does the freezing-thawing procedure induce sperm DNA damage?” Hamamah et al. [94] Acridine orange staining and Feulgen-DNA quantitative microspectrophotometry 10 Unspeciﬁed Yes Span`o et al. [44] SCSA + Acridine orange staining 19 Equilibration at 37◦C, freezing in liquid nitrogen vapour at −80◦C and then storage in liquid nitrogen at –196◦C Yes Hammadeh et al. [95] Acridine orange staining 59 Computerized slow-stage freezer + static liquid nitrogen vapour Yes Donnelly et al. [6] COMET assay 40 Equilibration at 37◦C, freezing in liquid nitrogen vapour at −80◦C and then storage in liquid nitrogen at –196◦C Yes Gandini et al. [96] Acridine orange staining 19 Equilibration at 37◦C, freezing in liquid nitrogen vapour at −80◦C and then storage in liquid nitrogen at –196◦C Yes de Paula et al. [40] TUNEL assay 77: (i) 30 normozoospermic (ii) 47 oligozoospermic Use of freezer at –20◦C, freezing in liquid nitrogen vapour, then storage in liquid nitrogen –196◦C Yes Petyim and Choavaratana [43] Acridine orange staining 50 Freezing with liquid nitrogen vapour + computerized program freezer Yes Nagamwuttiwong and Kunathikom [97] Acridine orange staining 20 Freezing with liquid nitrogen vapour Yes Dejarkom and Kunathikom [98] Acridine orange staining 20 Computerized controlled rate freezing Yes Thomson et al. [46] TUNEL assay 60 Use of programmable freezer Yes Thomson et al. [46] TUNEL assay 320 Sample frozen with and without cryoprotectant by slow-controlled-rate method using a programmable freezer Yes Zribi et al. 4. Cryopreservation and Reproductive Outcome [45] TUNEL assay 15 Equilibration at 37◦C, freezing in liquid nitrogen vapour at −80◦C, then storage in liquid nitrogen at –196◦C Yes Table 2: (a)–(c) Evaluation of DNA integrity after cryopreservation: description of the experimental design and conclusions. (a) 7 7 Advances in Urology (b) Authors Test to evaluate DNA integrity Number of samples Cryopreservation technique “Does the freezing-thawing procedure induce sperm DNA damage?” Donnelly et al. [6] COMET assay 57: (i) 17 fertile (ii) 40 infertile Equilibration at 37◦C, freezing in liquid nitrogen vapour at −80◦C, then storage in liquid nitrogen at –196◦C Yes, but semen from fertile men appears to be more resistant to freezing damage Kalthur et al. [49] COMET assay + Acridine orange staining 44 Equilibration at 37◦C, static cooling at 4◦C, cooling vapour phase, then storage in liquid nitrogen at –196◦C Yes, but morphologically abnormal sperms seems to be less resistant to freezing damage Ahmad et al. [99] COMET assay 196: (i) 30 normospermic (ii) 166 infertile Freezing with static-phase vapour cooling procedure Yes, but the sperm DNA integrity of frozen samples of fertile men is higher (c) Authors Test to evaluate the DNA integrity Number of samples Cryopreservation technique “Does the freezing-thawing procedure induce sperm DNA damage?” Høst et al. [100] Immunoperoxidase detection of digoxigenin-labelled genomic DNA 53: (i) 20 fertile (ii) 33 infertile Conventional cryopreservation No Steele et al. [101] COMET assay 21: (i) 9 control (ii) 12 with obstructive azoospermia Freezing in liquid nitrogen vapour No Duru et al. [41] TUNEL assay + annexin V 21 Equilibration at 37◦C, freezing in liquid nitrogen vapour at −80◦C, then storage in liquid nitrogen at –196◦C No Isachenko et al. [42] COMET assay 18 Programmable slow freezing + vitriﬁcation No Paasch et al. [50] TUNEL assay + ﬂow cytometric kit for apoptosis 84 Freezing at –20◦C, freezing in liquid nitrogen vapor at –100◦C, then storage in liquid nitrogen at –196◦C No COMET assay quality, implantation rate, clinical pregnancy rate, and ongoing pregnancy rate) between ICSI cycles with fresh or cryopreserved testicular spermatozoa from the same patients, comparing all ICSI cycles performed with fresh (25 cycles) and thawed (14 cycles) testicular spermatozoa, respectively. 5. Conclusion Today, sperm cryopreservation is widely used to store donor and partner spermatozoa before assisted reproduction treat- ments, to preserve spermatozoa before therapy for malignant diseases, vasectomy, or surgical infertility treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Therefore, sperm cryopreser- vation is an important component of fertility management, and much of its successful application seems to aﬀect the reproductive outcome of ART. 4.2. Epididymal Spermatozoa. On the other hand, some groups compared the results of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed epididymal spermatozoa. For example, Tournaye and colleagues [62] reported that the clinical pregnancy rate in ICSI cycles was comparable between fresh (157 cycles) and frozen-thawed (118 cycles) epididymal spermatozoa. Sukcharoen and col- leagues [63] performed a total of 53 ICSI cycles with fresh (40 cycles) and thawed (13 cycles) epididymal spermatozoa and came to the same conclusion; also Cayan and colleagues [64] supported the same opinion. In opposition Shibahara and colleagues [65] stated that there was a signiﬁcant diﬀerence in all reproductive parameters examined between ICSI cycles with fresh or cryopreserved epididymal spermatozoa, comparing ICSI cycles performed with fresh (5 cycles) and thawed (13 cycles) epididymal spermatozoa. p While the eﬀects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation aﬀects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. This suggests that, to date, it would be useful to perform a multicenter study with large numbers of semen specimens which could be processed using unique freezing protocols. Moreover, though further investigations are needed to fully understand the real inﬂuence of cryopreservation on sperm DNA integrity and the impact of the use of cryopreserved spermatozoa on the reproductive outcome, technical measures should be applied to provide maximum protection to the male gametes: appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seems to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates. 4.3. Ejaculated Spermatozoa. The majority of studies on cryopreservation and ICSI reproductive outcome were con- ducted using spermatozoa of testicular or epididymal origin. Only two major groups reported data on fertilization and pregnancy rates after ICSI comparing fresh and frozen- thawed human ejaculated spermatozoa. 4. Cryopreservation and Reproductive Outcome This hypothesis is supported by the study of some authors: Ben Rhouma and colleagues [58] performed a total of 60 ICSI cycles with fresh (32 cycles) and thawed (28 cycles) testicular spermatozoa; Habermann and colleagues [59] performed a total of 46 ICSI cycles with fresh (12 cycles) and thawed (34 cycles) testicular spermatozoa; Huang and colleagues [60] performed a total of 22 ICSI cycles with fresh (14 cycles) and thawed (8 cycles) testicular spermatozoa. All the authors reported the same results: the reproductive out- come of ICSI with frozen-thawed testicular spermatozoa is The considerations are diﬀerent for intracytoplasmic sperm injection (ICSI), because this procedure requires only a small number of motile spermatozoa for successful fertilization. Therefore, the current question is whether using fresh rather than cryopreserved sperm cells has the same eﬀect on reproductive outcome in ICSI. To date, only a few large-scale studies on ICSI reproductive outcome comparing fresh and frozen-thawed human ejaculated, testicular, or epididymal spermatozoa have been reported in the literature, and the results seem to diﬀer between the authors also depending on the origin of the employed spermatozoa. 4.1. Testicular Spermatozoa. Friedler and colleagues [57] reported no statistically signiﬁcant diﬀerences in all param- eters examined (fertilization rate, cleavage rate, embryo 8 8 Advances in Urology quality than that in patients with normal semen. However, once the oocyte is fertilized, implantation and pregnancy rates are similar in patients with or without sperm anomalies. comparable with the reproductive outcome of ICSI obtained with fresh testicular spermatozoa. In contrast, De Croo and colleagues [61] stated that fertilization, implantation, and live-birth rates per embryo transfer are signiﬁcantly lower after ICSI with frozen-thawed (35 cycles) than those with fresh (65 cycles) testicular spermatozoa. 5. Conclusion First, Kucznynski and colleagues [66] compared the reproductive outcome of 118 ICSI cycles using fresh spermatozoa and 122 ICSI cycles using frozen-thawed spermatozoa, all from oligoas- thenoteratozoospermic patients. The authors did not report of any statistically signiﬁcant diﬀerences in fertilization rate between the two groups of patients. Moreover, these data show that values of ongoing pregnancies are signiﬁcantly higher in ICSI patients when human sperm samples are cryopreserved. According to Ragni and his group [67], this suggests that properly performed cryopreservation selec- tively aﬀects defective rather than normal spermatozoa [44]. This observation seems to indicate that cryopreservation before ICSI might be helpful to eliminate senescent or deﬁcient spermatozoa, thus, improving reproductive out- come [62]. Borges and his group [68] also investigated sperm cryopreservation eﬀects on ICSI outcome. The author compared 61 and 79 ICSI cycles performed with cryopre- served and fresh ejaculated spermatozoa and, in particular, examined the reproductive outcome obtained using semen samples with decreased and with normal motility. Results demonstrated that (1) using semen with normal motility the reproductive outcome obtained using fresh or frozen- thawed spermatozoa is the same; (2) in semen with decreased motility the fertilization rate with fresh sperm was higher than that with the cryopreserved one, but no diﬀerences were detected in implantation and pregnancy. 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https://openalex.org/W4387149317	https://masujournal.org/store_file/archive/70-3-3-157-162.pdf	Maltese	null	CONTINUOUS CROPPING OF RICE-INFLUENCE OF TEMPERATURE ON UPTAKE OF N, P. K AND Zn IN RICE	Madras Agricultural Journal	1,983	cc-by	2	https://doi.org/10.29321/MAJ.10.A02595 https://doi.org/10.29321/MAJ.10.A02595
https://openalex.org/W2899367007	https://iimmun.ru/iimm/article/download/777/390	Russian	null	MECHANISMS OF INTERRACTION OF HELICOBACTER PYLORI WITH EPITHELIUM OF GASTRIC MUCOSA. I. PATHOGENIC FACTORS PROMOTING SUCCESSFUL COLONIZATION	Infekciâ i immunitet	2,018	cc-by	11,380	Reviews Russian Journal of Infection and Immunity = Infektsiya i immunitet 2018, vol. 8, no. 3, pp. 273–283 Инфекция и иммунитет 2018, Т. 8, № 3, с. 273–283 МЕХАНИЗМЫ ВЗАИМОДЕЙСТВИЯ HELICOBACTER PYLORI C ЭПИТЕЛИЕМ СЛИЗИСТОЙ ОБОЛОЧКИ ЖЕЛУДКА. I. ФАКТОРЫ ПАТОГЕННОСТИ, СПОСОБСТВУЮЩИЕ УСПЕШНОЙ КОЛОНИЗАЦИИ О.К. Поздеев1, А.О. Поздеева1, Ю.В. Валеева2, П.Е. Гуляев3 О.К. Поздеев1, А.О. Поздеева1, Ю.В. Валеева2, П.Е. Гуляев3 1 Казанская государственная медицинская академия — филиал ФГБОУ ДПО Российская медицинская академия непрерывного профессионального образования МЗ РФ, г. Казань, Россия 2 ФГАОУ ВО Казанский (Приволжский) федеральный университет, г. Казань, Россия 3 ФГБОУ ВО Казанский государственный медицинский университет МЗ РТ, г. Казань, Россия 1 Казанская государственная медицинская академия — филиал ФГБОУ ДПО Российская медицинска непрерывного профессионального образования МЗ РФ, г. Казань, Россия 2 ФГАОУ ВО Казанский (Приволжский) федеральный университет, г. Казань, Россия 3 ФГБОУ ВО Казанский государственный медицинский университет МЗ РТ, г. Казань, Россия Резюме. Helicobacter pylori — грамотрицательная, извитая, подвижная бактерия, способная колонизировать сли- зистую оболочку желудка (СОЖ) человека и выживать в этих крайне неблагоприятных условиях у более чем по- ловины населения нашей планеты. Показано, что микроорганизм может обитать на СОЖ в течение всей жизни хозяина, но при этом вызывает клинически выраженные заболевания лишь у незначительной группы инфици- рованных лиц. К причинам, способствующим развитию заболеваний, как правило, относят: сопутствующие ин- фекции желудочно-кишечного тракта, неправильную стерилизацию медицинских инструментов (как правило эндоскопов), несоблюдение правил личной гигиены, длительный контакт с инфицированными или носителя- ми, в том числе с членами семьи, и ряд других факторов. Хорошо известно, что персистирование H. pylori имеет этиологическое значение в развитии широкого спектра болезней желудочно-кишечного тракта, включая хрони- ческий гастрит, язвенную болезнь желудка и двенадцатиперстной кишки, аденокарциному желудка и MALT- лимфомы. Глобальное распространение носительства H. pylori позволяет предположить наличие у бактерии «умных» стратегий, способствующих ее адаптации к агрессивной среде желудка и пожизненному персистирова- нию в организме человека. Даже спустя 34 года после открытия микроорганизма остается множество вопросов, не имеющих своего ответа, в том числе о роли факторов патогенности, способствующих выживанию микроорга- низма в суровых условиях микробиома СОЖ. Изучение и понимание механизмов, способствующих колонизации и персистированию H. pylori, позволит оптимизировать прогноз повышенного риска тяжелых заболеваний у лиц, колонизированных этим микроорганизмом. Сложившийся в ходе эволюции долгосрочный баланс между чело- веком и H. pylori определяет микробное постоянство в микробиоме желудка, но в ряде случаев это совместное со- существование приводит к риску развития вышеперечисленных тяжелых патологий. В данном обзоре предпри- нято обсуждение характера взаимоотношений H. pylori с эпителием, участие факторов патогенности бактерии (уреазы, ЛПС, комплекса поверхностных белков, токсинов и протеаз, способствующих инвазии) в колонизации и длительном персистировании на СОЖ. Приведенная информация о механизмах, связанных с колонизацией Адрес для переписки: Поздеев Оскар Кимович 420012, Россия, г. Казань, ул. Бутлерова, 36, Казанская государственная медицинская академия. МЕХАНИЗМЫ ВЗАИМОДЕЙСТВИЯ HELICOBACTER PYLORI C ЭПИТЕЛИЕМ СЛИЗИСТОЙ ОБОЛОЧКИ ЖЕЛУДКА. I. ФАКТОРЫ ПАТОГЕННОСТИ, СПОСОБСТВУЮЩИЕ УСПЕШНОЙ КОЛОНИЗАЦИИ Тел.: 8 919 693-02-04 (моб.). E-mail: pozdeevoskar@rambler.ru Contacts: Oskar K. Pozdeev 420012, Russian Federation, Kazan, Butlerova str., 36, Kazan State Medical Academy. Phone: +7 919 693-02-04 (mobile). E-mail: pozdeevoskar@rambler.ru Библиографическое описание: Поздеев О.К., Поздеева А.О., Валеева Ю.В., Гуляев П.Е. Механизмы взаимодействия Helicobacter pylori c эпителием слизистой оболочки желудка. I. Факторы патогенности, способствующие успешной колонизации // Инфекция и иммунитет. 2018. Т. 8, № 3. С. 273–283. doi: 10.15789/2220-7619-2018-3-273-283 Citation: Pozdeev O.K., Pozdeeva A.O., Valeeva Yu.V., Gulyaev P.E. Mechanisms of interraction of Helicobacter pylori with epithelium of gastric mucosa. I. Pathogenic factors promoting successful colonization // Russian Journal of Infection and Immunity = Infektsiya i immunitet, 2018, vol. 8, no. 3, pp. 273–283. doi: 10.15789/2220-7619-2018-3-273-283 © Поздеев О.К. и соавт., 2018 DOI: http://dx.doi.org/10.15789/2220-7619-2018-3-273-283 Contacts: Oskar K. Pozdeev 420012, Russian Federation, Kazan, Butlerova str., 36, Kazan State Medical Academy. Phone: +7 919 693-02-04 (mobile). E-mail: pozdeevoskar@rambler.ru Contacts: Oskar K. Pozdeev 420012, Russian Federation, Kazan, Butlerova str., 36, Kazan State Medical Academy. Phone: +7 919 693-02-04 (mobile). E-mail: pozdeevoskar@rambler.ru Адрес для переписки: Поздеев Оскар Кимович 420012, Россия, г. Казань, ул. Бутлерова, 36, Казанская государственная медицинская академия. Тел.: 8 919 693-02-04 (моб.). E-mail: pozdeevoskar@rambler.ru Адрес для переписки: Поздеев Оскар Кимович 420012, Россия, г. Казань, ул. Бутлерова, 36, Казанская государственная медицинская академия. Тел.: 8 919 693-02-04 (моб.). E-mail: pozdeevoskar@rambler.ru Библиографическое описание: Поздеев О.К., Поздеева А.О., Валеева Ю.В., Гуляев П.Е. Механизмы взаимодействия Helicobacter pylori c эпителием слизистой оболочки желудка. I. Факторы патогенности, способствующие успешной колонизации // Инфекция и иммунитет. 2018. Т. 8, № 3. С. 273–283. doi: 10.15789/2220-7619-2018-3-273-283 Обзоры Обзоры Reviews MECHANISMS OF INTERRACTION OF HELICOBACTER PYLORI WITH EPITHELIUM OF GASTRIC MUCOSA. I. PATHOGENIC FACTORS PROMOTING SUCCESSFUL COLONIZATION P d O K P d A O V l Y V b G l P E Abstract. H. pylori is a Gram-negative, crimp and motile bacterium that colonizes the hostile microniche of the human stomach roughly one half of the human population. Then persists for the host’s entire life, but only causes overt gastric disease in a subset of infected hosts. To the reasons contributing to the development of diseases, usually include: con- comitant infections of the gastrointestinal tract, improper sterilization of medical instruments, usually endoscopes, non- observance of personal hygiene rules, prolonged contact with infected or carriers, including family members and a number of other factors. Clinically, H. pylori plays a causative role in the development of a wide spectrum of diseases including chronic active gastritis, peptic and duodenal ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Due to the global distribution of H. pylori, we are able to conclude that smart strategies are contributing to adaptation of the bacterium in an aggressive environment of a stomach and lifelong permanent circulation in its host. Thirty-four years after the discovery of this bacterium, there are still many unanswered questions. For example, which strategies help the bacterium to survive in this inhospitable conditions? Understanding the mechanisms governing H. py- lori persistence will improve identification of the increased risk of different gastric diseases in persons infected with this bacterium. A well-defined and long-term equilibrium between the human host and H. pylori allows bacterial persistence in the gastric microniche; although this coexistence leads to a high risk of severe diseases the diseases which are listed above. In this review, we discuss the pathogenesis of this bacterium and the mechanisms it uses to promote persistent colonization of the gastric mucosa, with a focus on recent insights into the role of some virulence factors like urease, LPS, outer membrane proteins, cytotoxins, factors, promoting invasion. Information on the mechanisms related to H. pylori persistence can also provide the direction for future research concerning effective therapy and management of gastroduo- denal disorders. The topics presented in the current review are important for elucidating the strategies used by H. pylori to help the bacterium persist in relation to the many unfavorable features of living in the gastric microniche. Key words: Helicobacter pylori, pathogenic factors, gastric mucosa, colonization mechanisms, persistence, pathogenesis. Helicobacter pylori (H. pylori) — спиралевидная подвижная грамотрицательная микроаэро- фильная бактерия, способная колонизировать слизистую оболочку желудка и двенадцати- перстной кишки человека и различных живот- ных. MECHANISMS OF INTERRACTION OF HELICOBACTER PYLORI WITH EPITHELIUM OF GASTRIC MUCOSA. I. PATHOGENIC FACTORS PROMOTING SUCCESSFUL COLONIZATION P d O K P d A O V l Y V b G l P E Основным биотопом бактерий является антральная часть желудка, где отсутствуют об- кладочные (париетальные) клетки. Показано, что H. pylori обнаруживают у большей части (до 50–60%) популяции людей. В зависимости от региона проживания и возраста обследо- ванных лиц уровни инфицированности могут варьировать от 15 до 90%, что делает его одним из самых успешных микробов-комменсалов, выживающих в экстремальных условиях желуд- ка. При этом в большинстве случаев микробная колонизация протекает бессимптомно, но у 10– 15% лиц инфицирование сопровождается кли- ническими проявлениями, обусловленными развитием хронического воспаления слизистой оболочки желудка (СОЖ). В подобных ситуаци- ях наиболее часто наблюдают развитие атрофи- ческого гастрита типа В, реже язвенной болезни, а у 0,5–2% инфицированных H. pylori могут раз- виваться злокачественные заболевания — рак желудка и MALT-лимфомы [5, 64]. Факторы, обусловливающие столь различ- ные реакции организма хозяина на циркуля- цию H. pylori, остаются до конца не выяснеными. Результаты многочисленных исследований свя- зывают их с вариабельностью генотипов бак- терий по факторам патогенности, либо нали- чием вариантов полиморфных локусов генов, кодирую щих синтез ключевых медиаторов про- и противовоспалительных реакций организма хозяина [16, 21, 22, 43, 69]. MECHANISMS OF INTERRACTION OF HELICOBACTER PYLORI WITH EPITHELIUM OF GASTRIC MUCOSA. I. PATHOGENIC FACTORS PROMOTING SUCCESSFUL COLONIZATION Pozdeev O.K.a, Pozdeeva A.O.a, Valeeva Yu.V.b, Gulyaev P.E.с a Kazan State Medical Academy — Branch Campus of the Russian Medical Academy of Continuous Professional Education, Kazan, Russian Federation b Kazan (Volga region) Federal University, Kazan, Russian Federation с Kazan State Medical University, Kazan, Russian Federation MECHANISMS OF INTERRACTION OF HELICOBACTER PYLORI WITH EPITHELIUM OF GASTRIC MUCOSA. I. PATHOGENIC FACTORS PROMOTING SUCCESSFUL COLONIZATION Pozdeev O.K.a, Pozdeeva A.O.a, Valeeva Yu.V.b, Gulyaev P.E.с a Kazan State Medical Academy — Branch Campus of the Russian Medical Academy of Continuous Professional Education, Kazan Russian Federation b Kazan (Volga region) Federal University, Kazan, Russian Federation с Kazan State Medical University, Kazan, Russian Federation Citation: Pozdeev O.K., Pozdeeva A.O., Valeeva Yu.V., Gulyaev P.E. Mechanisms of interraction of Helicobacter pylori with epithelium of gastric mucosa. I. Pathogenic factors promoting successful colonization // Russian Journal of Infection and Immunity = Infektsiya i immunitet, 2018, vol. 8, no. 3, pp. 273–283. doi: 10.15789/2220-7619-2018-3-273-283 DOI: http://dx.doi.org/10.15789/2220-7619-2018-3-273-2 © Поздеев О.К. и соавт., 2018 273 О.К. Поздеев и др. Инфекция и иммунитет эпителия желудка H. pylori позволяет создать предпосылки для более эффективной терапии заболеваний гастро- дуоденальной зоны. Cведения, представленные в данном обзоре, важны для объяснения стратегий, используе- мых H. pylori для выживания в крайне неблагоприятных условиях микробиома желудка. Ключевые слова: Helicobacter pylori, факторы патогенности, слизистая оболочка желудка, механизмы колонизации, персистирование, патогенез. 2018, Т. 8, № 3 2018, Т. 8, № 3 не только действия желудочного сока и анти- бактериальных веществ в просвете желудка, но и эффектров гуморального и клеточного им- мунных ответов. Для последующей успешной колонизации H. pylori должен преодолеть слой слизи и вступить в контакт с эпителием СОЖ. Степень вязкости слизи определяет содержа- ние гликопротеинов и ее pH. В нейтральной среде макромолекулы гликопротеинов форми- руют жидкую фракцию, а при снижении рН среды менее 4, трансформируются в вязкий полимерный гель [8]. Уреаза H. pylori разлага- ет мочевину, содержащуюся в слизи, с образо- ванием NH3, что, в свою очередь, изменяет рН среды, способствуя перемещению бактерий [7]. Движению H. pylori в слое слизи также способ- ствует спиральная форма, обусловливающая «винто образное» движение со скоростью, на- много превышающей движение многих палоч- ковидных бактерий [36]. В настоящее время подвижность H. pylori рассматривают как важ- ный фактор патогенности. не только действия желудочного сока и анти- бактериальных веществ в просвете желудка, но и эффектров гуморального и клеточного им- мунных ответов. Для последующей успешной колонизации H. pylori должен преодолеть слой слизи и вступить в контакт с эпителием СОЖ. Степень вязкости слизи определяет содержа- ние гликопротеинов и ее pH. В нейтральной среде макромолекулы гликопротеинов форми- руют жидкую фракцию, а при снижении рН среды менее 4, трансформируются в вязкий полимерный гель [8]. Уреаза H. pylori разлага- ет мочевину, содержащуюся в слизи, с образо- ванием NH3, что, в свою очередь, изменяет рН среды, способствуя перемещению бактерий [7]. Движению H. pylori в слое слизи также способ- ствует спиральная форма, обусловливающая «винто образное» движение со скоростью, на- много превышающей движение многих палоч- ковидных бактерий [36]. В настоящее время подвижность H. pylori рассматривают как важ- ный фактор патогенности. При попадании в среду с низким рН бактерия увеличивает синтез уреазы, формамидазы, ар- гиназы и других ферментов, разлагающих суб- страты с образованием NH4 + и СО2, что спо- собствует образованию «аммиачного облака» вокруг бактериальной клетки с щелочным рН, нейтрализую щим кислую среду желудка. р у у р у у Уреаза представляет собой Ni2+-содержащий гексадимер и является одним из основных мар- керов колонизации H. pylori, и ее определение наиболее часто применяют при проведении диа- гностических тестов. Генный кластер уреазы со- держит 7 генов: 2 структурных гена ureA и ureB (кодируют структурные субъединицы уреазы); 4 добавочных гена ureE, ureF, ureG и ureH коди- руют дополнительные белки, необходимые для сборки и включения ионов Ni2+ в субъедини- цу В. 2018, Т. 8, № 3 Пятый добавочный ген ureI кодирует канал уреазы для Н+ и является транспортной систе- мой для перемещения мочевины в цитоплазму бактерии. Синтез фермента результируется од- новременной экспрессией структурных и доба- вочных генов, продукты которых изначально су- ществуют как интактные апопротеины. Для по- явления активной формы фермента необходимы белки UreF, UreG и UreH [62]. Уреаза H. pylori является несекретируемым цитоплазматичес- ким белком, регулирующим внутреннюю кон- центрацию протонов посредством повышения рН в периплазме и увеличения мембранного по- тенциала [35]. Белок UreI образует протон-акти- вируемый канал для мочевины, активируемый низким рН и регулирует активность уреазы через варьирование уровней внутриклеточного им- порта мочевины [95]. Дополнительно алифати- ческая амидаза и формамидаза образуют аммиак и органические кислоты посредством гидролиза короткоцепочечных аминокислот. Активность этих ферментов регулируется двухкомпонент- ной системой ArsSR, состоящей из гистидин ки- назы ArsS, распознающей снижение рН и OmpR- подобного регулятора ответа [65]. Факторы, способствующие выживанию и перемещению H. pylori в просвете желудка Факторы, способствующие выживанию и перемещению H. pylori в просвете желудка Для того, чтобы выживать в условиях агрес- сивной среды желудка, колонизировать СОЖ и вызывать хроническое воспаление, H. pylori использует комплекс адаптивных механизмов. 274 Факторы патогенности H. pylori Факторы адгезии H. pylori pylori прояв- ляют полиморфизм в связывании сиалосодер- жащих структур через SabA, что свидетельствует о способности бактерий адаптироваться к уров- ню экспрессии сиалосодержащих рецепторов на поверхности эпителия СОЖ [2]. Если для ранних этапов колонизации первостепенное значение имеет взаимодействие BabA с Льюис- подобными антигенами на эпителиоцитах, то с увеличением выраженности воспалительно- го ответа увеличивается экспрессия sLeХ на их поверхности. Таким образом, SabA увеличивает уровни колонизации Нр воспаленной СОЖ [99]. AlpA/B (adherence-associated lipoprotein A and B). У Нр идентифицированы 2 гомологичных гена AlpA и AlpB, кодирующие поверхностные ад- гезины AlpA иAlpB [56, 75]. Однако до настоя- щего времени клеточные рецепторы для этих адгезинов остаются неидентифицированными. Для секреции AlpAB во внешней мембране бак- терий формируются «β-бочкообразные» поры, внутренняя полость которых сформирована 14 трансмембранными белковыми мономера- ми. Штаммы H. pylori, дефектные по alpA и alpB, или содержащие только alpB, проявляют слабую адгеизвную активность в отношении ламини- на, свидетельствующую, что ламинин является мишенью для AlpB [56]. Показано, что взаимо- действие AlpA/B с эпителиоцитами стимулирует запуск провоспалительного сигнального каска- да. Мутантные штаммы H. pylori с делецией гена alpAB проявляли слабую колонизационную ак- тивность и слабо индуцировали секрецию IL-6 и IL-8 [14, 41]. SabA (sialic acid-binding adhesin). Роль белка SabA как адгезина H. pylori была впервые доказа- на у страдающих гастритами пациентов, колони- зированных штаммами, дефектными по babA1A2 или babA2 [44, 49]. Мишенью для SabA является димерный сиало-льюис Х-гликосфинголипид (sLeХ), экспрессирующийся на поверхности эпителия СОЖ [44]. Также SabA способен свя- зывать и другие сиаловые рецепторы, напри- мер, расположенные на поверхности ламинина и эрит роцитов [2, 94]. Штаммы H. pylori прояв- ляют полиморфизм в связывании сиалосодер- жащих структур через SabA, что свидетельствует о способности бактерий адаптироваться к уров- ню экспрессии сиалосодержащих рецепторов на поверхности эпителия СОЖ [2]. Если для ранних этапов колонизации первостепенное значение имеет взаимодействие BabA с Льюис- подобными антигенами на эпителиоцитах, то с увеличением выраженности воспалительно- го ответа увеличивается экспрессия sLeХ на их поверхности. Таким образом, SabA увеличивает уровни колонизации Нр воспаленной СОЖ [99]. AlpA/B (adherence-associated lipoprotein A and B) вязывающий белок, способный неспеци фически связываться с эритроцитами, буккальным эпи- телием и ламинином [15]. Но следует отметить, что до настоящего времени аргументированные подтверждения проявлений этих эффектов in vivo отсутствуют. Липополисахарид (ЛПС). Синтез ЛПС H. pylori кодируют не менее 27 генов, разбросанных по всему геному бактерии. Участие ЛПС в ад- гезии на клетках эпителия СОЖ обусловле- но наличием в боковых О-цепях ЛПС Льюис- подобных лигандов, аналогичных антигенам си- стемы Льюис (Leb) ABO группы крови человека. Факторы адгезии H. pylori Показано, что большая часть изолятов H. pylori (> 80%), выделенных в Европе, экспрессирует LeX и/или LeY 2 типа, тогда как < 10% штам- мов — LeА и LeВ 1 типа. При этом штаммы, изо- лированные в Восточной Азии, экспрессируют антигены 1 и 2 типов [50, 79]. Льюис-подобный антиген X O-цепи ЛПС участвует в адгезии H. pylori к эпителию антральной части желудка, взаимодействуя с мембранными β-галактозид- связывающим рецептором галектином-3 [102]. При этом, структура Льюис-подобных антиге- нов H. pylori может претерпевать изменения в ди- намике инфекции in vivo, а также в ходе культи- вирования in vitro. Эти фазовые вариации ЛПС адаптируют бактерии к противодействию фак- торов резистентности посредством имитации Льюис-фенотипа эпителия СОЖ и подобная антигенная мимикрия не дает возможности рас- познавать их как «чужеродные» [67] В то же вре- мя длительное персистирование H. pylori может индуцировать синтез антител, перекрестно реа- гирующих с β-субъединицей протонной помпы (H+,K+-ATФаза), дислоцированной в канальцах париетальных клеток, что, в конечном итоге, может приводить к атрофии слизистой желуд- ка. Штаммы H. pylori могут экспрессировать эпитопы LeХ, LeY, LeА и LeВ, а антитела к ним могут реагировать с аналогичными эпитопами эпителия СОЖ [52]. К факторам, облегчающим персис тенцию бактерий, также можно отнести относительно низкую иммуногенность ЛПС H. pylori, способствующую хроническому тече- нию инфекции [102]. AlpA/B (adherence-associated lipoprotein A and B). У Нр идентифицированы 2 гомологичных гена AlpA и AlpB, кодирующие поверхностные ад- гезины AlpA иAlpB [56, 75]. Однако до настоя- щего времени клеточные рецепторы для этих адгезинов остаются неидентифицированными. Для секреции AlpAB во внешней мембране бак- терий формируются «β-бочкообразные» поры, внутренняя полость которых сформирована 14 трансмембранными белковыми мономера- ми. Штаммы H. pylori, дефектные по alpA и alpB, или содержащие только alpB, проявляют слабую адгеизвную активность в отношении ламини- на, свидетельствующую, что ламинин является мишенью для AlpB [56]. Показано, что взаимо- действие AlpA/B с эпителиоцитами стимулирует запуск провоспалительного сигнального каска- да. Мутантные штаммы H. pylori с делецией гена alpAB проявляли слабую колонизационную ак- тивность и слабо индуцировали секрецию IL-6 и IL-8 [14, 41]. HopZ (helicobacter outer protein Z). Другим по- верхностным адгезином H. pylori является белoк HopZ. Показано, что мутантные по гену hopZ штаммы бактерий проявляли слабую адгезию на клетки AGS in vitro. До настоящего времени остается неидентифицированным поверхност- ный рецептор, с которым взаимодействует HopZ. Ген hopZ является фазовариабельным, что обу- словлено наличием СТ динуклеотидных после- довательностей в регионе, кодирующем сигналь- ную последовательность [63]. Транскрипцию гена hopZ также регулируют изменения рН [48] и контакты с эпителиоцитами СОЖ [25]. Факторы адгезии H. pylori Важнейшим этапом колонизации H. pylori СОЖ является адгезия бактерий к эпителию. Ее медиируют липополисахарид (ЛПС) бактерий и специализированные адгезины OMP (outer membrane proteins, белки поверхностной мем- браны). Геном H. pylori содержит 32 гена, коди- рующих OMP. Последние включают подгруппы Hop (Helicobacter outer membrane proteins) и Hor (Нор-related). В подгруппу Hop входят белки ад- гезии BabA, SabA, AlpA/B, HopZ и OipA [4]. При микроскопии окрашенных биоптатов слизистой СОЖ пациентов, инфицированных H. pylori, S.J. Hessey et al. (1990) установили, что на момент фиксации препарата не менее 20% бактерий оказались прикрепленными к эпите- лию [28]. Дальнейшее изучение влияния коло- низации бактерий на ульраструктуру эпителия СОЖ показало, что H. pylori располагается на вер- шине микроворсинок и индуцирует образование «пьедесталов», либо прочно связывается с мем- бранами эпителиоцитов через фимбриальные структуры [54]. В качестве первого лиганда, взаи- модействующего с клеточными рецепторами, был идентифицирован фибриллярный гемагглюти- нин, связывающий N-ацетилнейраминиллактозу, позднее обозначенный как HpaA (H. pylori adhesion). Белок HpaA дислоцирован по всей по- верхности бактерий, включая оболочку жгути- ков [6]. Кроме того, у H. pylori обнаружен допол- нительный комплекс факторов, проявляющих свойства адгезинов in vitro. В частности, показано, что бактерии способны распознавать ганглио- тетраозилцерамиды, ганглиотриаозилцерамиды и фос фатидилэтаноламиды через экзофермент-S- подобный адгезин с липид-связывающей специ- фичностью. Также идентифицирован железос- В желудке H. pylori сталкивается с непре- рывным тонким слоем слизистого геля, по- крывающим поверхность СОЖ. Он состоит из гликопротеинов толщиной 190–300 мкм, под которыми располагается слой бикарбонатов, прилежащий к поверхностному эпителию сли- зистой оболочки. Вместе они образуют слизис- то-бикарбонатный барьер, защищающий эпи- телий от агрессивной среды и химуса в просвете желудка. В составе слизи находится комплекс антимикробных факторов, таких как имму- ноглобулин A, лизоцим, лактоферрин и др. [3]. Таким образом, он представляет собой эффек- тивную преграду для диффузии различных веществ, а также проникновения различных инфекционных агентов, впрочем, исключая H. pylori. В определенной степени в отношении последнего слизь играет защитную роль, так как H. pylori, располагаясь в ее толще, избегает 275 О.К. Поздеев и др. Инфекция и иммунитет SabA (sialic acid-binding adhesin). Роль белка SabA как адгезина H. pylori была впервые доказа- на у страдающих гастритами пациентов, колони- зированных штаммами, дефектными по babA1A2 или babA2 [44, 49]. Мишенью для SabA является димерный сиало-льюис Х-гликосфинголипид (sLeХ), экспрессирующийся на поверхности эпителия СОЖ [44]. Также SabA способен свя- зывать и другие сиаловые рецепторы, напри- мер, расположенные на поверхности ламинина и эрит роцитов [2, 94]. Штаммы H. Факторы адгезии H. pylori Плотные межклеточные контакты формируют клеточный барьер, препятствующий свободному прохождению различных молекул, где мембраны соседних клеток максимально сближены и «сши- ты» специализированными белками (клаудины, окклюдины и др.) [97, 103]. Молекула CagA со- держит несколько остатков тирозина, пригодных для фосфорилирования, причем мутации этих сайтов способны предотвратить фосфорилиро- вание белка CagA и нарушать развитие фенотипа «колибри» (удлинение и уплощение клеток эпи- телия, делающее их похожими на длинный узкий клюв этих птиц) [96]. Исследования последних лет выяснили функ- циональные свойства белка CagL, также кодиру- емого в острове патогенности cag [10, 38, 77, 88]. Ранее CagL рассматривали как компонент систе- мы секреции IV типа, обеспечивающей транс- портировку CagA. Позднее было установлено, что в действительности CagL является компонентом пилей, обеспечивающим контакт между эпите- лием СОЖ и бактерией [38]. В частности, CagL усиливает связывание инъекционного аппарата IV типа секреции с a5β1-интегриновым рецепто- ром эпителиоцита [18, 34]. С ним также могут вза- имодействовать и другие белки Cag (CagY, CagI) [34]. В конечном итоге, все эти взаимодействия медиируют транспорт CagA в клетки. Факторы адгезии H. pylori BabA (blood-group associated binding adhesin). Мишенями для BabA являются остатки фукозы льюис-подобных антигенов типа В (LeВ), экс- прессируемых клетками желудочного эпителия. В настоящее время идентифицированы 3 аллели гена bab: babA1, babA2 и babB. Функционально активным является ген babA2, кодирующий об- разование BabA [31]. Связывание BabA Нр с LeВ антигенами на поверхности эпителиоцитов ак- тивирует контакт-зависимую систему секреции IV типа, обеспечивающую непосредственную доставку эффекторных белков в цитозоль эпи- телиоцитов через особую «инъекционную иглу», отдаленно напоминающую конъюгационную пилю [32]. OipA (outer inflammatory protein A). Первона- чально белок OipA, кодируемый геном hopH, был идентифицирован как промотор синтеза интер- лейкина-8 (IL-8) эпителиоцитами СОЖ, незави- 276 2018, Т. 8, № 3 Факторы патогенности H. pylori симый от активности системы IV типа секреции, так как изогенные мутантные штаммы, дефект- ные по гену oipA индуцировали слабый провос- палительный ответ эпителием СОЖ [100, 101]. Показано, что бактерии индуцируют секрецию IL-8 посредством прямого контакта с эпители- оцитами. В эпителии СОЖ регуляцию транс- крипции гена цитокина осуществляет белок, подобный стимулированному интерфероном элементу ответа (ISRE), активаторный белок (AP)-1 и ядерный фактор NF-κB [78]. Позднее было установлено, что OipA может регулировать синтез других провоспалительных медиаторов, выраженность нейтрофильной инфильтрации СОЖ с развитием интерстициальной метапла- зии, а также вызывать повреждение цитоскелета эпителиоцитов [37, 42, 53, 85, 98]. В частности, по- казано, что OipA подавляет секрецию IL-10 и со- зревание дендритных клеток, что способству- ет развитию персистирующей инфекции [49]. Влияние OipA на развитие стрессового ответа цитоскелета клеток реализуется через фосфори- лирование киназ фокальной адгезии (FAK), что, в свою очередь, активирует киназу стрессового ответа Erk и образование ориентированных ак- тиновых микрофиламент (стрессовых волокон). Cag A H. pylori индуцирует фосфорилирование FAK остатка Y407, тогда как OipA — фосфорили- рование остатков Y397, Y576, Y577, Y861 и Y925, что указывает на большее влияние OipA на фор- мирование стрессовых волокон [89]. они становятся субстратом для внутриклеточ- ных тирозинкиназ, через которые бактерия мо- жет вмешиваться в процессы фосфоририлирова- ния. Попав в цитоплазму клетки, молекулы CagA фосфорилируется тирозиновыми протеинкина- зами Src и Abl по Glu-Pro-Ile-Tyr-Ala (EPIYA) мо- тиву в карбокси-териминальном домене [68, 82]. Фосфорилирование CagA приводит к наруше- нию межклеточных контактов, полярности и по- вреждению цитоскелета эпителиоцитов, а так- же стимулирует активность париетальных кле- ток [96]. В норме однослойный призматический железистый эпителий СОЖ отличает апико-ба- зальная полярность, которую обеспечивают раз- личные виды плотных межклеточных контактов, контролируемых цитоскелетом эпителиоцитов. Факторы H. pylori, повреждающие эпителий СОЖ Помимо мощного аппарата адгезии, H. pylori обладает комплексом эффекторных белков, не- посредственно повреждающих эпителиоциты. Их кодируют более 30 генов, дислоцированных в «острове патогенности» Сag. Кроме того, в нем расположены гены, кодирующие синтез компо- нентов систем секреции III и IV типа, обеспечи- вающих перенос эффекторных молекул в клет- ки-мишени. Интерес представляет тот факт, что способность к непосредственному взаимо- действию с эпителием СОЖ проявляют не более 20% бактерий, что во многом определяет вариа- бельность клинических проявлений, ассоции- рованных с циркуляцией H. pylori [30]. Помимо белка CagA, по системе IV типа секре- ции в эпителий СОЖ могут транспортироваться компоненты клеточной стенки, в частности, пеп- тидогликан и муропептиды. Попав в цитозоль, пептидогликан H. pylori распознается лигандом NOD1 (nucleotide-olygomerization domain). NOD1 относится к группе паттерн-распознающих ре- цепторов, располагающихся в цитоплазме и рас- познающих молекулы пептидогликана грамот- рицательных и грамположительных бактерий в виде высококонсервативных фрагментов экзо- генных молекул, ассоциированных с патогенны- ми микроорганизмами PAMP (pathogen-associated molecular pattern). Связывание собственных ли- гандов с паттерн-распознающими рецепторами запускает внутриклеточные сигнальные каска- ды, приводящие к активации факторов транс- Cag А (citotoxin associated gene A). Криптический иммунодоминантный белок Cag А является, очевидно, самым известным фактором виру- лентности H. pylori и своеобразным маркером «острова патогенности» Сag. [55]. Ген cagA явля- ется уникальным, а его продукт не имеет гомо- логов среди других бактерий. Он присутствует не у всех штаммов H. pylori, и его наличие расце- нивают как фактор риска последующего разви- тия язвы и болезней злокачественного роста [86]. Молекулы CagA транспортируются в клетку-ми- шень с помощью аппарата IV типа секреции, где 277 О.К. Поздеев и др. Инфекция и иммунитет крипции, в том числе NF-κB, являющегося фак- тором, инициирующим синтез провоспалитель- ных цитокинов и хемокинов, активирующим фагоциты и т. д. [93]. эпителия желудка крыс с VacA нарушает обра- зование «стрессовых волокон» и приводит к раз- рушению микротрубочек [58]. Подобный эффект VacA имеет значение не только для реализации вакуолизации, но объясняет ингибирующий эффект токсина на пролиферативный ответ клеток [59, 71]. Действуя на митохондриальную мембрану, VacA вызывает освобождение цитох- рома С и индуцирует апоптоз эпителиоцитов [85]. Повреждение эпителиоцитов VacA запуска- ет воспалительный процесс в СОЖ через син- тез клетками провоспалительных цитокинов: IL-1β, IL-6, IL-8, фактора некроза опухолей аль- фа (TNFα) и др. В свою очередь, цитокины спо- собствуют повреждению эпителиоцитов желуд- ка [104]. VacA (vacuolating cytotoxin A). Вакуолизи- рующий цитотоксин VacA — уникальный экзо- токсин, аминокислотные последовательности которого не встречаются у других бактериальных или эукариотических белков. По уровню синтеза VacA является основным белком, секретируемым H. pylori. Факторы H. pylori, повреждающие эпителий СОЖ Изначально VacA экспрессируется как протоксин с молекулярной массой 140 kD состо- ящий из N-терминального сигнального пептида, центрального региона, образующего собственно токсин, и C-терминального домена-аутотранс- портера [66, 74, 76]. В ходе секреции по IV типу центральный регион (м.м. 88 kDа) конвертирует- ся в субъединицы А (VacAp33) и В (VacAp55). [33]. Мишенью для субъединицы В VacA являются рецептор-подобные протеины тирозинфосфата- зы RPTPα и RPTPβ, а также гликозилированные трансмембранные белки на поверхности эпи- телиоцитов. Эти контакты запускают механизм рецептор-опосредованного эндоцитоза, обуслов- ливающего попадание VacA в эпителио цит [70]. Перед проникновением в клетку-мишень, под действием низкого или высокого рН протоксин переходит с активную форму [47]. После интер- нализации молекула VacA локализуется в мем- бранных микровезикулах [23], а затем с помощью протонной помпы вакуолярного типа (АТФаза V типа) закачиваются в поздние эндосомы и лизо- сомы. В них в неизмененной форме токсин может сохраняться в течение нескольких дней [72, 81]. Активность VacA реализуется через образование анионных каналов в мембранах, обусловленное активацией АТФазы V-типа [61, 84], что пони- жает рН внутри вакуолей эпителиоцитов и, тем самым, обеспечивает поступление в них анионов (аммиака и др.) из внутриклеточного простран- ства [87, 92, 46]. Под действием разницы осмо- тического давления в вакуоли поступает вода, что приводит к их набуханию. Сливаясь друг с другом, вакуоли приводят к разрыву клеточной мембраны и гибели клетки [11]. При этом актив- ность цитотоксина возрастает по мере снижения рН желудочного сока [73]. По аналогии с класси- ческими двухкомпонентными экзотоксинами, предполагают, что субъединица В взаимодей- ствует с мембраной, а субъединица А образует каналы [46, 70]. A Уреаза. В слое слизи между просветом желуд- ка и эпителием СОЖ существует градиент рН, обусловленный секрецией бикарбонатов в эпи- телиоцитах, что обеспечивает оптимальный рН на поверхности клеток. На поверхности эпите- лия под слоем слизи рН равен 6,5–7,0, в просве- те желудка — 1,5–3,0. Слизь замедляет скорость обратной диффузии Н+, в это время бикарбонаты нейтрализуют ионы водорода. Секреция бикар- бонатов и слизи зависит от микроциркуляции и регулируется простагландинами, последние же постоянно синтезируются поверхностным эпи- телием. При усилении секреции HCl усиливает- ся и секреция слизи. Этот градиент препятствует повреждению клеток ионами Н+, поскольку слой слизи замедляет скорость их обратной диффу- зии, за это время НСО3 – успевает нейтрализовать Н+, формируя «слизисто-бикарбонатный ба- рьер» [12]. Второй защитный барьер составляют гликопротеиновые компоненты слизи, образую- щие непрерывный слой и защищающие эпите- лий желудка от действия пептических факторов. 2018, Т. 8, № 3 Факторы патогенности H. pylori Эндопротеаза HtrA (high temperature require- ment A). Эндопротеазы HtrA локализуются в пе- риплазме бактерий и участвуют в обеспечении устойчивости бактериальной клетки к высоким температурам. Проявляют свойства сериновых протеаз и участвуют в удалении белков, повреж- денных либо денатруированных в результате воздействия высокой температуры или оксида- тивного стресса. Эндопротеазы HtrA и гомоло- гичные им ферменты экспрессируются в виде протеаз и шаперонов у про- и эукариот [91]. Одним из субстратов протеаза HtrA H. pylori является Е-кадгерин [29, 40, 60]. Е-кадгерин представляет собой одноцепочечную транс- мембранную молекулу и является основным белком межклеточных контактов эпителиаль- ных клеток, обеспечивающим поддержание ба- рьерных свойств. Внеклеточная часть молеку- лы имеет пять гомологичных ЕС-доменов, уча- ствующих в Са2+-зависимом контакте с двумя молекулами Е-кадгерина на соседних клетках. Цитоплазматический домен связан с актиновым цитоскелетом через молекулы α- и β-катенина, что обеспечивает стабильность межклеточ- ных контактов [27]. Эндопротеаза HtrA H. pylori разрушает внеклеточные домены Е-кадгерина и, соответственно, межклеточные контакты, что позволяет бактериями проникать в межклеточ- ные пространства. NAP (neutrophil-activating protein). Фактор, ак- тивирующий нейтрофилы (NAP) был впервые выделен из водных экстрактов H. pylori [17, 24]. Являясь цитоплазматическим белком, NAP вы- свобождается при разрушении бактериальной клетки. Анализ последовательности аминокис- лот показал, что NAP гомологичен бактерио- ферритинам. Бактериоферритины представляют собой олигомерные белки, формирующие по- лость внутри белковой глобулы. В этой полости происходит накопление Fe3+ в виде нетоксич- ных для клетки ферригидритов, формирующих минеральное ядро. Они играют исключительно важную роль в поддержании внутриклеточного гомео стаза Fe3+, а также образуют прочные ком- плексы с ДНК. Другой важнейшей функцией бак- териоферритинов является инактивация свобод- норадикального окисления с использованием железа в качестве катализатора [80]. Установлено, что активность NAP H. pylori отличается от про- чих бактериоферритинов. Он спо собен прохо- дить через эпителий CОЖ и эндотелий капил- ляров, где стимулирует нейтрофилы и моноциты к образованию IL-12 и IL-23 [1, 13]. Эти цитоки- ны индуцируют синтез IFNγ T-клетками и игра- ют важную роль в развитии Th1-ответа [104]. NAP взаимодействуют с Toll-подобным рецептором TLR-2, распознающим компоненты клеточной стенки бактерий, но в отличие от прочих TLR-2 гонистов, NAP индуцирует образование IL-12 [1]. Синтез NPA и экспрессия эпителиоцитами IL-8 с последующим запуском всего провоспа- лительного каскада обусловливает развитие ней- трофильной инфильтрации, обнаруживаемой у 100% инфицированных лиц. Мигрирующие в СОЖ нейтрофилы повреждают эпителиальные клетки за счет выделения кислородных суперок- сидантов и выделения комплекса энзимов, а так- же продуцируют провоспалительные цитокины. 2018, Т. 8, № 3 В таких условиях на фоне прогрессирования вос- паления в одних случаях имеют место поврежде- ние и гибель эпителиоцитов с формированием эрозивных и язвенных дефектов, а в других по- степенно формируются атрофии, метаплазии и неоплазии СОЖ. С ( GGT (gamma-glutamyl transpeptidase). Сообще- ние о том, что гамма-глутамил транспептидаза является фактором патогенности H. pylori яви- лось полной неожиданностью, так как у дру- гих бактерий таковыми гамма-глутамилпеп- тидазы (GGT) никогда не рассматривалась [9]. В бактериальной клетке фермент располагает- ся в пузырьках внешней мембраны и участву- ет во взаимодействии H. pylori с эпителиоцита- ми [57]. GGT активирует NF-κB, стимулирует синтез IL-8, а также образование Н2О2 эпите- лиоцитами. Кроме того GGT H. pylori повыша- ет уровень 8-гидрокси-2-деоксигуанина, что указывает на оксидативное повреждение ДНК. Клиническую значимость GGT подтверждает высокая активность фермента у штаммов, выде- ленных от пациентов с язвами желудка, по срав- нению с изолятами, выделенными от пациен- тов с диспепсиями [26]. Также GGT опосредует деградацию глутатиона с образованием веществ с проокидантной актиностью, что способствует повреждению эпителия СОЖ [20]. Следствием прямого или непрямого (за счет действия растворимых продуктов) взаимодей- ствия H. pylori с эпителием СОЖ является актива- ция эпителиоцитов и усиленное образование ими сигнальных молекул, посредством которых запус- кается целый каскад защитных механизмов. 1. Amedei A., Cappon A., Codolo G., Cabrelle A., Polenghi A., Benagiano M., Tasca E., Azzurri A., D’Elios M.M., Del Prete G., de Bernard M. The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses. J. Clin. Invest., 2006, vol. 116, no. 4, pp. 1092–1101. doi: 10.1172/JCI27177 Факторы H. pylori, повреждающие эпителий СОЖ Но этот защитный слой содержит также моче- вину, поступающую посредством транссудации из плазмы крови и концентрирующуюся вблизи межклеточных промежутков [39]. Ряд авторов указывают на способность аммиака, высвобож- дающегося при гидролизе мочевины уреазой H. pylori, разрушать фосфолипидный монослой в слизи, тем самым истощать гидрофобный ба- рьер СОЖ, что позволяет H. pylori преодолевать эти барьеры [45, 51]. Аммиак также резко повы- шает рН внутри слизистой, что приводит к уве- личению содержания неионизированного ве- щества [90]. Показано, что неионизированный аммиак может проникать через фосфолипидные мембраны эпителиоцитов, при этом пропорцио- нально повышению рН увеличивается его про- никающая способность. В цитозоле неионизи- рованный аммиак превращается в NH4 + и ОН–, что повышает внутриклеточный и митохондри- альный рН, нарушая тем самым митохондри- альное и клеточное дыхание и, как следствие, энергетический метаболизм, а в конечном сче- те — жизнеспособность клеток [90]. Но этот защитный слой содержит также моче- вину, поступающую посредством транссудации из плазмы крови и концентрирующуюся вблизи межклеточных промежутков [39]. Ряд авторов указывают на способность аммиака, высвобож- дающегося при гидролизе мочевины уреазой H. pylori, разрушать фосфолипидный монослой в слизи, тем самым истощать гидрофобный ба- рьер СОЖ, что позволяет H. pylori преодолевать эти барьеры [45, 51]. Аммиак также резко повы- шает рН внутри слизистой, что приводит к уве- личению содержания неионизированного ве- щества [90]. Показано, что неионизированный аммиак может проникать через фосфолипидные мембраны эпителиоцитов, при этом пропорцио- нально повышению рН увеличивается его про- никающая способность. В цитозоле неионизи- рованный аммиак превращается в NH4 + и ОН–, что повышает внутриклеточный и митохондри- альный рН, нарушая тем самым митохондри- альное и клеточное дыхание и, как следствие, энергетический метаболизм, а в конечном сче- те — жизнеспособность клеток [90]. Кроме вакуолизации VacA оказывает и дру- гие эффекты. На уровне клеток желудочного эпителия он нарушает функциональную актив- ность эндосом и лизосом, индуцирует увеличе- ние внеклеточной секреции кислых гидролаз, ингибирует клеточную пролиферацию, по- вреждает митохондрии, а также дезорганизует цитоскелет эпителиоцитов [19, 73]. Механизмы повреждения цитоскелета остаются до конца не выясненными, но показано, что экспозиция 278 2018, Т. 8, № 3 2. 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Thomsen L., Tasman-Jones C., Morris A., Wiggins P., Lee S., Forlong C. Ammonia produced by Campylobacter pylori neutrali- zes H+ moving through gastric mucus. Scand. J. Gastroenterol., 1989, vol. 24, no. 6, pp. 761–768. doi: 10.3109/00365528909093119 91. Поступила в редакцию 14.02.2018 Принята к печати 22.06.2018 Authors: Pozdeev O.K., PhD, MD (Medicine), Professor, Head of the Department of Microbiology, Kazan State Medical Academy, Kazan, Russian Federation; Pozdeeva A.O., Assistant of the Department of Therapy and Family Medicine, Kazan State Medical Academy, Kazan, Russian Federation; Valeeva Yu.V., PhD (Medicine), Associate Professor, Department Emergency Medical Care and Simulatory Medicine, Kazan (Volga region) Federal University, Kazan, Russian Federation; Gulyaev P.E., Assistant of the Department of Microbiology, Kazan State Medical University, Kazan, Russian Federation. Список литературы/References 414–424. doi: 10.1053/gast.2002.34781 pp /g 101. Yamaoka Y., Kwon D.H., Graham D.Y. AM(r) 34,000 proinflammatory outer membrane protein (oipA) of Helicobacter pylori. Proc. Natl. Acad. Sci. USA, 2000, vol. 97, no. 13, pp. 7533–7538. doi: 10.1073/pnas.130079797 102 Y k S A K H hi S K b T F jii N Y k hi T H ib d H li b l i li l 102. Yokota S., Amano K., Hayashi S., Kubota T., Fujii N., Yokochi T. Human antibody response to Helicobacter pylori lipopolysac- charide: presence of an immunodominant epitope in the polysaccharide chain of lipopolysaccharide. Infect. Immun., 1998, vol. 66, no. 6, pp. 3006–3011. 103. Yokota S., Okabayashi T, Rehli M., Fujii N., Amano K. Helicobacter pylori lipopolysaccharides upregulate toll-like receptor 4 expression and proliferation of gastric epithelial cells via the MEK1/2-ERK1/2 mitogen-activated protein kinase pathway. Infect. Immun., 2010, vol. 78, no. 1, pp. 468–476. doi: 10.1128/IAI.00903-09 , , , , pp / 104. Zarrilli R., Ricci V., Romano M. Molecular response of gastric epithelial cells to Helicobacter pylori-induced cell damage. Cell. Microbiol., 1999, vol. 1, no. 2, pp. 93–99. doi: 10.1046/j.1462-5822.1999.00018.x Авторы: Поздеев О.К., д.м.н., профессор, зав. кафедрой микробиологии Казанской государственной медицинской академии — филиала ФГБОУ ДПО РМАНПО МЗ РФ, г. Казань, Россия; Поздеева А.О., ассистент кафедры терапии и семейной медицины Казанской государственной медицинской академии — филиала ФГБОУ ДПО РМАНПО МЗ РФ, г. Казань, Россия; Валеева Ю.В., к.м.н., доцент кафедры неотложной медицинской помощи и симуляционной медицины ФГАОУ ВО Казанский (Приволжский) федеральный университет, г. Казань, Россия; Гуляев П.Е., ассистент кафедры микробиологии, ФГБОУ ВО Казанский государственный медицинский университет МЗ РТ, г. Казань, Россия. Authors: Pozdeev O.K., PhD, MD (Medicine), Professor, Head of the Department of Microbiology, Kazan State Medical Academy, Kazan, Russian Federation; Pozdeeva A.O., Assistant of the Department of Therapy and Family Medicine, Kazan State Medical Academy, Kazan, Russian Federation; Valeeva Yu.V., PhD (Medicine), Associate Professor, Department Emergency Medical Care and Simulatory Medicine, Kazan (Volga region) Federal University, Kazan, Russian Federation; Gulyaev P.E., Assistant of the Department of Microbiology, Kazan State Medical University, Kazan, Russian Federation. Поступила в редакцию 14.02.2018 Принята к печати 22.06.2018 Received 14.02.2018 Accepted 22.06.2018 Авторы: Received 14.02.2018 Accepted 22.06.2018 Поступила в редакцию 14.02.2018 Принята к печати 22.06.2018 283
W4233586489.txt	https://www.degruyter.com/document/doi/10.1515/9783110693447-015/pdf	de		15 Fazit: Wissenschaftsurheberrecht – ein Recht für Nutzungsrechte und Nutzungsfreiheiten	De Gruyter eBooks	2,020	cc-by	4,241	15 Fazit: Wissenschaftsurheberrecht – ein Recht für Nutzungsrechte und Nutzungsfreiheiten Das Urheberrecht, insbesondere das Wissenschaftsurheberecht, hat systematisch ein dogmatisches und ein Problem in seiner Rechtsrealität – und damit auch ein Akzeptanzproblem bei den davon betroffenen Wissenschaftlern. Das erste Problem ist eins der Fundamente, das zweite ein Problem des Hauses, welches auf diesen Fundamenten errichtet wurde und an dem immer noch weiter gewerkelt wird. Zu den Fundamenten sind zu rechnen den Geist des 19. Jahrhunderts widerspie gelnde Pfeiler wie z. B. die Konzepte von Schöpfung/Schöpfer, Werk, Wissen als Immaterialgut, Monismus (Einheit von persönlichkeits- und vermögensrechtlichen Ansprüchen der Urheber), Eigentums- und Vergütungsansprüche. Wenn – um noch einmal auf die Skepsis von (Dreier/Hilty 2015) zurückzukommen – die Fundamente brüchig geworden sind, kann das Haus nicht länger Bestand haben. Die Fundamente des Urheberrechts mögen für eine gewisse Zeit im 19. Jahrhun dert sinnvoll gewesen sein – so der Schutz des Urhebers, des kreativen Schöpfers vor dem einschränkenden Zensuransinnen einer undemokratischen Obrigkeit im 19. Jahrhundert. Veränderte Rahmenbedingungen – technologische, soziale, politische Entwicklungen, andere Märkte und daraus entwickelte moralische Ein stellungen – lassen das Urheberrecht heute mit der Übernahme von Prinzipien aus dem 19. Jahrhundert und daraus abgeleiteten Regulierungen wie aus der Zeit gefallen erscheinen. Das Urheberrecht hält seine Systematik und Dogmatik durch eine Reihe von heute obsolet angenommenen Als-ob-Annahmen, Fiktionen, auf recht. Diese haben sich in den oben erwähnten Fundamenten verfestigt. Eine Weile kann über das Als-ob Verhalten – daran sei mit Vaihinger erinnert – aus Fiktionen durchaus etwas Nützliches entstehen. Aber sie können auch dysfunktional werden, wenn aus ihnen normative Verpflichtungen mit ungewollten negativen „Nebenfol gen“ entstehen. Das trifft besonders für die Fiktion der immateriellen Objekte zu, die, vor allem für Bildung und Wissenschaft, dysfunktional mit negativen Effekten geworden sind. (Peukert 2018) hat überzeugend nachgewiesen, dass dem „immateriellen Objekt“ gar keine ontologische Realität entspricht – was aber, als Sieg des das Ur heberrecht konstituierenden Als-ob-Verhaltens, die Juristen nicht daran gehindert hat, diese Fiktion als soziale Realität anzuerkennen und entsprechende rechtlich verbindliche Regelungen für den Umgang damit festzulegen. Wenn etwas zu einem Objekt erklärt wurde (sei es auch „nur“ als ein immaterielles Objekt) und wenn das allgemeine und damit soziale und politische Anerkennung gefunden hat, dann ist es auch gutsfähig. Das Gut „Wissen“ kann kommodifiziert und entsprechend kann mit ihm nach Marktprinzipien gehandelt werden. Das Urheberrecht tut so, Open Access. © 2020 Rainer Kuhlen This work is licensed under a Creative Commons Attri bution 4.0 License. https://doi.org/10.1515/9783110693447-015 382 \| 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten als ob es mit seinen Regelungen die Interessen der Urheber, der Ersteller von Wissensobjekten, schützt, während es tatsächlich immer mehr die Interessen der Verwerter unterstützt. Wenigen außerhalb der Rechtswissenschaft ist vermutlich bewusst, dass es diese Fiktionen sind, die dafür verantwortlich sind, dass das Haus im Urheberrecht so unkomfortabel für Bildung und Wissenschaft eingerichtet wurde. Die Metapher des Hauses bezieht sich auf die positiven Urheberrechtsge setze, die sich u. a. aus den erwähnten Konzepten ableiten. Kleine Reparaturen, Instandsetzungen, wie sie im Gesetz immer wieder unternommen werden (zuletzt durch das UrhWissG 2017/18), helfen nicht. Das derzeit geltende Urheberrecht ist für Bildung und Wissenschaft eher behin dernd, in weiten Teilen sogar überflüssig. Politiker und Institutionen wie Parteien, die das Urheberrecht formulieren, haben in den letzten 20 Jahren zu wenig auf die Transformationsprozesse durch die drei Regulierungsinstanzen, Technologie, Markt und Wertewandel und auf die dadurch entstandenen Leitideen für den Umgang mit Wissen und Information reagiert. Dass der deutsche Gesetzgeber, unterstützt auch von vielen Rechtswissenschaftlern, sich immer wieder auf die internationalen, urheberrechtlich relevanten Vorgaben bezogen hat, die ihm keinen weiteren Spielraum böten, kann nicht als Entlastung akzeptiert werden. Zum einen wäre der Spielraum viel größer gewesen, wenn man sich mehr um kreative als um affirmative Hermeneutik bemüht hätte. Auch die internationalen Vorgaben arbeiten mit unbestimmten Rechtsbegriffen, die auslegungsbedürftig und -fähig sind. Sie könnten ganz anders als bislang verstanden werden. Das trifft auch für programmatische Vorgaben wie den Drei-Stufen-Test zu, für den eine andere Interpretation nötig und möglich ist. Ebenso können und sollten manche anderen Begriffe in Schrankenregelungen des Urheberrechts neu verstanden werden. Wir haben das am Beispiel der Bibliotheken gezeigt, bei denen man zu ganz anderen Regulierungen käme, wenn sie nicht mehr als Gebäude, sondern als virtuelle Organisationen begriffen würden. Vor allem für Bildung und Wissenschaft sind die erwähnten Fundamente nicht tragfähig. Solange aus den alten, dysfunktional gewordenen Konzepten weiter Rechtsvorschriften abgeleitet werden, ist heute im 21. Jahrhundert für Bildung und Wissenschaft vom Urheberrecht wenig zu erwarten. Dem Recht ist es nicht gelungen bzw. der Gesetzgeber hat es auch nicht gewollt, der aus den alten Fundamenten sich ableitenden Kommodifizierung bzw. der daraus folgenden Ökonomisierung und damit der Einbindung des Urheberrechts in ein Handelsrecht Einhalt zu gebieten. Das Urheberrecht ist entsprechend weiter davon ausgegangen, die Informations wirtschaft schützen zu müssen, und hat immer weiter an Schrankenbestimmungen gebastelt, die sich nur durch Ausnahmen von den exklusiven Rechten der Rechts inhaber rechtfertigen und die an der Praxis und den Bedürfnissen in Bildung und Wissenschaft vorbeigehen. Es hat weiter darauf gesetzt, dass den Verwertern nicht 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten \| 383 nur für die Erstellung ihrer Informationsprodukte eine Vergütung zusteht, sondern auch über die in den Schrankenregelungen geregelten aktuellen Nutzungen. Die Probleme mit dem Urheberrecht spiegeln sich auch in der Benennung dieses Rechts wider. Ein Recht, das sich letztlich aus dem öffentlichen Interesse an Wissen und Information begründet, sollte nicht auf ein individuelles Recht, auf ein exklusives Recht der Urheber reduziert werden. Das Urheberrecht sollte vom Konzept des Urhebers als individueller Schöpfer befreit werden und damit von dem Recht, über diese „Schöpfungen“ exklusiv verwertend verfügen zu können. Die exklusive Rechtsverfügung des Urhebers – auch das Recht, alle Verwertungsrechte als Nutzungsrechte an kommerzielle Verwerter abgeben zu dürfen – ist die Ursache für die Kommodifizierung und Ökonomisierung auch von öffentlich finanziertem Wissen. Je stärker das Urheberrecht die Position des Urhebers macht, desto stärker, so paradox das klingt, unterstützt es die Interessen der Verwerter. Das ist gerade nicht im Interesse des wissenschaftlichen Urhebers. Das belegen die immer häufiger und stärker werdenden Widerstände aus der Wissenschaft gegen die kommer zielle Aneignung und Ausbeutung ihrer Wissensobjekte vor allem durch das big business großer internationaler Verlagskonsortien. Auch das Wissenschaftsurheber recht – und diese Einschätzung ist wohl heute keine vernachlässigungsbedürftige Fehlinterpretation – ist immer mehr zu einem Handelsrecht, zu einem Investiti onsschutzrecht geworden, zu einer Schutzfunktion des digitalen Kapitalismus der Gegenwart (vgl. Staab 2019). Maximilian Becker spricht von der „Zweckentfremdung des Urheberrechts als zentrales Wirtschaftsrecht des Internets“.⁶³⁸ Viele internationale Förderorganisationen ziehen daraus die Konsequenz und verlangen inzwischen, dass es nicht bei dem exklusiven freien Recht der wissenschaftlichen Autoren bleiben darf, welches den Weg zur verknappenden Verwertung freimacht, sondern dass Publikationen von öffentlich finanzierten Werken frei nach Open-Access-Prinzipien verfügbar sein müssen. In Deutsch land, anders als in vielen anderen Ländern, ist es noch für viele Betroffene, auch für Autoren, problematisch, wenn diese freie Verfügbarkeit über eine Mandatie rung erzwungen werden soll. Erfolgversprechender wäre es vielleicht, den Weg in Richtung einer umfassenden Open-Access-Transformation dadurch zu befördern, dass das individuelle Recht explizit um ein am Gemeinwohlinteresse orientiertes institutionelles Recht erweitert würde. Diese Position hat sich der deutsche Gesetz geber bislang nicht angeschlossen, obgleich der Anlass bei der Einführung eines Zweitverwertungsrechts dies nahegelegt hätte. Ein institutionelles Recht schließt den Schutz der Interessen der „Urheber“ in keiner Weise aus. Auch das Wissenschaftsurheberrecht, wie es hier vorgeschlagen 638 (Becker 2019) Von der Freiheit, rechtswidrig handeln zu können, S. 648. 384 \| 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten wird, garantiert die Persönlichkeitsrechte. Autoren haben weiter das erste, zur Wis senschaftsfreiheit zu rechnende exklusive Recht, ihr produziertes Wissen öffentlich zu machen. Daraus müssen aber keine individuellen exklusiven Verwertungsrechte folgen. Schon gar nicht muss ihnen das Recht zugestanden werden, die im Urhe berrecht ihnen zugesicherten Verwertungsrechte vollständig als Nutzungsrechte an kommerzielle Verwerter zu übertragen, ohne dass die sie tragenden und finanzie renden Hochschulen und Forschungseinrichtungen darüber ein Mitspracherecht haben – wie es im Patentrecht der Fall geworden ist. Zur Wissenschaftsfreiheit kann es nicht gehören, alle Nutzungsrechte vollständig an kommerzielle Verwer ter übertragen zu dürfen – mit dem Ergebnis, dass die Rechte der Öffentlichkeit (und damit der Wissenschaft) an der Nutzung von Informationsobjekten unbillig eingeschränkt werden. Nichts muss im Recht so bleiben, wie es ist – also auch nicht die Unterstüt zung oder Tolerierung der Kommodifizierung von Wissen durch das Urheberrecht. Gegenwärtig artikuliert sich auf vielen Gebieten von Wirtschaft und Gesellschaft der Widerstand gegen die ökonomische Dominanz und den staatlich unterstützten Privatisierungsansprüchen gegenüber materiellen und immateriellen Ressourcen, welche für das Wohlergehen aller Menschen oder sogar für Leben aller Menschen entscheidend sind. Genannt seien hier nur Wasser, Energie, Luft, Klima, Bildung, Gesundheit, Verkehr, Wohnen, Nahrung – und eben auch Wissen und Informa tion. Das Konzept der Commons (Gemein-/Allmendegüter), das sich auf diese unverzichtbaren Ressourcen bezieht, erlebt in den letzten Jahren weltweit eine Renaissance. Für den Umgang mit den letztlich commons-basierten Ressourcen haben sich seit einigen Jahren neue Leitideen entwickelt, die Auswirkungen auf die politischen, sozialen und ökonomischen Strukturen und eben auch auf das Recht haben (werden). Auch diese neuen Leitideen waren anfänglich nur Fiktionen oder sogar nur Träume (wie der zu Beginn in (FN 61) erwähnte Traum von Grötschel). Aber in ihrer institutionellen Verdichtung zu Leitideen haben sie durchaus eine pragmatische, konstruktive handlungsleitende Funktion – auch eine befreiende Wirkung von früheren Fiktionen, die damals auch die pragmatische Leistung der Befreiung von bevormundenden und kontrollierenden Systemen erbrachten, aber heute nicht mehr befreiend wirken. Für jedermann ersichtlich sind neue Leitideen bezüglich der Ressourcen Luft/ Klima/Energie. Der Zeitgeist ist eindeutig ökologisch bestimmt und wird auch in Politik, Gesellschaft und Wirtschaft akzeptiert und hat für die politischen, sozialen und ökonomischen Strukturen und Verhaltensweisen Konsequenzen. Wirtschaft ohne Ökologie ist nicht mehr denkbar. Ökologie allgemein war nicht das Thema dieser Darstellung. Aber es verfertigt sich in der Öffentlichkeit auch so etwas wie eine Wissensökologie für den Umgang mit immateriellen Objekten (ohne dass diese Benennung schon umfassend Eingang im Sprachgebrauch gefunden 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten \| 385 hat). Wissensökologie, der nachhaltige Umgang mit Wissen und Information, ist das Pendant zur allgemeinen Ökologie für den nachhaltigen Umgang mit materiellen Objekten.⁶³⁹ Die hier geübte Kritik an der Kommodifizierung von Wissen und Information und an dem Zögern von Rechtsetzung, der Gesetzgebung und Rechtsprechung ist Teil des gegenwärtigen Unbehagens an bzw. des Protestes gegen die Zerstörung der Ressourcen durch sich verselbständigende ökonomische Interessen. Das Reden über den Umgang mit Wissen und Information gestaltet sich in der Wissenschaft zunehmend als ein Reden über den offenen, nutzungs- und vergütungsfreien Zugriff auf das öffentlich entstandene und dann publizierte Wissen. Das ist die sich seit etwa 20 Jahren entwickelnde Leitidee.⁶⁴⁰ Darüber wird zunehmend in der Wissenschaft, aber immer mehr auch weltweit bei den politischen Instanzen gesprochen. Es bleibt dann nicht beim Reden. Das sich verfestigende Reden, das, was Peukert in Rückgriff auf Searle einen deklarativen Sprechakt genannt hat, wird zur sozialen Realität. Diese Realität gewinnt Gestalt im Handeln der davon Betroffenen, sich die Nutzungsfreiheit von Wissen und Information zu sichern. Konkretisiert und operationalisierbar wird inzwischen diese sozusagen institutionalisierte Leitidee mit Blick auf Bildung und Wissenschaft durch die allgemeine Anerkennung von Open-(Access)-Prinzipien. Open ist der Default nicht nur des Publizierens, sondern der Prozesse in Bildung und Wissenschaft allgemein. Die dadurch angestoßene Transformation der Informationsmärkte ist längst noch nicht abgeschlossen und schon gar nicht hat die Gesetzgebung für das Ur heberrecht dieser Transformation ausreichend Rechnung getragen. Ebenfalls ist es nicht sicher, ob durch diese Transformation der kommerziellen Verwertung auch von öffentlich produziertem Wissen Einhalt geboten werden kann. Ob als Dauerzustand oder nur als Übergang – derzeit zeigt sich die Öffentlichkeit durchaus bereit, die kommerzielle Wirtschaft auch im allgemeinen Open-Access-Paradigma auf den wissenschaftlichen Informationsmärkten nicht nur am Leben zu halten, 639 Vgl. (Kuhlen 2004e) Nachhaltigkeit muss nicht Verknappung bedeuten – in Richtung Wis sensökologie; (Kuhlen 2004f) Artikel Wissensökologie und (Kuhlen 2012b) Wissensökonomie und Wissensökologie zusammen denken. 640 Die Leitidee der Nutzungsfreiheit sollte keine Privilegierung von Bildung und Wissenschaft bedeuten – obgleich dies hier im Vordergrund stand –, sondern bezieht sich auch die Nutzung von Wissen in der Öffentlichkeit, also durch jedermann, für alle. Vgl. (Chan et al. 2019) Situating Open Science: “Contextualizing Openness aims to stimulate further research and debates about how to collectively design a knowledge system that is open and equitable for all.”; ebenso Nick Shockey, posted 25.10.2019 on International Open Access Week: For Whom? Prompting Ourselves to Center Equity Year-Round – https://bit.ly/2pjephH; vgl. das Plädoyer von Klaus Graf für eine Öffnung der elektronischen Ressourcen der Bibliotheken über einen für jedermann erwerblichen Bibliotheksausweis. Remote Access und Open Access. Archivalia 27.10.2019 – https://bit.ly/2BVSGib. 386 \| 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten sondern auch als weiter bestimmenden „Partner“ anzuerkennen – mit der Konse quenz, dass auch die Open-Access-Märkte entscheidend von den kommerziellen Informationsmärken dominiert werden. Die Bereitschaft von öffentlich finanzier ten Einrichtungen, für einen in einer (kommerziellen) Open-Access-Zeitschrift zu erscheinenden Artikel eine Publikationsgebühr zu zahlen – das geschah lange Zeit über eine APC –, lässt deutlich erkennen, dass die Leistung der kommer ziellen Verwerter nicht nur anerkannt wird, sondern dass diese Leistung durch finanzielle Unterstützung stabil gehalten werden soll. Einen Schritt noch weiter gehen Vereinbarungen wie der Deal im Rahmen von DEAL (ausführlich dazu in 14.8). Trotzdem, auch wenn einiges darauf hindeutet, dass die Verlagskonsortien sich bislang als Gewinner des deal sehen können, ist DEAL doch ein wichtiger Schritt in die vollständige Transformation der Publikationsmärkte in Open-AccessMärkte mit dem neuen Paradigma der Nutzungsfreiheit – zumal dann, wenn die DEAL-Vereinbarungen nur als Übergangslösung eingeschätzt würden. Wenn es Dauerlösungen sein sollten, kann aber durchaus die Frage gestellt werden, ob diese Transformation nicht besser und auf Dauer auch kostengünstiger erreicht werden könnte, wenn die umfänglichen, den Verlagen bereitgestellten finanziellen Mittel für die Entwicklung von Open-Access-Produkten und Dienstleis tungen aus der Wissenschaft selbst eingesetzt würden. Wissenschaftler bzw. die ihnen zugeordneten Organisationen (wie z. B. Bibliotheken, Fachgesellschaften) sind heute technisch und organisatorisch in der Lage – und es gibt auch genug Bei spiele dafür, dass sie es tun –, das Publizieren selbst in die Hand zu nehmen. Diese Möglichkeiten gelten nicht nur für das Publizieren von Forschungsergebnissen, sondern sie gelten auch für auf Lehre und Lernen bezogene Produkte. OER-Produkte sind realistische Alternativen zu den klassischen kommerziellen Lehrbüchern auch dadurch, dass sie auf Kollaboration und dynamische hypertextähnliche Weiterentwicklung setzen.⁶⁴¹ Die Frage stellt sich tatsächlich, ob durch die öffentliche Finanzierung nicht das am Leben gehalten wird, was für sich nicht mehr marktfähig ist, sondern 641 (Kreutzer/Lahmann 2019) Rechtsfragen bei Open Science weisen darauf hin, dass auch mit den über freie Lizenzen (wie Creative Commons) zur Verfügung gestellten Open-Produkten durchaus Geld verdient werden kann. Allerdings darf dann kein Entgelt für die Nutzung selbst verlangt werden. Als Beispiel führen sie an, dass auch OER-Repositories kostenpflichtig angeboten werden können: „Zugriffsgebühren für einen Online-Dienst sind keine Lizenzgebühren, sie werden für die Nutzung des Dienstes gezahlt und nicht für die Nutzungsrechte an den Inhalten, die dort verfügbar sind. OER-Repositorien können daher kostenpflichtig angeboten werden, ohne dass dies den Prinzipien von OER oder gar den hierbei eingesetzten offenen Lizenzen widersprechen würde.“ Die Autoren stellen aber durchaus in Frage, ob so etwas „sinnvoll oder ratsam“ sei. Vor allem bei öffentlich finanzierten Organisationen wie Bibliotheken etc. sollte diese Frage wohl verneint werden. 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten \| 387 quasi auf öffentliche „Subventionen“ angewiesen ist. Vielleicht waren die wis senschaftlichen Informationsmärkte nie im klassischen Sinne Märkte – immer waren die öffentlich finanzierten Bibliotheken die gesicherten Hauptabnehmer der Produkte –, aber lange gab es dazu keine Alternativen. Diese wären heute gegeben. Wahrscheinlich wird es auch in Zukunft weiter kommerzielle Produkte und Dienstleistungen auf den Informationsmärkten geben. Diese werden sich aber abnehmend auf die Umwandlung der von den Urhebern verfassten Wissensobjekte in veröffentlichungs- und nutzungsfähige Informationsobjekte, also auf das blo ße Publizieren beziehen. Ob neue kommerzielle Produkte mit informationellen Mehrwertleistungen und Wissenschaft unterstützende Dienstleistungen, wie sie sich derzeit über Konzepte wie Research Intelligence Provider (REP) von Elsevier abzeichnen (vgl. 14.8), über das Urheberrecht geschützt werden sollen/müssen, ist eine eher negativ zu beantwortende Frage. Entsprechend der bisherigen Systematik könnte das möglicherweise über spezielle Leistungsschutzrechte geregelt werden. Konsequenzen für das Urheberrecht Wissenschaft entwickelt sich schneller als das Recht in der Lage ist, auf diese Entwicklungen zu reagieren. Fast systematisch zwingend ist Wissenschaft immer mit den aktuell geltenden rechtlichen Vorschriften für den Umgang mit Wissen und Information unzufrieden. Eine Weile kann Wissenschaft noch mit unzulänglichen rechtlichen Regelungen leben, sei es durch schlichtes Ignorieren der bestehenden Vorschriften – in der bislang berechtigten Erwartung, dass im Wissenschaftsbereich kein Rechtsinhaber Verstöße gegen bestehende Urheberrechtsregelungen vor Gericht bringen wird – oder sei es durch erfindungsreiche Ersatzlösungen, durch die, als Voraussetzung für Wissenschaftsfreiheit, die freie Einsicht und freie Nutzung des öffentlich gemachten Wissens gesichert wird. Soll das Urheberrecht dabei eine wichtige Rolle spielen, wäre dafür eine Bezeichnung wie „Nutzungsrechte und Nutzungsfreiheiten für Wissen und Information“ angemessener als „Urheberrecht“. Um kein Missverständnis aufkommen zu lassen – mit „Nutzungsrechten“ sind nicht in erster Linie die Nutzungsrechte von Verlagen gemeint, durch die sie die Wissensobjekte der Urheber zur Verwertung nutzen zu dürfen. „Nutzungsrechte“ haben in dem Vorschlag „Nutzungsrechte und Informationsfreiheiten für Wissen und Information“ eine fünffache Bedeutung: – Nutzungsrechte beziehen sich zunächst auf Autoren selbst. Diese nutzen ihr Wissen für Veröffentlichungen und haben das Recht zu entscheiden, ob, wann und wie sie ihr erarbeitetes Wissen öffentlich machen wollen. – Nutzungsrechte sind auch das Recht von Personen, das Wissen Anderer frei zu nutzen – vor allem um in der Wissenschaft selbst dadurch Autor werden zu können. 388 \| 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten – – – Nutzungsrechte sind auch das institutionelle Nutzungsrecht der die Wissen schaftler tragenden, finanzierenden und bedienenden Organisationen. Nutzungsrechte sind auch das Recht der Öffentlichkeit an der freien Nutzung der veröffentlichten und i. d. R. auch von ihr finanzierten Werke: Schließlich sind Nutzungsrechte auch auf Verleger bezogen als das zu er werbende Recht auf kommerzielle Verwertung der von Autoren produzierten Wissensobjekte. Die hier vorgeschlagene Bezeichnung „Nutzungsrechte und Nutzungsfreiheiten für Wissen und Information“ könnte in einer Präambel des Urheberrechts – § 1 ersetzend oder erweiternd – verwendet werden. In einem solchen Recht gelten weiter die durch das jetzige Urheberrecht uneingeschränkt geschützten und nicht durch Dritte in Frage zu stellenden Persönlichkeitsrechte.⁶⁴² Aber angesichts der in allen Disziplinen, aber besonders in den STM-Fächern erkennbaren Tendenz zum kollaborativen Arbeiten in der Wissenschaft ist es wenig sinnvoll, exklusiv an dem individuellen Autor festzuhalten. Niemand aber kann und niemand will Autoren/Autorengruppen das Recht nehmen zu entscheiden, ob (und damit wann) und in welcher Form sie ihre erstellten Werke publizieren wollen. Das einklagbare Recht auf Anerkennung und Nennung der Autor-/Urheberschaft und der Schutz vor Entstellungen werden gerade in elektronischen Umgebungen keineswegs unwichtiger. Anders sieht es bei den im Urheberrecht vorgesehenen Verwertungsrechten aus. Diese bislang exklusiv den Urhebern zustehenden Rechte werden durch das Open-Access-Paradigma bzw. durch die Leitidee der Nutzungsfreiheit überflüssig bzw. gegenstandslos. Mit einem Open Access gestellten Werk kann jeder weltweit genehmigungs- und vergütungsfrei das tun, was ursprünglich als Verwertungs rechte den Autoren exklusiv zustand, z. B. vervielfältigen, verbreiten, öffentlich zugänglich machen.⁶⁴³ Dies konsequent zu Ende gedacht, könnte das Wissen schaftsurheberrecht bzw. das Recht „Nutzungsrechte und Nutzungsfreiheiten für Wissen und Information“ im Open-Paradigma weitgehend auf die Verwertungsrech te und Teile des Urhebervertragsrechts verzichten. Dadurch würden tatsächlich all die bisherigen Schrankenregelungen mit ihren kleinteiligen Nutzungsvorschriften überflüssig. Entscheidend für die Nutzung sollte alleine der Zweck der in Bildung und Wissenschaft vorzunehmenden Handlungen sein. Dem kann ganz knapp, 642 Ob es möglich und sinnvoll ist, wie es in der Creative-Commons CC0-Lizenz vorgesehen ist, nämlich dass Autoren, die ihr Werk gänzlich in die Public domain stellen wollen, auf alle ihre Rechte, also auch auf ihre Persönlichkeitsrechte von sich verzichten können, kann hier nur als Frage gestellt werden. 643 Vgl. (Sandberger 2017) Die Zukunft wissenschaftlichen Publizierens. 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten \| 389 ohne Schranken, z. B. über die hier vorgeschlagene ABWK entsprochen werden. Die von Thomas Dreier 2019 gestellte Frage (Dreier 2019b), ob Schranken mit Blick auf Wissenschaft überhaupt nötig sind, wird hier, anders als von ihm, verneint. Vielleicht müssen Übergangslösungen erhalten bleiben, weil der Großteil der weltweit (analog oder elektronisch) verfügbaren Artikel und Bücher früher rein kommerziell produziert wurde und diese entsprechend über die bisherigen Urheberrechtsregelungen geschützt sind. Auch wenn deren Bedeutung für viele wissenschaftliche Fächer abnehmen wird, sind diese doch Teil des kulturellen Erbes, das zugriffs- und nutzungsfähig gehalten werden muss. Ob das weiter durch das Urheberrecht geregelt werden muss oder durch eine Erweiterung von Verträgen wie DEAL, welche die Finanzierung der Erstellung von Informationsobjekten an die Freistellung des „Lesens“ dieser Objekte bindet, muss sich zeigen. Zuweilen braucht es Revolutionen, um ganz neue politische Realitäten zu schaffen. Kopernikus hatte die Umlaufbahnen der Planeten um die Sonne in seinem Werk „De revolutionibus orbium coelestium“ (1543) beschrieben: Nicht mehr dreht sich die Sonne um die Erde, sondern die Erde um die Sonne. Das war dann tatsäch lich eine Revolution, die unser Weltbild änderte. Revolutionen im Urheberrecht sind eher unwahrscheinlich. Vielleicht ist die Metapher der Kopernikanische Wende passender. Darunter wird, mit Kant, jede grundsätzlich andere Sicht auf bislang als unumstößlich angesehene Beschreibungen von Objekten und Sachverhalten in der Welt verstanden – so heute auch für eine grundlegende Reform des Urheber rechts.⁶⁴⁴ Julia Reda, Grüne Politikerin im EU-Parlament bis 2018, übernahm die Metapher der „Kopernikanischen Wende“, als sich (2015) über ihren umfassenden Vorschlag⁶⁴⁵ zumindest noch die Chance zu eröffnen schien, ein neues zeitgemäßes Urheberrecht durch eine neue EU-Richtlinie zu entwickeln. Vorbilder für solche Wenden hat es aus der jüngeren Vergangenheit durchaus auch aus der Politik gegeben. 2010 versuchte es Till Steffen, Justizminister für Bündnis 90/Die Grünen im Senat der Freien und Hansestadt Hamburg in Hamburg ab 2008 (und dann wieder ab 2015). Er legte gemeinsam mit Netzpolitikern aus den Grünen ein Diskussionspapier „für ein nutzerorientiertes Urheberrecht“ vor.⁶⁴⁶ Darin werden auch Überlegungen in der Dissertation von Gerd Hansen aufgegrif 644 (Kuhlen 2015a) Kopernikanische Wende in der EU-Urheberrechtsdebatte? 645 (Reda 2019) EU-Urheberrechtsreform: Der Kampf war nicht umsonst. 646 (Steffen 2010) Diskussionspapier „Nutzerorientierte Ausrichtung des Urheberrechts“. Dieser Text konnte nicht mehr über das Web ausfindig gemacht werden. 2010 habe ich mich in net ethics ausführlich mit diesem Diskussionspapier auseinandergesetzt – https://bit.ly/2sBILgY. Die zentralen Formulierungen von Steffen, wie die hier im Text zitierten, sind dort umfänglich wieder gegeben; kritisch zu Steffens Vorschlägen: (Mönikes 2010) Anmerkungen zum Diskussionspapier „Nutzerorientierte Ausrichtung des Urheberrechts“ – https://bit.ly/2m36K4S. 390 \| 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten fen,⁶⁴⁷ in der dieser die These vertrat, dass das Urheberrecht nur dann akzeptabel bleiben kann, wenn es den Nutzerschutz stärker berücksichtigt. Hier einige Zitate aus diesem Steffen Papier: Das Urheberrecht, so wie es jetzt aussieht, passt nicht mehr zu den technischen Möglich keiten elektronischen Umgebungen und der darin entwickelten Nutzungsgewohnheiten. [. . . ] Das individualistische Begründungsmodell des Urheberrechts ist überholt. [. . . ] Im Internet entstehen kreative Nutzergewohnheiten und Formate, die es schwierig machen, auch bei unterstelltem guten Willen fremde Urheberrechte zu beachten. [. . . ] In der bisherigen Entwicklung des Urheberrechts sind diese mit der digitalen Entwicklung neu entstandenen Nutzungserwartungen weitgehend unberücksichtigt geblieben. [. . . ] Die Rechtspolitik ist aufgerufen, den „Schrankenbegünstigten“ eine Rechtsposition zu verschaffen, die der Grund rechtsrelevanz der Schranken Rechnung trägt und die dem Recht des Urhebers ein Recht auf Nutzung an die Seite stellt. Schließlich wird im Diskussionspapier von Steffen am Ende vorgeschlagen, im UrhG den Titel von § 1 „Allgemeines“ durch „Zweck des Gesetzes“ zu ersetzen. Damit käme ein bislang im deutschen Urheberrecht eher vernachlässigtes utilitaristisches und damit die institutionellen Rechte unterstützendes Moment ins Spiel. Weiter sollte der bisherigen Formulierung in § 11 UrhG, durch die bislang nur der Urheber (Satz 1) und dessen Recht auf Vergütung (Satz 2) geschützt wird, durch einen Satz ergänzt werden: „Zugleich trägt es den Bedürfnissen der Werknutzenden an der Teilnahme am kulturellen und geistigen Leben Rechnung.“ Dann sollte, so Steffen, dann gleich der gesamte Name des Gesetzes geändert werden in „Gesetz über Urheberrechte, verwandte Schutzrechte und Nutzungsfreiheiten (UrhG)“. Das erinnert schon sehr an den hier vorgeschlagenen Titel „Nutzungsrechte und Nutzungsfreiheiten für Wissen und Information. Mittelfristig, aber ganz sicher langfristig kann sich das Recht nicht gegen sich entwickelnde Leitideen mit neuen Werten und neuen Verhaltensformen für Wissen und Information behaupten. Das macht die anfänglich vertretene These der Priorität von Ethik, Informationsethik, gegenüber den anderen Regulierungsinstanzen wie Technologien, Markt aber auch Recht aus. Die Priorität legitimiert sich dadurch, dass das Verhalten der Akteure für den Umgang mit Wissen und Information sich an normativ begründete Leitideen orientiert. Ein anderes Wort für „Verhalten“ ist in der Sprache der Commons das Commoning. Commoning ist die Verständigung der betroffenen Akteure (hier in Bildung und Wissenschaft) zum einen auf die den Leitideen zugrundeliegenden Werte wie Informationsautonomie, Nutzungsfreiheit, Gemeinwohl, Gerechtigkeit, Nachhaltigkeit und zum anderen auf Regeln, auch Sanktionen gegen Verstöße und Verhaltensformen wie Teilen und Kollaboration. 647 (Hansen 2009) Warum Urheberrecht? 15 Fazit: Wissenschaftsurheberrecht – Nutzungsrechte und -freiheiten \| 391 Die Ausführungsbestimmungen der Open-Access-Erklärungen sind Hinweise auf solches Commoning. Die Prioritätsthese zugunsten der Ethik hat nichts mit moralisierender Politik zu tun. Priorität, bedeutet nicht, dass, entsprechend Platons erstem Vorschlag für eine gerechte Politik, die (informationsethischen) Philosophen „Könige“ werden sollen, aber vielleicht doch im Sinne seines zweiten Vorschlags, dass die „Könige“ sich informationsethisches Denken zu eigen machen bzw. die sich entwickelnde Leitidee des freien Umgangs mit Wissen und Information als handlungsrelevant annehmen und dies in die Sprache und Formalitäten des Rechts umsetzen. Kurzfristige „Siege“ der Informationsethik sind nicht zu erwarten. Aber ein Recht für Nutzungsrechte und Nutzungsfreiheiten sollte sich realisieren lassen. Eine Utopie – so wird es Alphonse de Lamartine zugeschrieben – ist eine Idee, deren Zeit noch nicht gekommen ist. Aber Nutzungsfreiheit in der Wissenschaft ist keine Utopie, sondern ist in der Zeit angekommen, und nichts – so wird es Victor Hugo zugeschrieben – ist so stark wie eine Idee, deren Zeit gekommen ist.
https://openalex.org/W609182043	https://irpa.is/index.php/irpa/article/download/c.2010.6.2.5/pdf_189	Icelandic	null	Elías Snæland Jónsson: Möðruvallahreyfingin - baráttusaga	Stjórnmál og stjórnsýsla	2,010	cc-by	786	MIKILVÆGUR ÞÁTTUR Í ÍSLENSKRI STJÓRMÁLASÖGU Bókin um Möðruvallahreyfinguna er í frásögn eins forystumans hreyfingarinnar, sem fylgdist með allt frá byrjun og stóð í miðri atburðarásinni allan tímann á meðan hreyfingin starfaði. Í bókinni er skýrður aðdragandi hreyfingarinnar, farið yfir tímabilið sem hún starfaði og loks endalok hennar. Höfundurinn hefur haldið saman miklum fjölda gagna, úrklippum og minnisblöðum frá þeim tíma þegar atburðirnir áttu sér stað. Að þessu öllu er mikill fengur sem gerir bókina að skyldulesningu fyrir hvern þann sem vill kynna sér íslensk stjórnmál á seinni hluta 20. aldar. Um langa hríð báru íslenskir jafnaðarmenn, hvar í flokki sem þeir stóðu, þá von í brjósti að á Íslandi risi upp flokkur, stærstur stjórmálaflokka, sambærilegur við þá jafnaðarmannaflokka sem starfað hafa svo áratugum skiptir annars staðar á Norðurlöndum. Í stuttu máli var það tilgangur Möðruvallahreyfingarinnar og nokkurra annarra hreyfinga síðar. Sú vegferð hefur verið ærið löng og margir orðið vegmóðir á leiðinni. Í hugum margra var með stofnun Samfylkingarinnar loksins það skref stigið. Um langa hríð báru íslenskir jafnaðarmenn, hvar í flokki sem þeir stóðu, þá von í brjósti að á Íslandi risi upp flokkur, stærstur stjórmálaflokka, sambærilegur við þá jafnaðarmannaflokka sem starfað hafa svo áratugum skiptir annars staðar á Norðurlöndum. Í stuttu máli var það tilgangur áratugum skiptir annars staðar á Norðurlöndum. Í stuttu máli var það tilgangur Möðruvallahreyfingarinnar og nokkurra annarra hreyfinga síðar. Sú vegferð hefur verið ærið löng og margir orðið vegmóðir á leiðinni. Í hugum margra var með stofnun Samfylkingarinnar loksins það skref stigið. Eitt þeirra mála sem kom alla tíð í veg fyrir að flokkarnir gætu starfað saman var spurningin um her í landi og aðildina að Nató. Eru þessu gerð góð skil í bókinni og er ekki ósennlegt að dómur sögunnar um það mál eigi eftir að verða sá að þar hafi menn látið eitt afmarkað málefni ráða för og þannig hindrað þær þjóðfélagsumbætur sem flokkarnir hefðu getað náð saman um. Þjóðfélagsumbætur sem hinir sömu flokkar, a.m.k. í orði kveðnu, töldu mikilvægar fyrir íslenskan almenning. Er þetta enn ein áþreifanleg sönnun um hversu grátt kalda stríðið lék íslensk stjórnmál. Í bókinni er varið miklu rými í frásagnir af fundum, stórum og smáum, mikilvægum og öðrum minna mikilvægum. Erfitt er fyrir þá sem ekki þekkja mikið til atburðarásarinnar að átta sig á mikilvægi einstakra funda en þetta er í samræmi við upplegg bókarinnar sem byggist mikið á minnisblöðum höfundarins. Möðruvallahreyfingin - baráttusaga Bókarhöfundur: Elías Snæland Jónsson Bókarheiti: Möðruvallahreyfingin - baráttusaga Gagnrýnandi: Bolli Héðinsson, hagfræðingur Útgáfa: Hergill, Kópavogi 2010, 464 bls. Möðruvallahreyfingin - baráttusaga Bókarhöfundur: Elías Snæland Jónsson Bókarheiti: Möðruvallahreyfingin - baráttusaga Gagnrýnandi: Bolli Héðinsson, hagfræðingur Útgáfa: Hergill, Kópavogi 2010, 464 bls. Möðruvallahreyfingin - baráttusaga Bókarhöfundur: Elías Snæland Jónsson Bókarheiti: Möðruvallahreyfingin - baráttusaga Gagnrýnandi: Bolli Héðinsson, hagfræðingur Útgáfa: Hergill, Kópavogi 2010, 464 bls. Í umsögn gagnrýnanda kemur meðal annars eftirfarandi fram: „Bók Elíasar Snæland er afar fróðleg og upplýsandi frásögn um sögulegt tímabil í þjóðarsögunni og um hreyfingu sem á sér ekki hliðstæðu. Greint er frá undirmálum og ótrúlegum vinnubrögðum innan Framsóknarflokksins sem gera hvern mann orðlausan. Bókin er mikill fengur fyrir þá sem rannsaka íslenska stjórnmálasögu á síðustu öld. Þar er nákvæmni í frásögnum af atburðum og tímasetningu þeirra sem þakka má fádæma elju höfundar við að halda saman gögnum og skrá hjá sér atburði líðandi stundar.“ það verður sagan einn dómari. MIKILVÆGUR ÞÁTTUR Í ÍSLENSKRI STJÓRMÁLASÖGU Flestir tengjast fundirnir Framsóknarflokknum og þá helst ungliðahreyfingu flokksins, eðli málsins samkvæmt. Að þessu öllu er mikill fengur fyrir sagnfræðinga og stjórnmálafræðinga framtíðarinnar. Ekki er víst að svo ítarlegt efni höfði endilega til almennra lesenda, annarra en þeirra sem voru þátttakendur eða áhorfendur að atburðunum þegar þeir áttu sér stað, en ber aftur á móti vott um vönduð vinnubrögð af hálfu höfundarins. Stjórnmál eru ekki bara hugsjónir og fræði eins og ráða má af lestri bókarinnar heldur snúast þau ekki síst um einstaklingana, persónurnar sem fyrir hugmyndum fara að ekki sé talað um persónurnar sem gegn hugmyndunum standa. Slíkum málum er ekki auðvelt að gera viðunandi skil í bók af því tagi sem bókin um Möðruvallarhreyfingin er. Þetta skiptir þó ekki minna máli þegar ris og fall hreyfingarinnar er skoðað heldur en málefnin sem lagt var upp með og tekist var á um. Athyglisvert er að lesa um starfshætti Framsóknarflokksins í Reykjavík á þessum árum, virðingarleysi fyrir lýðræðislegum starfsháttum, umgengni um félagaskrár og annað sem lýtur að vinnubrögðum félagasamtaka sem vilja vera vönd að virðingu sinni. Bókin um Möðruvallahreyfinguna verður þegar fram líða stundir sennilega grundvallarrit þeirra sem munu reyna að rekja sig í gegnum þá flokka, flokksbrot og samtök sem síðar leiddu til stofnunar Samfylkingarinnar. Hvort með Samfylkingunni hafi raungerst draumur íslenskra jafnaðarmanna um norrænan jafnaðarmannaflokk og Samfylkingin rísi undir þeim væntingum er of snemmt að segja. Um það verður sagan einn dómari.
https://openalex.org/W1994308248	https://research.bond.edu.au/files/31147325/Patients_expectations_of_acute_low_back_pain_management.pdf	English	null	Patients’ expectations of acute low back pain management: implications for evidence uptake	BMC family practice	2,013	cc-by	6,468	General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. * Correspondence: thoffman@bond.edu.au 1Associate Professor of Clinical Epidemiology, Centre for Research in Evidence-Based Practice, Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland 4229, Australia 2Division of Occupational Therapy, School of Health and Rehabilitation Sciences, The University of Queensland, Brisbane, Queensland, Australia Full list of author information is available at the end of the article Abstract Background: In many countries, general practitioner (GP) care of acute low back pain often does not adhere to evidence-based clinical guidelines. There has been little exploration of this deviation from evidence-based care from the patients’ perspective, particularly in relation to patients’ care expectations. The aim of this study was to explore the care expectations in patients who present to their GP with acute low back pain, influences on expectation development, and congruence of these expectations with clinical guideline recommendations. Methods: Qualitative study in an inner urban general practice in Brisbane, Australia. Semi-structured interviews were conducted with 11 patients who presented to their GP with acute low back pain. Results: Patients had a biomechanical understanding of back pain, how it should be tested and treated, and a poor understanding of its natural history. Most expected x-rays, believing they were necessary to identify the “cause of the pain” without belief of any downsides to x-rays. Patients’ expectations were primarily influenced by the experiences of family and friends, their own previous experiences of low back pain care, and comments from other health professionals they were consulting. The GP-patient relationship was important in influencing patient satisfaction of care provided. Most patient expectations, and some of the care that they reported receiving, were incongruent with guideline recommendations. Conclusions: A biomechanical approach to management rather than an awareness of empirical evidence was evident in patients’ expectations. Communication and education by the GP that includes specifically enquiring about patients’ expectations, provides an opportunity to correct misperceptions, explain acute low back pain natural history, and the rationale for test and treatment recommendations. rds: Low back pain, Doctor-patient communication, Patient expectations, Clinical practice guideline care Keywords: Low back pain, Doctor-patient communication, Patient expectations, Clinical practice guidelines, Primary health care © 2013 Hoffmann 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. Patients’ expectations of acute low back pain management: implications for evidence uptake Tammy C Hoffmann1,2, Chris B Del Mar3, Jenny Strong2 and Juliana Mai2 Tammy C Hoffmann1,2, Chris B Del Mar3, Jenny Strong2 and Juliana Mai2 Bond University Research Repository Patients' expectations of acute low back pain management Implications for evidence uptake Hoffmann, Tammy C.; Del Mar, Chris B.; Strong, Jenny; Mai, Juliana Published in: BMC Family Practice DOI: 10.1186/1471-2296-14-7 Licence: CC BY Link to output in Bond University research repository. Recommended citation(APA): Hoffmann, T. C., Del Mar, C. B., Strong, J., & Mai, J. (2013). Patients' expectations of acute low back pain management: Implications for evidence uptake. BMC Family Practice, 14, Article 7. https://doi.org/10.1186/1471- 2296-14-7 Bond University Research Repository Patients' expectations of acute low back pain management Implications for evidence uptake Link to output in Bond University research repository. Recommended citation(APA): Hoffmann, T. C., Del Mar, C. B., Strong, J., & Mai, J. (2013). Patients' expectations of acute low back pain management: Implications for evidence uptake. BMC Family Practice, 14, Article 7. https://doi.org/10.1186/1471- 2296-14-7 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. For more information, or if you believe that this document breaches copyright, please contact the Bond University rese coordinator. Download date: 24 Oct 2024 Download date: 24 Oct 2024 Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 © 2013 Hoffmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creativ 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. Data collection f h One of the authors (JM) completed a semi-structured telephone interview with each patient. Interviews were audio-recorded, with an average duration of interviews of 20 minutes. An interview guide was used, based on research on barriers to evidence-based practice from the patient perspective, including barriers to guideline imple- mentation. The guide focused on topics such as patients’ expectations of testing and treatment procedures for low Participants Patients were eligible for inclusion if they: were 18 years or older; presented to their GP with an episode of acute low back pain (defined as an episode of pain present for less than three months [2]); and had adequate English, cognition and communication "as determined by the treating GP" to provide written informed consent and participate in a telephone interview. Background examinations unless ‘red flags’ are identified [2-4]. Although patients’ health will benefit from their clini- cians’ adherence to these guidelines, in many coun- tries, usual GP care of acute low back pain does not typically adhere to guidelines [5]. Low back pain consultations are among one of the most common reasons for general practice appointments in many developed countries [1,2]. Key recommendations in evidence-based clinical practice guidelines include patients staying active and avoiding bed rest, trying para- cetamol as the first choice of analgesic, and general practi- tioners (GPs) providing education, reassurance of likely positive prognosis, and not routinely ordering radiological Various reasons for non-adherence to guidelines have been reported. [6-8] In addition to clinician-related bar- riers such as knowledge and beliefs, patients are them- selves a powerful influence on GPs’ behaviour [8,9]. GPs report accommodating patient demands, such as for x-rays [9] and that the care they provide is sometimes influenced by very indirect information, such as their perceptions of patients’ expectations [8]. The problem of GPs’ deviation from evidence-based care from the patient perspective in relation to patients’ Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 Page 2 of 6 actual expectations has been insufficiently explored. We aimed to explore the expectations of the management of patients presenting to primary care with acute low back pain, influences on the development of these expectations, and their congruence with guideline recommendations. back pain and how these expectations were conceived. The interview guide (available from the corresponding au- thor on request) was used flexibly so that the interviewer could explore patients’ comments further if needed. Data saturation was reached when no new ideas or constructs arose in two consecutive interviews [10,13]. Procedure A convenience sample of patients was recruited from an inner urban general practice in Brisbane, Australia, be- tween April and July 2011. The medical centre is located in a suburb with a bimodal socio-economic profile, with large proportions of both high income and low income individuals [12]. Patients can typically see their usual GP if appointments are made at least one day in advance. All 10 GPs working there were invited to recruit all eli- gible patients during the study timeframe; participants were recruited by four GPs. In the consultation, the GP explained the study and obtained patients’ consent to provide their contact details to the research team. For those who agreed, further written information was pro- vided by the medical centre staff, written informed con- sent obtained, and their telephone number provided to the research team. A convenient time for a telephone interview was scheduled. Results Data saturation was reached when no new themes were identified in two consecutive interviews at interview 11, completing the recruitment phase. Of three additional patients invited to participate in the study, two declined (one too busy to participate and one not interested in being interviewed), and one was excluded because the primary reason for her consultation was not acute low back pain. Ten women and one man, with an average age of 52 years (SD =57, range 22 to 72) were inter- viewed. Three of the participants had work-related acute low back pain. Table 1 lists participants’ demographic characteristics. The themes which emerged could be framed around three areas: patient expectations about the testing and treatment arising during their consultation; influences on patients’ expectations; and the importance of the re- lationship between the patient and the GP. Table 2 con- tains the themes and illustrative quotations. Data analysis E h i t i Each interview was transcribed verbatim, using pseudo- nyms, prior to inductive thematic content analysis [14]. Two of the authors (JS, JM) independently read and re- read the transcripts and identified themes using colour coding, Post-it notes and annotation. In a face to face meeting, they discussed the themes until consensus was reached. A third author (TH) then coded the transcripts using the extracted themes, and was able to allocate text chunks to the aforementioned themes. The themes and illustrative quotes were then agreed to by all authors. This investigator triangulation added to the rigor of the study. Three of the authors are experienced evidence- based practice academics, and thus had particular views on the importance of using clinical guidelines to guide clinical practice. The fourth author (JM) was a research student whose opinions were less well shaped. She inter- viewed all patients, to ensure both consistency and bracketing of any bias. Design We chose a qualitative design using a descriptive phe- nomenological approach [10,11] and purposive sampling and interviews to enable a clearer understanding of patients’ perspectives in the consultation experience. Methods This study received ethical approval from the Behav- ioural and Social Sciences Ethical Review Committee of The University of Queensland. Patient expectations Test expectations Most patients expected their GP to refer them for an x- ray, particularly patients who felt that their pain was se- vere (Table 2, quote A). Many patients believed that an x-ray would enable the cause of the pain to be deter- mined (quote B), and that the problem would be able to Page 3 of 6 Page 3 of 6 Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 Table 1 Participant demographics Study ID Age (years) Gender First episode of back pain Saw usual GP Occupation Highest level of education completed 1 22 Female Yes Yes Student and receptionist TAFEa 2 69 Female No Yes Retired High school 3 60 Female No No Office worker High school 4 48 Female No Yes Office worker University 5 69 Female No Yes Retired registered nurse TAFE 6 33 Female Yes No Restaurant manager; group fitness instructor TAFE 7 46 Female Yes No University lecturer University 8 72 Male No Yes Retired High school 9 37 Female No Yes Fitness instructor High school 10 51 Female No Yes Office management University 11 67 Female Yes No Retired High school aA Technical and Further Education (TAFE) Institute -offers predominately vocational tertiary education courses. Table 1 Participant demographics be seen (quote C). Patients felt that x-rays played an im- portant role in providing reassurance as well as confirm- ation of their GPs’ diagnosis (quotes D-F). Many patients believed that by identifying the cause of their pain through x-ray, the GP would then know how to ‘fix it’ (quote G). Very few patients expected a physical examination to be performed and a few patients had no expectations about what the tests their GP would per- form/request. be seen (quote C). Patients felt that x-rays played an im- portant role in providing reassurance as well as confirm- ation of their GPs’ diagnosis (quotes D-F). Many patients believed that by identifying the cause of their pain through x-ray, the GP would then know how to ‘fix it’ (quote G). Very few patients expected a physical examination to be performed and a few patients had no expectations about what the tests their GP would per- form/request. few patients felt that no treatment may be suitable for some people, but not for them (quote R). Patients were advised to use, or prescribed, anti-inflammatory drugs and analgesics. Influences on patients’ expectations Patients’ expectations about diagnostic investigations had been influenced by family, friends and/or other health professionals (particularly osteopaths and chiro- practors) (quotes S, T). Prior experiences of care for low back pain were an influence for those who had experi- enced it before (quote U). However many patients could identify no specific influences on their expectations, in- stead reiterating their biomechanical model. Those who expected an x-ray assumed it was part of standard pro- cedure (quote V) and those experiencing no improve- ment expected a change in management (quote W). Of the patients who had articulated particular treatment expectations, these were predominantly influenced by prior experiences of family and friends (quote X). When patients held a biomechanical model, the assumed cause of their low back pain determined their treatment expec- tations (quotes Y, Z). Most patients, including both those that did and did not have an expectation of an x-ray, believed that there were no downsides associated with x-rays (quote H). A few expressed minor concerns about radiation and time inconveniences. However, these patients reported that the usefulness of an x-ray outweighed these potential negatives (quotes I, J), assuming that their GP had already balanced these benefits and harms. Treatment expectations h There was variation among patients regarding the treat- ments they were expecting from their GP. Many did not know what to expect (quote K); some expected a referral to another health professional (e.g. physiotherapist) (quote L); others expected analgesics (quotes M, N). When asked about the option of ‘no treatment’ for low back pain with the exception of analgesics, most believed in a biomechanical approach of needing to find the problem and fix it in a timely manner (quotes O-Q). A Patient expectations Test expectations Some patients reported being told to avoid vigorous activities; others were told to stay active, including specific muscle strengthening exercises. Other treatments that patients reported receiving from their GP were referrals to allied health professionals, and recom- mendations for orthotics. There was no expressed dissatis- faction with GP treatment. Some patients reported receiving the testing proce- dures they had anticipated (namely, an x-ray referral), while some stated their GP provided procedures they had not expected to receive (including a physical exam- ination). Patients reported satisfaction with their GP’s care, apart from one who wanted to have an MRI and was given an x-ray referral instead. Importance of the GP-patient relationship (P5) BB I went and saw my doctor again because I was happy with past results and she knew my history (P8) Because it . . . was pretty bad, so I figured. . . need [an x-ray] to see what it was.....They [x-rays] can isolate the problem (P1) Because it . . . was pretty bad, so I figured. . . need [an x-ray] to see what it was.....They [x-rays] can isolate the problem (P1) I thought it [x-ray] might show the cause in my spine. I think it helps (P4) I guess [an x-ray] was to establish whether, from my perspective, whether it was just a pulled muscle or whether it was called herniated disc or whatever. (P7) I guess [an x-ray] was to establish whether, from my perspective, whether it was just a pulled muscle or whether it was called herniated disc or whatever. (P7) I knew there was something wrong and I thought well, I was just guessing and without actually seeing (P8) I knew there was something wrong and I thought well, I was just guessing and without actually seeing (P8) . . .gave me a physical examination and she said well clearly there was a problem and maybe we should confirm her findings with an x-ray. . . (P5) . . .gave me a physical examination and she said well clearly there was a problem and maybe we should confirm her findings with an x-ray. . . (P5) . . .the whole thing sort of confirmed what we knew, that I had wear and tear (P11) . . .the whole thing sort of confirmed what we knew, that I had wear and tear (P11) No, just positive reassurance I think. (P10) I think they’re like the last resort because it’s like radiation on your body, like if it’s not necessary and plus it’s the hours of your whole day that you’ve got to take out to get it done and wait. (P1) I think they’re like the last resort because it’s like radiation on your body, like if it’s not necessary and plus it’s the hours of your whole day that you’ve got to take out to get it done and wait. (P1) The only downside maybe . . . was the radiation, but that was the only negative. Importance of the GP-patient relationship Importance of the GP-patient relationship When asked how their GP knew what management to adopt, most patients identified their experience (quote AA). Some thought care was guided by the GP’s know- ledge of the patient and his/her medical history (quote Page 4 of 6 Page 4 of 6 Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 Table 2 Themes and illustrative quotes Quote letter (used in text) Illustrative quote (participant ID in brackets) Patients’ expectations of tests and beliefs about x-rays A Because it . . . was pretty bad, so I figured. . . need [an x-ray] to see what it was.....They [x-rays] can isolate the problem (P1) B I thought it [x-ray] might show the cause in my spine. I think it helps (P4) C I guess [an x-ray] was to establish whether, from my perspective, whether it was just a pulled muscle or whether it was called herniated disc or whatever. (P7) D I knew there was something wrong and I thought well, I was just guessing and without actually seeing (P8) E . . .gave me a physical examination and she said well clearly there was a problem and maybe we should confirm her findings with an x-ray. . . (P5) F . . .the whole thing sort of confirmed what we knew, that I had wear and tear (P11) G . . . to find out what it is and try to fix it (P7) H No, just positive reassurance I think. (P10) I I think they’re like the last resort because it’s like radiation on your body, like if it’s not necessary and plus it’s the hours of your whole day that you’ve got to take out to get it done and wait. (P1) J The only downside maybe . . . was the radiation, but that was the only negative. I think the less radiation yo can be exposed to the better, but that having being said the results justified that risk. I would rely on my doctor’s advice. (P8) Patients’ treatment expectations K I had no expectations. (P5 and P8) L I thought she’d say go see a physiotherapist, and she was going to give me a letter yesterday but she said she’ll get the results of the x-ray first. Importance of the GP-patient relationship (P2) M I didn’t think that a doctor could do anything apart from give me pain relief as in medication (P3) N I had no idea, so I guess, a painkiller obviously. The other thing, um the anti-inflammatory, I guess I didn’t expect an anti-inflammatory but it worked really well (P7) O I think [the GP] should always try to find out what the cause is and take steps to eliminate that if possible, even if it’s just a simple exercise or even if it’s just lying down, I think it’s important that they try and find a way of helping the situation. (P4) P Because if you don’t get something treated it can impact on other parts of your body. (P9) Q I kind of felt either I’d left it too long and it’d got to the stage where it just needed something. (P10) R It depends, I suppose. In my case it was quite severe and I had, there was no option but to get something done. (P8) Influences on patients’ expectations S I’d heard about friends having MRIs and the osteopath suggested that I have an MRI to look at the soft tissue, and [the doctor] suggested the x-rays and I did have the x-rays and the verdict is that there is wear and tear (P11) T [The chiropractor] said to me ‘Before I touch you I want you to get an x-ray done.’ (P10) U Because that’s what’s happened before (P8) V Probably because I’d never had one [x-ray]done, and that’s the usual, the doctor probably wants to see what’s happening. (P4) W Physio wasn’t working. . .it wasn’t getting better. . . and I knew that I needed to sort of have something furth checked out (P11) X Well in most cases that I’m aware of your doctors just tend to put a patient on anti-inflammatories and away you go. (P9) Y I assumed I probably would get some sort of anti-inflammatories. . . because it was a muscle. . . (P5) Z Anti-inflammatories. . . aren’t they like strong antibiotics that will deal with the infection? (P11) Importance of the GP-patient relationship AA I suppose it’s like any clinical part of what a doctor does, it’s the experience, the amount of patients they see, that they find the tried and true methods and that’s what happened. I had no expectations. (P5 and P8) I thought she’d say go see a physiotherapist, and she was going to give me a letter yesterday but she said she’ll get the results of the x-ray first. (P2) I didn’t think that a doctor could do anything apart from give me pain relief as in medication (P3) I had no idea, so I guess, a painkiller obviously. The other thing, um the anti-inflammatory, I guess I didn’t expect an anti-inflammatory but it worked really well (P7) I think [the GP] should always try to find out what the cause is and take steps to eliminate that if possible, even if it’s just a simple exercise or even if it’s just lying down, I think it’s important that they try and find a way of helping the situation. (P4) Because if you don’t get something treated it can impact on other parts of your body. (P9) kind of felt either I’d left it too long and it’d got to the stage where it just needed something. (P10) It depends, I suppose. In my case it was quite severe and I had, there was no option but to get something done. (P8) It depends, I suppose. In my case it was quite severe and I had, there was no option but to get something done. (P8) I’d heard about friends having MRIs and the osteopath suggested that I have an MRI to look at the soft tissue, and [the doctor] suggested the x-rays and I did have the x-rays and the verdict is that there is wear and tear (P11) [The chiropractor] said to me ‘Before I touch you I want you to get an x-ray done.’ (P10) Probably because I’d never had one [x-ray]done, and that’s the usual, the doctor probably wants to see what’s happening. (P4) Physio wasn’t working. . .it wasn’t getting better. . . and I knew that I needed to sort of have something further checked out (P11) Well in most cases that I’m aware of your doctors just tend to put a patient on anti-inflammatories and away you go. (P9) I assumed I probably would get some sort of anti-inflammatories. . . because it was a muscle. . . (P5) Anti-inflammatories. . . aren’t they like strong antibiotics that will deal with the infection? Importance of the GP-patient relationship I think the less radiation you can be exposed to the better, but that having being said the results justified that risk. I would rely on my doctor’s advice. (P8) I had no expectations. (P5 and P8) (P11) I suppose it’s like any clinical part of what a doctor does, it’s the experience, the amount of patients they see, that they find the tried and true methods and that’s what happened. (P5) I went and saw my doctor again because I was happy with past results and she knew my history (P8) I’m sure he’s got guides as to what to use when, so, and experience. (P7) Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 Page 5 of 6 Table 2 Themes and illustrative quotes (Continued) DD Yeah, a lot depends on sort of your relationship with your GP. I think in this case the doctor’s advice is good (P10) EE I knew she would zero in on what the problem was. . . I was quite happy to leave it in her hands. I would rely on my doctor’s advice (P7) FF [My doctor] has been treating me for quite a long time, and she has been extremely proficient at diagnosing things I have, and. . . she knew what she was doing. She had come across this before. . . and immediately zeroed in on the problem. Her experience was a factor, and my experience with her as a doctor immediately made me go straight to her and say look, I need help here. (P8) Table 2 Themes and illustrative quotes (Continued) Table 2 Themes and illustrative quotes (Continued) Yeah, a lot depends on sort of your relationship with your GP. I think in this case the doctor’s advice is good (P10) Yeah, a lot depends on sort of your relationship with your GP. I think in this case the doctor’s advice is good (P10) I knew she would zero in on what the problem was. . . I was quite happy to leave it in her hands. I would rely on my doctor’s advice (P7) I knew she would zero in on what the problem was. . . I was quite happy to leave it in her hands. I would rely on my doctor’s advice (P7) [My doctor] has been treating me for quite a long time, and she has been extremely proficient at diagnosing things I have, and. . . she knew what she was doing. She had come across this before. . . and immediately zeroed in on the problem. Summary of main findings Key findings were that patient expectations included: al- most universally that x-rays were standard; a wide var- iety of treatments; a biomechanical understanding was necessary to dictate management; and a naivety about contemporary empirical clinical evidence on best prac- tice for acute low back pain. Patients in this study expressed a number of mispercep- tions including that severity of the pain is an important in- dicator and that the cause of the pain needs to be identified and is necessary for guiding treatment. Any (even incidental) findings on x-ray were thought to indi- cate the cause of the pain. Patients were referred for x-ray, despite guideline recommendations that this is unneces- sary for most in primary care. In Australia, 25% of acute low back pain primary care patients are referred for im- aging [5]. Patients were not aware of the limited diagnostic value of x-rays for acute low back pain. Nor did most con- sider x-rays to have drawbacks, or, at least assuming that if they did, the benefits outweighed the risks. Patients assumed that clinicians will only make recommendations such as ordering x-rays if they believed them necessary, safe and effective. Strengths and limitations of the study Our study approached a well-known problem from a per- spective that has been rarely studied. It provides insights into patients’ management expectations when consulting a GP for acute low back pain. These insights can be used to guide the interaction that GPs have with patients with acute low back pain. We did not recruit a random sample of par- ticipants as is appropriate for qualitative research, but our sample was a convenience sample, relatively small, with an unequal gender distribution, and participants from only one medical centre. However, our study is strengthened by including patients from a range of ages, occupations, and education levels, and consulting either their usual GP or another. Self-reported patient recall imposes some concerns about the accuracy of patients’ recall of events from the consultation and patients’ expectations may have been influenced as a result of the consultation. I had no expectations. (P5 and P8) Her experience was a factor, and my experience with her as a doctor immediately made me go straight to her and say look, I need help here. (P8) BB). One patient mentioned a guide, although it is not clear whether this referred to a research-based guide (quote CC). Patients’ level of trust and confidence in their GP, particularly in patients who had seen their usual GP, influenced their overall satisfaction about the consultation, and confidence in their GP’s management (quotes DD-EE). Those reporting discrepancies between their expectations of care and that actually provided by the GP, described satisfaction with the consultation if they had a strong, and previously successful, relationship with the GP (quotes FF). rather than paracetamol when this is the recommended first choice of analgesic [2], perhaps helping to explain why Aus- tralian GPs recommend non-steroidal anti-inflammatory drugs (NSAIDs) most often for acute low back pain [5]. Patients commonly saw “painkillers” and anti-inflammatory drugs as having distinct actions, without appreciating the overlap. Author details 1 1Associate Professor of Clinical Epidemiology, Centre for Research in Evidence-Based Practice, Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland 4229, Australia. 2Division of Occupational Therapy, School of Health and Rehabilitation Sciences, The University of Queensland, Brisbane, Queensland, Australia. 3Centre for Research in Evidence-Based Practice, Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland, Australia. Competing interests Competing interests The authors declared that they have no competing interest. , 15. Tarrant C, Stokes T, Baker R: Factors associated with patients’ trust in their general practitioner: a cross-sectional survey. Br J Gen Pract 2003, 53:798–800. 16. Epstein R, Alper B, Quill T: Communicating evidence for participatory decision making. JAMA 2004, 291:2359–2366. 15. Tarrant C, Stokes T, Baker R: Factors associated with patients’ trust in their general practitioner: a cross-sectional survey. Br J Gen Pract 2003, 53:798–800. 16. Epstein R, Alper B, Quill T: Communicating evidence for participatory decision making. JAMA 2004, 291:2359–2366. 16. Epstein R, Alper B, Quill T: Communicating evidence for participatory decision making. JAMA 2004, 291:2359–2366. Implications for future research Future enhancements to this area of research could include interviewing patients prior to the consultation, and record- ing the consultation so that the actual care received is cap- tured to further explore the doctor-patient encounter. 5. Williams CM, Maher CG, Hancock MJ, McAuley JH, McLachlan AJ, Britt H, Fahridin S, Harrison C, Latimer J: Low back pain and best practice care: a survey of general practice physicians. Arch Intern Med 2010, 170:271–277. 6. Dahan R, Borkan J, Brown J-B, Reis S, Hermoni D, Harris S: The challenge of using the low back pain guidelines: a qualitative research. J Eval Clin Pract 2007, 13:616–620. 6. Dahan R, Borkan J, Brown J-B, Reis S, Hermoni D, Harris S: The challenge of using the low back pain guidelines: a qualitative research. J Eval Clin Pract 2007, 13:616–620. Authors’ contributions TH, JS, and CDM conceptualised the study. JM was responsible for data collection. JM, JS, and TH were responsible for most of the data analysis, with some input from CDM. All authors read and approved the final manuscript. 17. Buchbinder R, Jolley D, Wyatt M: Population based intervention to change back pain beliefs and disability: three part evaluation. BMJ 2001, 322:1516–1520. 18. Buchbinder R, Jolley D: Population based intervention to change back pain beliefs: three year follow up population survey. BMJ 2004, 328:321. References 1. Britt H, Miller GC, Charles J, Henderson J, Bayram C, Harrison C, Valenti L, Fahridin S, Pan Y, O’Halloran J: General practice activity in Australia 2007–08. Canberra: Australian Institute of Health and Welfare2008; 2008. Contract No.: Cat. no. GEP 22. 2. National Health and Medical Research Council: Evidence-based management of acute musculoskeletal pain. Canberra, Australia: Australian Academic Press Pty. Ltd; 2003. 2. National Health and Medical Research Council: Evidence-based management of acute musculoskeletal pain. Canberra, Australia: Australian Academic Press Pty. Ltd; 2003. 3. Waddell G, Feder G, McIntosh A, Lewis M, Hutchinson A: Low Back Pain Evidence Review. London, England: Royal College of General Practitioners; 1996. 4. van Tulder M, Becker A, Bekkering T, Breen A, del Real MTG, Hutchinson A, Koes B, Laerum E, Malmivaara A, COST B13 Working Group on Guidelines for the Management of Acute Low Back Pain in Primary Care: European guidelines for the management of acute nonspecific low back pain in primary care. Eur Spine J 2006, 15:S169–S191. Comparison with existing literature f d h h Patients were satisfied with their GP consultation, even when their management expectations were not met - pos- sibly because of a strong doctor-patient relationship. This is fundamentally important in promoting patients’ trust [15], perhaps even more so when expectations are not met. That GPs are keen not to fail their patient’s expectations has been previously identified as a major barrier to implement- ing low back pain guidelines [8]. In facilitating patient-centred care and shared decision making, a fundamental step is for clinicians to elicit patients’ expectations; discuss, and correct any of their mis- perceptions, expectations or fears [16]. However patients in this study appeared to have been provided with little educa- tion about ordering, or not ordering, x-rays, their down- sides, and the natural history of acute low back pain, including its likely favourable prognosis. As this was an Patients felt that their back pain needed to receive ac- tive treatment, particularly to prevent it from getting worse. They demonstrated little understanding of its nat- ural prognosis. A key guideline recommendation is that patients should receive education and reassurance [2], yet patients did not perceive this as sufficient, wanting “something done” in addition. Patients expected “painkillers” Hoffmann et al. BMC Family Practice 2013, 14:7 http://www.biomedcentral.com/1471-2296/14/7 Page 6 of 6 Page 6 of 6 exploratory study eliciting patients’ perspectives and we did not audit the care that patients actually received, it is pos- sible that more education was provided than reported. Al- though if so, patients were not able to convey this information to the interviewer, possibly because the infor- mation was not understood, retained, or accepted by the patients. Received: 31 July 2012 Accepted: 26 December 2012 Published: 8 January 2013 Received: 31 July 2012 Accepted: 26 December 2012 Published: 8 January 2013 Received: 31 July 2012 Accepted: 26 December 2012 Published: 8 January 2013 Not unexpectedly, patients’ expectations were predom- inately influenced by their previous experiences of low back pain care and advice received from family, friends, colleagues and health professionals. But the messages from other health professionals were alarmingly inconsist- ent, as noted previously [8] and further support the need for clinicians to explore patients’ expectations early in the consultation so that misperceptions can be addressed. Patients in our study also lacked awareness that GPs should use the latest empirical research evidence to guide their practice. Conclusion 2010. Available from: http://www.qualitative-research.net/index.php/fqs/article/view/1428/3027. 14. Braun V, Clarke V: Using thematic analysis in psychology. Qual Res Psychol 2006, 3:77–101. 14. Braun V, Clarke V: Using thematic analysis in psychology. Qual Res Psychol 2006, 3:77–101. Conclusion 7. Wilkinson E, Bosanquet A, Salisbury C, Hasler J, Bosanquet N: Barriers and facilitators to the implementation of evidence-based medicine in general practice: a qualitative study. Eur J Gen Pract 1999, 5:66–70. 7. Wilkinson E, Bosanquet A, Salisbury C, Hasler J, Bosanquet N: Barriers and facilitators to the implementation of evidence-based medicine in general practice: a qualitative study. Eur J Gen Pract 1999, 5:66–70. Patients’ biomechanical understanding of back pain, their subsequent test and treatment expectations, poor understanding of the natural history of low back pain, and message inconsistency from health professionals suggest a need for public education about the appropri- ate management of acute low back pain. A public educa- tion campaign that ran in the mass media in Victoria, Australia for 22 months improved back pain beliefs in both the general public and GPs [17], with the effects sustained 3 years after the campaign ended [18]. In the absence of such a campaign, GPs are encouraged to spe- cifically enquire about patients’ expectations to correct common misperceptions, and better educate patients about the prognosis of acute low back pain, the role of imaging, and management recommendations with their rationale. 8. Chenot JF, Scherer M, Becker A, Donner-Banzhoff N, Baum E, Leonhardt C, Keller S, Pfingsten M, Hildebrandt J, Basler H-D, Kochen MM: Acceptance and perceived barriers of implementing a guideline for managing low back in general practice. Implementation Sci 2008, 3:1–6. 8. Chenot JF, Scherer M, Becker A, Donner-Banzhoff N, Baum E, Leonhardt C, Keller S, Pfingsten M, Hildebrandt J, Basler H-D, Kochen MM: Acceptance and perceived barriers of implementing a guideline for managing low back in general practice. Implementation Sci 2008, 3:1–6. 9. Schers H, Wensing M, Huijsmans Z, Van Tulder M, Grol R: Implementation barriers for general practice guidelines on low back pain. Spine 2001, 26:E348–E353. 9. Schers H, Wensing M, Huijsmans Z, Van Tulder M, Grol R: Implementation barriers for general practice guidelines on low back pain. Spine 2001, 26:E348–E353. 10. Patton M: Qualitative research and evaluation methods. 3rd edition. Thousand Oaks: Sage Publications; 2002. 11. Speziale H, Carpenter D: Qualitative research in nursing: advancing the humanistic imperative. Philadelphia: Lippincott, Williams and Wilkins; 2007. 12. Brisbane City Council: Indooroopilly: Weekly individual income 2006. 2006. [cited 2011 17 October]; Available from: http://profile.id.com.au/Default.aspx? id=327&pg=124&gid=780&type=enum. 13. Mason M: Sample size and saturation in PhD studies using qualitative interviews. Forum Qual Soc Res [serial on the Internet]. 11th edition. doi:10.1186/1471-2296-14-7 Cite this article as: Hoffmann et al.: Patients’ expectations of acute low back pain management: implications for evidence uptake. BMC Family Practice 2013 14:7. Acknowledgements We thank all staff from the medical practice involved and all participants for taking part in the interviews. We also thank Prof Geoff Mitchell for his support of the project and Prof Paul Glasziou for his helpful comments during preparation of the manuscript. Tammy Hoffmann is supported by a National Health and Medical Research Council (NHMRC) of Australia/PHCRED Career Development Fellowship with funding provided by the Department of Health and Ageing. doi:10.1186/1471-2296-14-7 Cite this article as: Hoffmann et al.: Patients’ expectations of acute low back pain management: implications for evidence uptake. BMC Family Practice 2013 14:7.
https://openalex.org/W4235713915	http://ncr-journal.bear-land.org/uploads/b2b496ad6de0d3ff237a1619aae747ea.pdf	Russian	null	Editorial	Nature conservation research. Zapovednaâ nauka	2,017	cc-by	650	DEAR COLLEAGUES! of plants and animals: the Dzeren or Mongolian Ga zelle (Procapra gutturosa), Russian Desman (Des mana moschata), Manul or Pallas’s Cat (Otocolobus manul), Long-tailed Goral (Naemorhedus caudatus), Lady’s Slipper Orchid (Cypripedium calceolus) and Fairy Slipper (Calypso bulbosa). The extinction of each of them in future could disrupt the integrity and functional state of natural ecosystems. Articles, pre sented in this volume, concern several conservation aspects for species included in the Red Data Book of the Russian Federation and regional Red Data Books: e.g., the organisation of species’ protection, development of measures and scientific programmes for conservation of their habitats, determining of factors most crucially affecting the status of popula tions of these species. You may find this information and much more in this volume. We wish you a lot of reading pleasure. of plants and animals: the Dzeren or Mongolian Ga zelle (Procapra gutturosa), Russian Desman (Des mana moschata), Manul or Pallas’s Cat (Otocolobus manul), Long-tailed Goral (Naemorhedus caudatus), Lady’s Slipper Orchid (Cypripedium calceolus) and Fairy Slipper (Calypso bulbosa). The extinction of each of them in future could disrupt the integrity and functional state of natural ecosystems. Articles, pre sented in this volume, concern several conservation aspects for species included in the Red Data Book of the Russian Federation and regional Red Data Books: e.g., the organisation of species’ protection, development of measures and scientific programmes for conservation of their habitats, determining of factors most crucially affecting the status of popula tions of these species. You may find this information and much more in this volume. We wish you a lot of reading pleasure. In your hands you have a thematic volume of the journal «Nature Conservation Research». It is devoted to plants and animals included in the Red Data Book of the Russian Federation. Currently, they are in need of protection on federal level as these species are very threatened in Russia. Some plants and animals are considered (or will be con sidered soon) as Critically Endangered. The present volume consists of two groups of articles. The first group presents reviews of diversity and status of populations of plants and animals with in some federal Protected Areas (Mordovia State Na ture Reserve, Central Forest State Natural Biosphere Reserve, Oka State Nature Biosphere Reserve, Rus sian Arctic National Park) and regions (Krasnodar Krai, Republic of Mordovia) of the Russian Federa tion. Nature Conservation Research. Заповедная наука 2017. 2(Suppl. 1): 1 Nature Conservation Research. Заповедная наука 2017. 2(Suppl. 1): 1 DOI: 10.24189/ncr.2017.025 УВАЖАЕМЫЕ КОЛЛЕГИ! Республики Мордовия. Вторая группа статей посвящена изучению отдельных редких и ис чезающих видов растений и животных. Среди них – дзерен, русская выхухоль, манул, горал, венерин башмачок настоящий, калипсо луко вичный. Исчезновение каждого из этих видов в будущем может нарушить целостность и функциональное состояние природных эко систем. Представленные в выпуске статьи за трагивают многие аспекты сохранения видов, включенных в Красную книгу России и ее ре гионов: организация их охраны, разработка мер и научных программ по сохранению мест их обитания, выявление факторов, наиболее остро влияющих на состояние популяций этих видов. Обо всем этом можно узнать на страни цах этого выпуска. Вы держите в руках тематический выпуск журнала «1DWXUH�&RQVHUYDWLRQ�5HVHDUFK�Ɂ��ɨ Nature�&RQVHUYDWLRQ�5HVHDUFK�Ɂ��ɨ &RQVHUYDWLRQ�5HVHDUFK�Ɂ��ɨ Conservation�5HVHDUFK�Ɂ��ɨ 5HVHDUFK�Ɂ��ɨ Research�Ɂ��ɨ . Запо ведная наука». Он посвящен видам растений и животных, занесенным в Красную книгу Российской Федерации. В настоящее время их численность в России настолько мала, что их охрана осуществляется на федеральном уров не. Некоторые виды находятся или скоро будут находиться на грани исчезновения. Данный выпуск содержит несколько групп материалов. Первая – это обзоры разнообра зия и состояния популяций видов растений и животных на территории некоторых феде ральных ООПТ и регионов РФ: Мордовского заповедника, Центрально-Лесного заповедни ка, Окского заповедника, национального пар ка «Русская Арктика», Краснодарского края, С уважением, Редакционная коллегия журнала Nature Conservation Research. Заповедная наука DEAR COLLEAGUES! The second group consists of articles devoted to the study of certain rare and endangered species Best regards, Editorial Board of the journal Nature Conservation Research 1
https://openalex.org/W2914441724	https://repository.arizona.edu/bitstream/10150/632967/1/journal.pgen.1007942.pdf	English	null	Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage	PLOS genetics	2,019	cc-by	17,274	Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage Item Type Article Authors Pond, Kelvin W; de Renty, Christelle; Yagle, Mary K; Ellis, Nathan A Citation Pond KW, de Renty C, Yagle MK, Ellis NA (2019) Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage. PLoS Genet 15(2): e1007942. https:// doi.org/10.1371/journal.pgen.1007942 DOI 10.1371/journal.pgen.1007942 Publisher PUBLIC LIBRARY SCIENCE Journal PLOS GENETICS Rights © 2019 Pond et al. This is an open access article distributed under the terms of the Creative Commons Attribution License. Download date 24/10/2024 03:59:35 Item License https://creativecommons.org/licenses/by/4.0/ Version Final published version Link to Item http://hdl.handle.net/10150/632967 Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage Item Type Article Authors Pond, Kelvin W; de Renty, Christelle; Yagle, Mary K; Ellis, Nathan A Citation Pond KW, de Renty C, Yagle MK, Ellis NA (2019) Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage. PLoS Genet 15(2): e1007942. https:// doi.org/10.1371/journal.pgen.1007942 DOI 10.1371/journal.pgen.1007942 Publisher PUBLIC LIBRARY SCIENCE Journal PLOS GENETICS Rights © 2019 Pond et al. This is an open access article distributed under the terms of the Creative Commons Attribution License. Download date 24/10/2024 03:59:35 Item License https://creativecommons.org/licenses/by/4.0/ Version Final published version Link to Item http://hdl.handle.net/10150/632967 RESEARCH ARTICLE Editor: Robert M. Brosh, Jr., NIA-NIH, UNITED STATES Editor: Robert M. Brosh, Jr., NIA-NIH, UNITED STATES Received: June 19, 2018 Accepted: January 7, 2019 Published: February 8, 2019 Copyright: © 2019 Pond 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. Received: June 19, 2018 Accepted: January 7, 2019 Published: February 8, 2019 Copyright: © 2019 Pond 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. Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage Kelvin W. Pond1, Christelle de RentyID2, Mary K. YagleID2, Nathan A. EllisID1,2* Kelvin W. Pond1, Christelle de RentyID2, Mary K. YagleID2, Nathan A. EllisID1,2* 1 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona, United States of America, 2 University of Arizona Cancer Center, University of Arizona, Tucson, Arizona, United States of America 1 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona, United States of America, 2 University of Arizona Cancer Center, University of Arizona, Tucson, Arizona, United States of America 1 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona, United States of America, 2 University of Arizona Cancer Center, University of Arizona, Tucson, Arizona, United States of America * naellis@email.arizona.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of col- lapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2- deficient cells. Double strand breaks accumulate during rescue by converging forks in nor- mal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of col- lapsed forks by converging forks is dependent on NSMCE2. OPEN ACCESS Citation: Pond KW, de Renty C, Yagle MK, Ellis NA (2019) Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage. PLoS Genet 15(2): e1007942. https://doi. org/10.1371/journal.pgen.1007942 Author summary DNA damage encountered by the replication fork causes fork stalling and is a major source of mutations when not adequately repaired. Fork stalling can lead to fork collapse, that is, a state of the fork in which normal DNA synthesis cannot be resumed at the site of stalling. Collapsed forks must be rescued by replication forks initiated nearby, but little is known about the rescue mechanism by which an active fork merges with a collapsed fork. We used an inhibitor of DNA replication to generate collapsed replication forks and then studied genetic control of collapsed-fork rescue. We found that NSMCE2, which is a gene product that is known to regulate repair responses to replication stress, is required for cells to effectively rescue collapsed replication forks in order to complete DNA synthesis. DNA double strand breaks that are associated with normal collapsed-fork rescue do not accumulate in cells that are deficient for NSMCE2, suggesting that DNA breakage is part of the rescue and repair mechanism. Failure to rescue collapsed forks leads to DNA Introduction Replication-associated DNA damage is common in human cells and can lead to the develop- ment of somatic mutations. DNA damage during replication can be induced by DNA lesion- producing chemicals, proteins bound to the DNA, DNA polymerase inhibitors, or nucleotide limitation [1]. Hydroxyurea (HU) triggers fork stalling due to nucleotide limitation through inhibition of ribonucleotide reductase [2]. Human cells exposed to HU for up to six hours are capable of restarting 80% of their replication forks [3–6]. However, forks that are stalled for 16 to 24 h are unable to restart [7], indicating they have collapsed. Collapsed replication forks must be rescued by active forks initiated at dormant origins to complete genome duplication. In both human cells and yeasts, the induction of a double strand break (DSB) is associated with repair of collapsed forks [8, 9]. Although factors essential for the formation of DSBs dur- ing fork collapse have been identified [10], the mechanism generating DSBs when the new rep- lication forks converge with the collapsed forks is unknown. The DNA helicase mutated in Bloom’s syndrome BLM possesses multiple functions in DNA replication fork stabilization and homologous recombination (HR), which is a mecha- nism that operates in the repair of replication-associated DSBs [11]. Recruitment of BLM to replication forks is part of the mechanism that stabilizes forks in both unperturbed and replica- tion-stressed cells [12, 13]. Excessive DSBs accumulate in BLM-deficient cells released from replication blockade after prolonged fork stalling [14], suggesting that BLM plays a role in col- lapsed-fork rescue. In the absence of BLM, after fork collapse, under-replicated DNA and unresolved HR intermediates persist into mitosis where they cause DNA damage [15, 16]. The E3 SUMO ligase NSMCE2 is a component of the SMC5/6 complex, which is present at stalled replication forks and a key component of the stalled fork proteome [17]. In budding yeast, deletion of the NSMCE2 homolog MMS21 is lethal; however, sumoylation-deficient hypomorphs are viable and have defects in replication-specific DNA repair [18]. These cells also accumulate excess RAD51-dependent recombination intermediates during replication stress and are deficient in HR [18, 19]. During fork stalling, MMS21 undergoes auto-sumoyla- tion during replication stress, and it then recruits the BLM homolog Sgs1 via SUMO binding sites on Sgs1 [20, 21]. Once recruited, Sgs1 resolves HR intermediates generated during repair of damaged replication forks. NSMCE2 and collapsed-fork rescue Competing interests: The authors have declared that they have no competing interests. damage in mitosis and DNA damage in the following cell cycle. Our work highlights a unique role for NSMCE2 in rescue of collapsed replication forks. Data Availability Statement: All relevant data are within the paper and its Supporting Information files Data Availability Statement: All relevant data are within the paper and its Supporting Information files Funding: Research reported in this publication was supported by the University of Arizona Cancer Center and the National Cancer Institute of the National Institutes of Health (https://www.cancer. gov) under award numbers R01CA140804 (NAE) and P30 CA023074 (NAE) and by funds from the University of Arizona. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Results We used two different siRNAs to deplete NSMCE2 in HeLa cells, resulting in an approximately 80% reduction in both RNA and protein levels (S1A Fig). To corroborate key NSMCE2-defi- cient phenotypes, we also prepared NSMCE2-/- clones of HEK293T cells in which we targeted a single site in exon 2 of NSMCE2 and isolated two clones with different frameshift mutations containing no detectable NSMCE2 protein (S1B Fig). Hereafter, we refer to cells in which we have knocked down NSMCE2 as NSMCE2-deficient cells and we refer to NSCME2-/- cells as NSMCE2 null cells. NSMCE2 is required for BLM sumoylation and its localization to stalled replication forks Experiments in yeast suggested that NSMCE2 is required for efficient sumoylation of BLM [20]. To measure SUMO-BLM levels, we used human U2OS cells that express a His-tagged SUMO2 to carry out pull down assays. Analysis of SUMO-conjugates revealed that sumoylated BLM levels increased approximately eight fold after prolonged fork stalling by treatment with 2 mM HU for 16 hours (Fig 1A and 1B). We then tested if sumoylation of BLM is dependent on NSMCE2 by knockdown of endogenous NSMCE2. Depletion of NSMCE2 using two differ- ent siRNAs resulted in a 60% decrease in sumoylated BLM in HU-treated cells compared to HU-treated control cells (Fig 1A and 1B). These data indicate that BLM sumoylation is depen- dent on NSMCE2. Residual SUMO-BLM could result from the incomplete depletion of NSMCE2 or from residual sumoylation catalyzed by other E3 SUMO ligases. We previously reported that a BLM protein mutated at its preferred sumoylation sites K317R and K331R is recruited normally to stalled replication forks [14]; consequently, we hypothesized that BLM localization at stalled forks might be normal in NSMCE2-deficient cells. On the contrary, siRNA-mediated depletion in HeLa cells led to three-fold reduction in BLM foci in cells treated with HU for 24 h compared to HU-treated control cells (Fig 1C). NSMCE2 depletion was not associated with a change in the levels of BLM protein in NSMCE2-deficient HeLa cells (S2A Fig), and overexpression of siRNA-resistant NSMCE2 in depleted HeLa cells substantially rescued the defect in localization of BLM at stalled forks (S3A Fig; representative low power images of BLM localization are shown in S4A Fig). These data show that efficient recruitment to or retention of BLM at collapsed replication forks is depen- dent on NSMCE2. NSMCE2 and collapsed-fork rescue Here we studied the role of NSMCE2 in repair and rescue of collapsed replication forks. We found that NSMCE2 is essential for formation of DSBs during collapsed-fork rescue. Inter- estingly, lack of DSBs during collapsed-fork rescue is associated with hyper-accumulation of RAD51 and impaired sister chromatid recombination. Defects in the rescue of collapsed repli- cation forks in NSMCE2-deficient cells lead to DNA damage in mitosis. Introduction In human cells, forks adopt a RAD51-dependent structure during stalling, which resem- bles a Holliday junction [22]. RAD51 is required to prevent replication-induced DSBs, and RAD51 levels increase at stalled forks as they transition from a restart-competent state to a collapsed state [7]. BLM regulates the exchange of RAD51 recombinase for RPA [23, 24], and in previous work we showed that sumoylation of BLM regulates a switch between BLM’s pro- and anti-recombinogenic functions [14]. If negative regulators of RAD51 such as BLM and the recently described RADX are ablated, excess RAD51 is loaded at stalled forks and excess DSBs accumulate [25]. In other situations, however, induction of excessive RAD51 can instead trigger inhibition of HR repair [26]. Because NSMCE2 regulates BLM recruit- ment and RAD51-dependent HR intermediates accumulate in yeast mms21 mutant cells, we hypothesized that NSMCE2 may be a critical regulator of RAD51 function at collapsed repli- cation forks. 2 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 NSMCE2 prevents excessive accumulation of RAD51 and promotes HR BLM can promote dissociation of RAD51 recombinase from ssDNA [23]. Because BLM’s role in dissociation of RAD51 at stalled forks could be defective in NSMCE2-deficient cells, we tested whether NSMCE2 plays a role in regulation of RAD51 accumulation. NSMCE2-defi- cient cells, both untreated cells and cells treated with HU for 24 hours, exhibited increases in the number, intensity, and size of RAD51 foci compared to controls (Fig 1D and 1E; S3B Fig; representative low power images of RAD51 localization are shown in S4C Fig). Western blot analysis showed that total cellular RAD51 protein levels were similar in NSMCE2-deficient and control cells (S2A Fig). Because over 90% of stalled replication forks are unable to restart PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 3 / 25 NSMCE2 and collapsed-fork rescue depleted in HeLa cells with two different siRNAs, and cells were treated or not with 2 mM HU for 24 hours. Box and whisker plots represent cell distributions of BLM foci per cell. Numerical labels represent the median value of at least 10,000 BLM foci in each experimental condition. Three independent experiments were performed. (D) Quantitative analysis of the number of RAD51 foci per cell. Plots and analysis performed as in panel C. RAD51 and RPA staining were performed in the same experiment. Three independent experiments were performed. (E) Quantitative analysis of the median fluorescence intensity of RAD51 per cell. (F) Quantitative analysis of the number of RPA foci per cell. (G) Representative immunofluorescence images of RAD51 and RPA foci in cells analyzed in (D-F), showing colocalization of RAD51 and RPA. Scale bars represent 10 microns. (H) SCEs in HeLa cells exposed to control or NSMCE2 siRNA and treated or not with 2 mM HU for 24 hours. Mean and standard deviation of the number of SCEs/metaphase is shown. Three independent experiments were performed. Asterisks represent statistical analysis by paired t-test (A,H) or Mann-Whitney test (C-F) ( = p<0.05, = p<0.01, = p<0.001, = p<0.0001). https://doi.org/10.1371/journal.pgen.1007942.g001 after 24 hours of HU treatment [7, 27], we tested whether the excess RAD51 was localized to collapsed replication forks. To test this, we labeled nascent DNA synthesis with 10 μM EdU for 12 min prior to HU treatment, treated cells with HU for 24 hours, and then analyzed RAD51 foci. As expected, RAD51 co-localized with EdU foci in HU-treated control and NSMCE2-de- ficient cells (S3C Fig). These data show that NSMCE2 is required to prevent over-accumula- tion of RAD51 at forks under conditions that lead to their collapse. p RAD51 is normally loaded onto ssDNA by exchange with ssDNA binding protein RPA [28]. We therefore tested whether the excess RAD51 accumulation in NSMCE2-deficient cells might correlate with a diminished accumulation of RPA at stalled forks. For this experiment, we measured the accumulation of chromatin-bound RPA after nucleoplasmic extraction of cells treated with HU for 24 hours. After siRNA-mediated depletion of NSMCE2 and HU treatment, cells displayed 40% fewer RPA foci in both HeLa (Fig 1F and 1G; representative low power images of RPA localization are shown in S4B Fig) and U2OS cells (S3D Fig) compared to control cells. NSMCE2 and collapsed-fork rescue Fig 1. NSMCE2 is required for normal accumulations of BLM, RAD51, and RPA at stalled replication forks. (A) Depletion of NSMCE2 is associated with a reduction in SUMO2-BLM. Quantitation of SUMO2-BLM in U2OS cells normalized to controls (NC1). SUMO2-conjugates were pulled down from U2OS cells that stably overexpress His-tagged SUMO2 protein. NSMCE2 was depleted with two different siRNAs, and cells were treated or not with 2 mM HU for 16 hours. The levels of SUMO2-BLM pulled down were normalized to the amounts of SUMO2. Shown are the mean and standard deviation of five independent experiments. (B) Representative images of western blots quantitated in (A). (C) Quantitative analysis of the number of BLM foci in HeLa cells. NSMCE2 was Fig 1. NSMCE2 is required for normal accumulations of BLM, RAD51, and RPA at stalled replication forks. (A) Depletion of NSMCE2 is associated with a reduction in SUMO2-BLM. Quantitation of SUMO2-BLM in U2OS cells normalized to controls (NC1). SUMO2-conjugates were pulled down from U2OS cells that stably overexpress His-tagged SUMO2 protein. NSMCE2 was depleted with two different siRNAs, and cells were treated or not with 2 mM HU for 16 hours. The levels of SUMO2-BLM pulled down were normalized to the amounts of SUMO2. Shown are the mean and standard deviation of five independent experiments. (B) Representative images of western blots quantitated in (A). (C) Quantitative analysis of the number of BLM foci in HeLa cells. NSMCE2 was PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 4 / 25 https://doi.org/10.1371/journal.pgen.1007942.g001 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 NSMCE2 is required for DSBs and RAD51 resolution during collapsed- fork rescue Because HU-induced SCEs were suppressed in NSMCE2-deficient cells, we hypothesized that the excess RAD51 leads to a defect in DSB formation during rescue of collapsed forks. To test this possibility, we measured DSB accumulation in control and NSMCE2-deficient cells after prolonged exposure to HU. In HeLa cells exposed to control siRNA, a 16-hour treatment with HU did not induce DSBs; however, a 48-hour treatment with HU led to an accumulation of DSBs detectable by pulsed-field gel electrophoresis (PFGE) (Fig 2A). Interestingly, we found that NSMCE2-deficient cells failed to produce a detectable increase in DSBs after a 48-h expo- sure. Ionizing radiation with 4 Gy followed by a 30-min repair period results in equal levels of DSBs in both control and NSMCE2-deficient cells, indicating that NSMCE2-deficient cells are not defective in DSB formation per se but in replication stress-induced DSBs. Because NSMCE2-deficient cells are defective in DSB formation at stalled forks after pro- longed HU treatment, we tested whether the DNA damage response was also reduced. We measured γ-H2AX levels by analysis of DNA damage foci and flow cytometry. We found by both measures that NSMCE2-deficient cells accumulate two- to three-fold less γ-H2AX after prolonged HU treatment (S3J and S3K Fig) despite normal levels of phosphorylation of CHK1 and of RPA32 (S2A Fig). Substantial rescue of the levels of γ-H2AX foci was observed by over- expression of siRNA-resistant NSMCE2 (S3K Fig). To investigate the ability of cells to generate DSBs during collapsed-fork rescue, we mea- sured the kinetics of accumulation of DSBs over time after release from HU. After release from the HU block, normal cells linearly accumulated DSBs, whereas NSMCE2-deficient cells failed to accumulate DSBs four and eight hours after release (Fig 2A). The accumulation appears to be replication-dependent, because normal cells released into 10 μM aphidicolin after HU arrest did not accumulate similar levels of DSBs (S5A Fig). Flow cytometry confirmed that control and NSMCE2-deficient cells show similar cell-cycle distributions 6 and 12 hours after release from HU, suggesting that differences in cell-cycle progression after release from HU do not explain these results (S5B Fig). Moreover, no significant differences in the levels of apoptosis were observed in control and NSMCE2-deficient cells after release from HU, ruling out apo- ptosis as a confounder of differences in DSBs (S5D Fig). NSMCE2 and collapsed-fork rescue PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Overexpression of siRNA-resistant NSMCE2 in depleted HeLa cells rescued the defect in RPA foci accumulation (S3E Fig). In addition, HU-treated HEK293T NSMCE2 null cells displayed reduced levels of chromatin-bound RPA compared to control normal cells (S3F and S3G Fig). We conclude that RAD51 accumulates in excess over RPA in NSMCE2-de- ficient and NSMCE2 null cells, perhaps due to a failure to recruit BLM to stalled forks. The lower levels of RPA foci suggest that there are lower levels of ssDNA. To test this possi- bility, we measured the levels of ssDNA by incorporation of BrdU for two cell divisions prior to HU treatment, followed by immunodetection with anti-BrdU antibodies in non-denaturing conditions. Unlike control cells, which displayed at least a two-fold increase in BrdU foci after treatment with 2 mM HU for 24 hours, NSMCE2-deficient cells displayed no induction of BrdU foci after HU treatment (S3H Fig; representative low power images of BrdU localization are shown in S4D Fig). Thus, the lower levels of focal RPA in HU-treated NSMCE2-deficient cells are evidence of lower levels of ssDNA detectable by anti-BrdU antibodies at collapsed rep- lication forks. Because anti-BrdU antibodies cannot detect BrdU in the ssDNA-RAD51 nucle- oprotein filament [29], these results do not rule out the possibility that the excess RAD51 is bound to ssDNA. To test whether the RAD51-bound chromatin in NSMCE2-deficient cells is competent for HR, we measured the frequency of sister chromatid exchanges (SCEs) after prolonged fork stalling by 24-hour treatment with HU. The SCE assay measures crossovers generated after resumption of DNA synthesis that can be detected in the subsequent mitosis. NSMCE2-defi- cient cells had a 45% reduction in the number of HU-induced SCEs/metaphase compared to control cells (Fig 1H). Thus, the excess RAD51 observed at stalled forks is not associated with increased sister chromatid recombination. Contrary to previous reports using murine cells [30], we found that basal levels of SCEs in HEK293T NSMCE2 null cells were similar to nor- mal HEK293T cells (S3I Fig). PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 5 / 25 NSMCE2 is required for DSBs and RAD51 resolution during collapsed- fork rescue Levels of DSBs in untreated HEK293T NSMCE2 null cells were higher than in untreated normal HEK293T cells; however, similar to the results obtained with NSMCE2 depletion with siRNAs, after treatment with HU and dur- ing release into normal medium we observed a defect in accumulation of DSBs in NSMCE2 null cells (S5C Fig). Similar to NSMCE2-deficient HeLa cells, NSMCE2 null cells were also defective in their γ-H2AX response after HU treatment (S5E Fig). Collectively, the results sug- gest that in the absence of NSMCE2 the levels of DSBs that are normally generated during col- lapsed-fork rescue are reduced. We next tested whether NSMCE2-deficient cells have a defect in the dynamics of RAD51 localization during collapsed-fork rescue. Because RAD51 protein accumulates during HU treatment, we hypothesized that converging forks displace the RAD51 over time. We therefore released cells from prolonged fork stalling and measured levels of the RAD51 foci at collapsed replication forks in a time course. Two, four, and eight hours after release from HU, control HeLa cells exhibited a steady decrease in RAD51 foci whereas NSMCE2-deficient cells retained them (Fig 2B). RAD51 foci increased in normal cells between 8 and 12 h after release from HU, possibly due to RAD51-dependent DNA repair in late S or G2 phase. In addition, we also observed a persistence of RAD51 foci at stalled forks in NSMCE2 null cells compared to nor- mal cells after release from HU treatment (S6A Fig). In both normal and NSMCE2 null HEK293T cells, RAD51 foci co-localized with RPA and γ-H2AX foci (S6B Fig). PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 6 / 25 NSMCE2 and collapsed-fork rescue Fig 2. DSB formation and resolution of RAD51 foci at collapsed forks is dependent on NSMCE2. (A) Cleavage of collapsed forks is dependent on NSMCE2. Quantitative analysis (top panel on left) and representative image (bottom panel on left) of DSBs in control and NSMCE2-deficient HeLa cells treated or not with HU for 24 hours then released for 16 or 48 hours. As a control, cells were irradiated with 4 Gy and allowed to recover for 30 min. DSBs were visualized by PFGE. Three independent experiments were performed. (top panel on right) Quantitative analysis of DSBs at different times after release from HU block. Mean and standard deviation is shown. Different time courses were performed, including release times of 4 hours and 8 hours; 12 hours and 24 hours; and 4 hours, 8 hours, 12 hours, and 24 hours. In each time course, the no-treatment sample was used to normalize the data. A minimum of two independent experiments were analyzed for the 4-hour and 8-hour and the 12-hour and 24-hour time courses. (bottom panel on right) Image of a representative PFGE experiment. (B) Persistence of RAD51 foci after release from HU block in NSMCE2-deficient cells. (top panel) Quantitative analysis of number of RAD51 foci per cell at different times after release from HU block. Mean and standard error are shown. At least 10,000 RAD51 foci were analyzed in each experimental condition. Three independent experiments were performed. (bottom panel) Three representative images from the experiment. Scale bars represent 10 microns. (C) Similar replication dynamics in normal and NSMCE2-deficient cells treated with HU. DNA combing analysis by maRTA of HeLa cells exposed to control and NSMCE2 siRNAs and treated or not with 2 mM HU for 5 hours or 16 hours. Two independent experiments were performed. Four panels show quantitative analysis of new origin firing, replication fork re-start, fork collapse, and replication fork speed. The three panels below the bar graph in (D) show representative images from the maRTA. (D) Cell-cycle analysis by flow cytometry of untreated HeLa cells pulsed with 20 μM BrdU for 40 min. Mean and standard deviation is shown. Three independent experiments were performed. https://doi.org/10.1371/journal.pgen.1007942.g002 https://doi.org/10.1371/journal.pgen.1007942.g002 We considered the possibility that persistence of excessive RAD51 at collapsed replication forks might disturb replication dynamics in S phase after release from HU. To measure repli- cation fork dynamics, we performed microfluidics-assisted replication track analysis (maRTA) [31]. We found that replication fork speed, fork restart, and dormant origin firing were similar in NSMCE2-deficient cells compared to control cells, after either 5 or 16 hours of HU treat- ment (Fig 2C). These data indicate that both the replication dynamics of unperturbed forks and of dormant origin activation in replication-stressed cells are not adversely affected by NSMCE2 deficiency. NSMCE2 and collapsed-fork rescue PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 7 / 25 https://doi.org/10.1371/journal.pgen.1007942.g002 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 NSMCE2 and collapsed-fork rescue Fig 3. NSMCE2 prevents mitosis-dependent DNA damage. (A) Schematic for treatment and enrichment of anaphase cells for analysis of UFBs and FANCD2 foci. (B) Representative deconvoluted, maximum intensity projection images of z-stacks showing multiple PICH positive UFBs, (C) BLM/PICH colocalization, (D) FANCD2 foci flanking UFBs, and (E) EdU/FANCD2 co-localization in anaphase cells. Scale bars represent 5 microns. (F) Quantitative analysis of PICH- stained UFBs in NSMCE2-deficient and control cells, processed as in A. 30–50 anaphase z-stacks were analyzed in each experimental condition. At least three independent experiments were performed. (G) Quantitation of number of FANCD2 foci per cell in anaphase cells independent of UFBs. 30–50 anaphase z- stacks were analyzed in each experimental condition. (H) Quantitation of EdU positive anaphase cells represented as the percent of FANCD2 foci localized in regions with EdU signal. At least 100 FANCD2 foci were analyzed in each experiment in cells positive for EdU signal only. Mean and standard deviation is shown. Three independent experiments were performed. See Fig 1 for definition of asterisks. https://doi org/10 1371/journal pgen 1007942 g003 Fig 3. NSMCE2 prevents mitosis-dependent DNA damage. (A) Schematic for treatment and enrichment of anaphase cells for analysis of UFBs and FANCD2 foci. (B) Representative deconvoluted, maximum intensity projection images of z-stacks showing multiple PICH positive UFBs, (C) BLM/PICH colocalization, (D) FANCD2 foci flanking UFBs, and (E) EdU/FANCD2 co-localization in anaphase cells. Scale bars represent 5 microns. (F) Quantitative analysis of PICH- stained UFBs in NSMCE2-deficient and control cells, processed as in A. 30–50 anaphase z-stacks were analyzed in each experimental condition. At least three independent experiments were performed. (G) Quantitation of number of FANCD2 foci per cell in anaphase cells independent of UFBs. 30–50 anaphase z- stacks were analyzed in each experimental condition. (H) Quantitation of EdU positive anaphase cells represented as the percent of FANCD2 foci localized in regions with EdU signal. At least 100 FANCD2 foci were analyzed in each experiment in cells positive for EdU signal only. Mean and standard deviation is shown. Three independent experiments were performed. See Fig 1 for definition of asterisks. Fig 3. NSMCE2 prevents mitosis-dependent DNA damage. (A) Schematic for treatment and enrichment of anaphase cells for analysis of UFBs and FANCD2 foci. (B) Representative deconvoluted, maximum intensity projection images of z-stacks showing multiple PICH positive UFBs, (C) BLM/PICH colocalization, (D) FANCD2 foci flanking UFBs, and (E) EdU/FANCD2 co-localization in anaphase cells. NSMCE2 prevents mitotic DNA damage resulting from replication stress NSMCE2-deficient cells maintained normal cell-cycle progression in the absence of HU treat- ment (Fig 2D); however, after release from prolonged HU treatment, NSMCE2-deficient cells displayed an arrest in the next G1 phase (S7A Fig). Defects in collapsed-fork rescue could lead to under-replicated DNA in S phase and DNA damage in mitosis. Similar to previously reported results [30, 32], we found increases in the frequencies of abnormal anaphases, micro- nuclei, and G1 53BP1 nuclear bodies after release from HU block in NSMCE2-deficient cells compared to controls indicating that excess mitotic DNA damage is induced in NSMCE2-defi- cient cells (S7B–S7F Fig; representative low power images of 53BP1 localization are shown in S4E Fig). To investigate further the nature of the mitotic damage invoked in HU-treated, NSMCE2- deficient cells, we measured the frequency of ultra-fine bridge (UFB) formation in cells under- going mitosis. In order to obtain a sufficient number of cells in anaphase, cells were pretreated or not with HU for 24 hours; they were then blocked in G2 with the CDK1 inhibitor RO-3306 at 7.5 μM for 15 hours and then finally released into metaphase for 1 hour prior to fixation (Fig 3A). Flow cytometry confirmed effective G2 arrest by RO-3306 treatment (S7G Fig). We visualized UFBs using the PICH repair helicase, which localizes to UFBs and DNA under ten- sion [3]. The number of UFBs measured by PICH staining was increased after HU treatment in NSMCE2-deficient cells but not control cells (Fig 3B–3D and 3F). PICH-positive UFBs were also positive for BLM (Fig 3C), indicating that localization of BLM to these structures is not dependent on NSMCE2. The crosslink repair protein FANCD2 is sometimes associated with the ends of UFBs, and has been used as a marker for under-replicated DNA persisting into mitosis [3,16]. FANCD2-flanked, PICH-positive UFBs (Fig 3D) were infrequently observed in both NSMCE2-deficient and control cells. Thus, the excess UFBs produced in NSMCE2-deficient cells are not equivalent to the UFBs produced in cells treated with low- dose aphidicolin [15, 16]. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 8 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Epistasis relationships among NSMCE2, BLM, and RAD51 during collapsed-fork rescue Because NSMCE2 is essential for proper localization of BLM to stalled replication forks but displays phenotypes distinct from BLM-deficient cells, we asked whether NSMCE2 is epistatic to BLM during rescue of collapsed forks. In these experiments, we used siRNAs to deplete BLM in NSMCE2 null and control cells (Fig 4A). In HU-treated normal HEK293T cells depleted for BLM, levels of phosphorylated RPA and γ-H2AX were similar to levels in HU- treated control cells as evidence by Western blot analysis. In contrast, in HU-treated NSMCE2 nulls cells depleted for BLM, the levels of phosphorylated RPA and γ-H2AX were reduced in comparison to HU-treated control cells, but they were similar to levels in HU-treated NSCME2 null cells. The data for phosphorylated H2AX were confirmed by analysis of focal γ-H2AX and flow cytometry with γ-H2AX antibodies (Fig 4B, 4D and 4E). Prolonged HU treatment of BLM-deficient normal HEK293T cells resulted in a 2.6 fold increase in the levels of SCEs from 8.2 to 21.1 SCEs/metaphase, whereas prolonged HU treatment of BLM-deficient NSMCE2 null cells resulted in an only small increase in SCEs from 4.6 to 6.3 SCEs/metaphase (Fig 4C). Consistent with the suppression of HU-induced SCEs, the levels of HU-induced DSBs were also suppressed in BLM-depleted NSMCE2 null cells relative to BLM-deficient nor- mal cells. These data indicate that NSMCE2 is epistatic to BLM with respect to HU-induced phenotypes. Because DSB accumulation is suppressed in NSMC2-deficient cells, we tested whether depletion of RAD51 would restore HU-induced DSB levels to normal in NSMCE2 null cells. After transfection of control and RAD51 siRNAs, we treated cells with 2 mM HU for 24 hours, then released into normal medium for 6 hours and quantitated the DSB marker γ-H2AX by flow cytometry and measured DSBs by PFGE. In HU-treated, control-depleted normal HEK293T cells, γ-H2AX levels increased approximately 10 fold compared to baseline after release into normal medium for 6 hours (S10A and S10C Fig). Contrary to expectation, RAD51 depletion in normal cells resulted in much smaller induction of γ-H2AX, similar to the levels observed in HU-treated control-depleted NSMCE2 null and HU-treated, RAD51-de- pleted NSMCE2 null cells. Consistent with the γ-H2AX results, in HU-treated, control- depleted normal HEK293T cells, DSB levels increased approximately three fold compared to baseline after release into normal medium for 6 hours (S10B Fig). NSMCE2 and collapsed-fork rescue immunofluorescence (S8A and S8B Fig). HU-treated NSMCE2 null cells exhibited a nearly 50% increase in median fluorescence intensity of chromosome-associated γ-H2AX in phos- pho-H3-positive cells compared to HU-treated HEK293T normal cells. For the analysis of chromosome aberrations, cells were treated or not with 2 mM HU for 24 hours and then meta- phase chromosomes were prepared and analyzed by fluorescence microscopy (S9A Fig). We identified increased frequencies of chromatid arm breaks, telomere fusions, and secondary constrictions in NSMCE2 null cells compared to control cells. Chromatid arm breaks and sec- ondary constrictions were induced by HU treatment (S9B Fig). The increase in secondary con- strictions in HU-treated NSMCE2 nulls cells is consistent with increased under-replicated DNA and the increase in chromatid breaks could arise from chromosome breakage in mitosis or under-replication as seen at common fragile sites. Scale bars represent 5 microns. (F) Quantitative analysis of PICH- stained UFBs in NSMCE2-deficient and control cells, processed as in A. 30–50 anaphase z-stacks were analyzed in each experimental condition. At least three independent experiments were performed. (G) Quantitation of number of FANCD2 foci per cell in anaphase cells independent of UFBs. 30–50 anaphase z- stacks were analyzed in each experimental condition. (H) Quantitation of EdU positive anaphase cells represented as the percent of FANCD2 foci localized in regions with EdU signal. At least 100 FANCD2 foci were analyzed in each experiment in cells positive for EdU signal only. Mean and standard deviation is shown. Three independent experiments were performed. See Fig 1 for definition of asterisks. https://doi.org/10.1371/journal.pgen.1007942.g003 https://doi.org/10.1371/journal.pgen.1007942.g003 We then tested whether mitotic DNA damage originated from collapsed forks in the previ- ous S phase. As a marker for damaged DNA and repair in anaphase cells, we counted the num- ber of FANCD2 foci and found a 1.8 fold increase in NSMCE2-deficient cells treated with HU compared to control cells (Fig 3E and 3G). In order to monitor the location of collapsed forks generated by prolonged treatment with HU, we labeled cells with EdU for 20 min before treat- ment with HU. FANCD2 foci co-localized with the EdU label at a greater frequency in HU- treated, NSMCE2-deficient cells compared to control cells (Fig 3H), indicating that the recruit- ment of FANCD2 observed in mitosis had occurred in regions of chromatin where replication forks had previously stalled and collapsed. These data suggest that defective collapsed-fork res- cue in NSMCE2-deficient cells leads to increased under-replicated DNA persisting into mito- sis, which results in mitotic DNA damage. In order to measure DNA damage in metaphase cells, we measured the levels of γ-H2AX associated with metaphase chromosomes and the levels of chromosome aberrations detectable at metaphase. For the analysis of γ-H2AX levels, normal HEK293T and NSMCE2 null cells were treated with 2 mM HU for 24 hours, released into medium with RO-3306 to block them in G2, then released into normal medium and prepared for analysis of γ-H2AX levels by PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 9 / 25 Epistasis relationships among NSMCE2, BLM, and RAD51 during collapsed-fork rescue In contrast, RAD51 deple- tion in normal cells resulted in almost no induction of DSBs compared to baseline, which again was similar to the levels of DSBs in HU-treated control-depleted NSMCE2 null and HU- treated, RAD51-depleted NSMCE2 null cells. These data suggest that, despite the fact that RAD51 has hyper-accumulated at collapsed replication forks, in the absence of NSMCE2, the RAD51 at collapsed forks is nonfunctional. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 10 / 25 Discussion DNA combing experiments in many mammalian cell lines have shown that, after prolonged fork stalling due to HU exposure, DNA synthesis does not normally resume at the site where the fork stalled (see reference 2 and Fig 2). These data rule out rescue mechanisms, such as HR-mediated restart or break-induced replication, in which replication is re-established at the site of fork stalling. The majority of collapsed forks must therefore be rescued by converging forks initiated at dormant origins after release from prolonged replication arrest. DSBs have been previously associated with resumption of DNA synthesis after release from prolonged HU block [7, 14], but the mechanism by which these breaks are generated is not well under- stood. Here we show that normal rescue of collapsed replication forks is dependent on NSMCE2. After resumption of DNA synthesis, NSMCE2-deficient cells do not accumulate normal numbers of DSBs. The large increase in UFBs in mitosis indicates that many forks fail to complete DNA replication. NSMCE2 deficiency is associated with higher levels of RAD51 foci at collapsed forks and persistence of foci after release from replication arrest. These results are consistent with results obtained in yeast mutants of the SMC5/6 complex and of MMS21, in which excess RAD51-de- pendent recombination intermediates accumulate at stalled forks [19, 33, 34]. Pathological accumulations of RAD51 have been associated with DNA repair defects [26, 35]. We do not know the structure of the RAD51-bound DNA in NSMCE2-deficient cells, and the excess RAD51 could be at the fork itself, associated with ssDNA gaps behind the fork, or associated with other abnormal structures. There are established roles for RAD51 at stalled replication forks that do not involve DSBs. For instance, RAD51’s role in reversal of the replication fork is epistatic to its BRCA2-mediated fork protection function [36, 37]. Our experiments with RAD51 depletion in normal cells suggest that reversed fork structures, catalyzed by RAD51, are required for the formation of DSBs during fork rescue, very likely as substrates for nucle- ases such as MUS81. Biochemical studies suggest that BLM performs a quality-control func- tion on stressed replication forks by dissociating nonfunctional, ADP-bound RAD51 from the nucleoprotein filament [23]. Because BLM does not accumulate normally at stalled forks in the absence of NSMCE2, it is possible that the RAD51 that accumulates excessively in NSMCE2- defcient cells is in the nonfunctional ADP-bound state. NSMCE2 and collapsed-fork rescue Fig 4. NSMCE2 is epistatic to BLM for HU-induced phenotypes. Analysis of HU-induced phenotypes in normal HEK293T and NSMCE2 null cells in which BLM levels were reduced or not by siRNA-mediated depletion. (A) Western blot analysis of levels of phosphorylation of H2AX and RPA. Black line indicates separate blots using lysates from the same experiment. The experiment was performed two times. (B) Analysis of focal concentrations of γ-H2AX after treatment with HU for 24 hours and after release into normal medium for 6 hours. Box and whisker plots representing at least 10,000 γH2AX foci in two independent experiments. Medians are shown above the graph. (C) Levels of HU- induced SCEs. Box and whisker plots representing distribution of SCE per metaphase. Medians of the combined data are shown above the graph. Two independent experiments were performed. Unpaired t-test was performed to analyze the distributions. (D) Analysis by flow cytometry of fluorescence intensity of γ-H2AX after treatment with HU for 24 hours and after release into normal medium for 6 hours. Median florescence intensity was measured from a minimum of 10,000 events. The bar graph represents the fold change in median fluorescent intensity normalized to untreated normal HEK293T cells exposed to control siRNA from a single experiment. Two independent experiments were performed. (E) Representative distributions of fluorescence intensity of cells in selected conditions from the flow cytometry data. (F) Analysis by PFGE of DSBs after treatment with HU for 24 hours and after release into medium for 6 hours. The bar graph represents fold change in DSBs detected by PFGE normalized to untreated normal NSMCE2 cells exposed to control siRNA. The error bars represent the SEM of three independent experiments. The gel below the bar graph contains the results from one experiment. See Fig 1 for definition of asterisks. https://doi.org/10.1371/journal.pgen.1007942.g004 NSMCE2 and collapsed-fork rescue 71/journal pgen 1007942 February 8 2019 11 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 11 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Normally (top part of model), after exposure to HU, uncoupling of the replicative helicase from the replicative DNA polymerase at the fork leads to production of ssDNA, which is immediately bound by ssDNA binding protein RPA (red circle). BLM (yellow circle) is one of the first factors to be recruited to the stalled replication fork and this accumulation of BLM is dependent on the NSMCE2 protein (green rectangle) on the SMC5/6 complex (purple ring). The localization of BLM at the stalled fork assists in maintaining functional RAD51 (teal rhombus) exchange during fork reversal. Given enough time in the reversed configuration, the fork is unable to restart (fork collapse). After resumption of DNA synthesis at dormant origins, an active fork converges upon the collapsed fork. The convergence is associated with RAD51 dissociation and a DSB at the collapsed fork, followed by DSB repair by HR via the canonical RAD51-mediated pathway. In NSMCE2-deficient cells (bottom part of model), BLM is no longer recruited to the stalled fork, and there is a failure in the regulation of RAD51 loading at the fork, shown here as a loss of dynamic exchange of RAD51 and RPA resulting in excess, hyper-stable RAD51. After resumption of DNA synthesis at dormant origins, the aberrant RAD51 structure prevents convergence of the active fork with the collapsed fork, resulting in a double fork stall instead of a DSB. The double fork stall persists into mitosis where it results in an ultra-fine DNA bridge and mitotic DNA damage. https://doi.org/10.1371/journal.pgen.1007942.g005 definitive tests of this proposition are required to rule out other possible models. For example, it could be informative to use molecular combing to monitor the replication dynamics of forks from newly fired origins as they converge upon collapsed replication forks. We found that BLM sumoylation is dependent on the presence of NSMCE2. We and others [32] have shown that BLM does not accumulate normally at stalled forks in NSMCE2-deficient cells. BLM has multiple functions in the resolution of recombination intermediates during rep- lication stress, normally ensuring that recombination intermediates are resolved without exchange. Yet the levels of SCEs are low in the absence of NSMCE2, suggesting additional roles of NSMCE2 in promotion of crossover events when BLM is absent. Our evidence sug- gests that RAD51-depenedent intermediates in NSMCE2-deficient cells are not resolved until M phase, whereas RAD51 foci are normally resolved during S phase. Discussion That said, the epistasis experiments indicate that NSMCE2 controls other factors besides BLM that contribute to the function of RAD51 at collapsed forks. We propose a model in which the excess nonfunctional RAD51 pre- vents DSB formation during the convergence of active replication forks with collapsed forks, leading to excess under-replicated DNA that is detectable at anaphase (Fig 5). However, more PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 12 / 25 NSMCE2 and collapsed-fork rescue Fig 5. Model for collapsed-fork rescue by NSMCE2. Normally (top part of model), after exposure to HU, uncoupling of the replicative helicase from the replicative DNA polymerase at the fork leads to production of ssDNA, which is immediately bound by ssDNA binding protein RPA (red circle). BLM (yellow circle) is one of the first factors to be recruited to the stalled replication fork and this accumulation of BLM is dependent on the NSMCE2 protein (green rectangle) on the SMC5/6 complex (purple ring). The localization of BLM at the stalled fork assists in maintaining functional RAD51 (teal rhombus) exchange during fork reversal. Given enough time in the reversed configuration, the fork is unable to restart (fork collapse). After resumption of DNA synthesis at dormant origins, an active fork converges upon the collapsed fork. The convergence is associated with RAD51 dissociation and a DSB at the collapsed fork, followed by DSB repair by HR via the canonical RAD51-mediated pathway. In NSMCE2-deficient cells (bottom part of model), BLM is no longer recruited to the stalled fork, and there is a failure in the regulation of RAD51 loading at the fork, shown here as a loss of dynamic exchange of RAD51 and RPA resulting in excess, hyper-stable RAD51. After resumption of DNA synthesis at dormant origins, the aberrant RAD51 structure prevents convergence of the active fork with the collapsed fork, resulting in a double fork stall instead of a DSB. The double fork stall persists into mitosis where it results in an ultra-fine DNA bridge and mitotic DNA damage. htt //d i /10 1371/j l 1007942 005 Fig 5. Model for collapsed-fork rescue by NSMCE2. Normally (top part of model), after exposure to HU, uncoupling of the replicative helicase from the replicative DNA polymerase at the fork leads to production of ssDNA, which is immediately bound by ssDNA binding protein RPA (red circle). BLM (yellow circle) is one of the first factors to be recruited to the stalled replication fork and this accumulation of BLM is dependent on the NSMCE2 protein (green rectangle) on the SMC5/6 complex (purple ring). The localization of BLM at the stalled fork assists in maintaining functional RAD51 (teal rhombus) exchange during fork reversal. Given enough time in the reversed configuration, the fork is unable to restart (fork collapse). After resumption of DNA synthesis at dormant origins, an active fork converges upon the collapsed fork. The convergence is associated with RAD51 dissociation and a DSB at the collapsed fork, followed by DSB repair by HR via the canonical RAD51-mediated pathway. In NSMCE2-deficient cells (bottom part of model), BLM is no longer recruited to the stalled fork, and there is a failure in the regulation of RAD51 loading at the fork, shown here as a loss of dynamic exchange of RAD51 and RPA resulting in excess, hyper-stable RAD51. After resumption of DNA synthesis at dormant origins, the aberrant RAD51 structure prevents convergence of the active fork with the collapsed fork, resulting in a double fork stall instead of a DSB. The double fork stall persists into mitosis where it results in an ultra-fine DNA bridge and mitotic DNA damage. Fig 5. Model for collapsed-fork rescue by NSMCE2. Normally (top part of model), after exposure to HU, uncoupling of the replicative helicase from the replicative DNA polymerase at the fork leads to production of ssDNA, which is immediately bound by ssDNA binding protein RPA (red circle). BLM (yellow circle) is one of the first factors to be recruited to the stalled replication fork and this accumulation of BLM is dependent on the NSMCE2 protein (green rectangle) on the SMC5/6 complex (purple ring). The localization of BLM at the stalled fork assists in maintaining functional RAD51 (teal rhombus) exchange during fork reversal. Given enough time in the reversed configuration, the fork is unable to restart (fork collapse). After resumption of DNA synthesis at dormant origins, an active fork converges upon the collapsed fork. The convergence is associated with RAD51 dissociation and a DSB at the collapsed fork, followed by DSB repair by HR via the canonical RAD51-mediated pathway. In NSMCE2-deficient cells (bottom part of model), BLM is no longer recruited to the stalled fork, and there is a failure in the regulation of RAD51 loading at the fork, shown here as a loss of dynamic exchange of RAD51 and RPA resulting in excess, hyper-stable RAD51. After resumption of DNA synthesis at dormant origins, the aberrant RAD51 structure prevents convergence of the active fork with the collapsed fork, resulting in a double fork stall instead of a DSB. The double fork stall persists into mitosis where it results in an ultra-fine DNA bridge and mitotic DNA damage. Fig 5. Model for collapsed-fork rescue by NSMCE2. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 https://doi.org/10.1371/journal.pgen.1007942.g005 NSMCE2 and collapsed-fork rescue Our results here agree with previous results showing lower levels of SCEs in NSMCE2-defi- cient cells [42]. Hypomorphic NSMCE2 mutation in humans is associated with a syndrome characterized by short stature and acanthosis nigricans [32]. Cells derived from these patients display increased micronuclei, nuceloplasmic bridges at cytokinesis, and binucleated cells. Despite a defect in BLM localization at replication forks in patient cells, untreated cells have normal SCE levels, and UV treatment induces only a small increase in SCEs [32]. We found that cells deficient for both NSMCE2 and BLM exhibit reduced levels of HU-induced DSBs and SCEs, indicating that NSMCE2 is epistatic to BLM during collapsed-fork rescue. In con- trast to results with human cells, murine cells that are null for Nsmce2 exhibit increased SCEs, and Blm knockdowns in the murine Nsmce2 null cells display an additive increase in SCE lev- els [30]. The explanation for these different outcomes of NSMCE2 deficiency between humans and mice is unknown. We used HU to generate and study collapsed forks and to block repair-coupled DNA syn- thesis (e.g., break-induced replication, gap filling, lesion bypass, etc.). We therefore cannot rule out the possibility that NSMCE2 plays other roles during the unperturbed cell cycle or in situations where template switching can bypass DNA damage during replication, such as in cells treated with methyl methanesulfonate or UV irradiation. We observed higher levels of DSBs in untreated NSMCE2 null cells (S4 Fig), but observed no increase in basal SCE levels (Fig 4C). Our analysis using maRTA indicated that deficiency of NSMCE2 did not alter repli- cation dynamics in untreated cells, which is consistent with results reported in yeast [43]. We suggest that the increased DSBs in untreated NSMCE2-deficient cells may originate from incomplete replication at common fragile sites, leading to DNA breakage in mitosis and the formation of micronuclei in the next cell cycle. Micronuclei are known to be prone to replica- tion-associated DNA breakage [44]. HR-directed DSB repair is dependent on NSMCE2 [45, 46]; consequently, these breaks would most likely be repaired by non-homologous end joining. Studies in both mammalian cells [30, 47] and yeasts [48] indicate that HR can be increased in NSMCE2-deficient cells under some conditions, emphasizing the general complexity of NSCME2’s roles in maintenance of genomic integrity. The importance of mechanisms that regulate RAD51 protein levels is underscored by stud- ies that have identified increased RAD51 protein levels as a negative predictor of patient out- come in several cancer types [49]. The present work has uncovered a connection between NSMCE2 and the formation of DSBs at collapsed replication forks during rescue. The identifi- cation of NSMCE2 as a potential controller of HR-mediated fork rescue highlights NSMCE2’s potential as a new therapeutic target for combinatorial therapy of HR-dependent cancers. Because BLM localization to UFBs is not dependent on NSCME2, BLM could have a role in resolving RAD51-depene- dent intermediates in mitosis. Our findings that excess γ-H2AX accumulated on metaphase chromosomes but not during S phase suggest that under-replicated DNA is not resolved until G2/M phase. For example, prometaphase DNA repair [3] or mitotic resolvases [38] could dec- atenate under-replicated DNA to permit disjunction of inter-linked sister chromatids. We cannot rule out the possibility that the loss of NSMCE2 in cells affects the function of the SMC5/6 complex. However, in agreement with previous results in human U2OS and DT40 cells [39, 40], we found SMC5 levels were normal in NSMCE2-deficient HeLa cells (S2B Fig). These data suggest that, unlike in S. cerevisiae [41], SMC5 levels are not dependent on the presence of NSMCE2 in human cells. Whether human NSMCE2 plays a structural role in col- lapsed-fork rescue and other repair processes remains to be determined. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 13 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Genome editing for NSMCE2 Genome editing was carried out with Integrated DNA Technologies (IDT) ALT-R CRISPR-- Cas9 system with crRNA guide (GTCCATACCAGAGTTGATAC) targeting the first coding exon. Ribonucleoprotein particles were introduced into HEK293T cells using FuGENE HD (Promega). After 48 hours, single cells were deposited in a 96-well plate by flow cytometry. After four to six weeks, clones were analyzed by the T7 endonuclease assay (New England Bio- labs), and clones that scored positively were PCR sequenced. >50 clones were analyzed in the first screen and a single heterozygous NSMCE2 null mutant was obtained. This clone was genome edited again to obtain two NSMCE2 null mutants. Western blot analysis Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, and 50 mM Tris-HCl, pH 8.0) supplemented with 5 mM EDTA, 1 mM EGTA, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethane sulfonyl fluoride (PMSF) in 1x EDTA-free Halt protease inhibitor (Thermo Scientific). Protein concentration was measured using Pierce BCA Protein Assay. 30–50 μg of total protein from cell lysates was separated by electrophoresis through 4–20% gradient polyacrylamide gels and transferred onto Hybond nitrocellulose membranes by semi-dry transfer. Before addition of primary antibodies, mem- branes were probed with Ponceau S (Sigma) for 7 min, imaged, and washed with 1% glacial acetic acid in water. Membranes were blocked for 1 hour in Tris-buffered saline with 0.1% polysorbate 20 (TBST) containing 5% Bio-Rad Blotting-Grade Blocker, then incubated with primary antibody in 3% BSA in TBST overnight at 4˚C. 3 5’-rGrArUrCrGrGrCrArArCrUrArArArCrCrArUrUrArUrGrUdAdA-3’ siNSMCE2-6: 5’-rUrArUrArUrUrCrArCrUrArCrUrCrArCrUrUrCrArGrUrCrUrGrArC- 3’ 5’-rCrArGrArCrUrGrArArGrUrGrArGrUrArGrUrGrArArUrAdTdA-3’ siBLM: 5’-rArUrUrCrUrUrGrArGrArGrCrArGrUrArUrCrCrCrGrGrGrArUrU-3’ 5’-rUrCrCrCrGrGrGrArUrArCrUrGrCrUrCrUrCrArArGrAdAdAdT3’ siRAD51: 5’-rGrArGrCrUrUrGrArCrArArArCrUrArCrUrU-3’ 5’-rCrArCrCrUrUrGrArArGrUrArGrUrUrUrGrU-3’ NSMCE2 and collapsed-fork rescue 10; Cell Signaling; mouse monoclonal 9706). siRNAs for NSMCE2, BLM, and RAD51 were as follows: siNSMCE2-2: 5’-rUrUrArCrArUrArArUrGrGrUrUrUrArGrUrUrGrCrCrGrArUrCrCrA- 3’ 3’ 5’-rGrArUrCrGrGrCrArArCrUrArArArCrCrArUrUrArUrGrUdAdA-3’ siNSMCE2-6: 5’-rUrArUrArUrUrCrArCrUrArCrUrCrArCrUrUrCrArGrUrCrUrGrArC- PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Antibodies and siRNAs Antibodies for immunofluorescence and western blots were obtained as follows: anti-RPA2 (Abcam; mouse monoclonal ab2175), anti-RAD51 (Abcam; rabbit monoclonal ab133534), anti-NSMCE2 (OriGene; mouse monoclonal TA501632), anti-BLM [50], anti-RANGAP (Thermo Fisher Scientific; rabbit polyclonal PA1-5866), anti-histone H3 (Cell Signaling Tech- nology; rabbit polyclonal 9715), anti-PCNA (OriGene; mouse monoclonal TA800875), anti- SMC5 (Bethyl; rabbit polyclonal A300-236A), anti-HSP90 (OriGene; mouse monoclonal TA500494), γ-H2AX (BioLegend; 613406; or Upstate; mouse monoclonal 05–636), anti-bro- modeoxyuridine (BrdU) (Bio-Rad; mouse monoclonal OBT0030), anti-RPA p70 (Santa Cruz; mouse monoclonal SC-53497), anti-PML (Santa Cruz; mouse monoclonal SC-966), Phalloi- din-546 (Thermo Fisher Scientific; Alexa Fluor A22283), and anti-phospho-histone H3 (serine PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 14 / 25 NSMCE2 and collapsed-fork rescue urea, 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100, 2 mM β-mercaptoethanol). The beads were transferred to 1.5 ml microfuge tubes for one more wash with urea wash buffer and proteins were eluted for 1 hour at room temperature with elution buffer (63 mM Tris-HCl pH 6.8, 2% SDS, 200 mM imidazole, 1.5% β-mercaptoethanol, 10% glycerol, with bromophe- nol blue). The eluates were boiled for 5 min and cleared by centrifugation prior to loading on a 10% polyacrylamide gel. The whole cell lysates were lysed in RIPA buffer. Laemmli buffer was added to 1X in aliquots representing 10% of the eluate, then boiled for 5 min prior to gel loading. Analysis of chromatin-bound RPA The protocol from Mendez and Stillman for chromatin isolation by small-scale fractionation was followed [52]. HEK293T normal and NSMCE2 null cells treated or not with 2 mM HU for 16 hours were harvested by scraping, centrifuging, and washing twice with PBS. The cells were resuspended such that there were 1 x 107 cells per 200 μl of Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, and 1x Protease Inhibitor Cocktail). Triton X-100 was added to a concentration of 0.05%, and the cells were incubated for 5 min on ice. Nuclei were collected by low-speed centrifugation (4 min, 1300 x g at 4˚C) and the supernatant was reserved as the cytoplasmic fraction. The nuclei were washed once in Buffer A, and then lysed in 100 μl Buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 1x Protease Inhibitor Cocktail). Nucleoplasmic proteins were separated from chromatin-bound proteins by centrifugation (5 min, 1700 x g at 4 oC). Nucleoplasmic fractions were collected in the supernatant. The chromatin pellet was resuspended in 250 μl Laemmli buffer and the material was sonicated. The cytoplasmic and nucleoplasmic fractions were clari- fied by high-speed centrifugation (5 min, 20,000 x g at 4˚C). The proteins in the fractions were analyzed by Western blot. Initially, cytoplasmic and nucleoplasmic fractions were analyzed separately; however, because the cytoplasmic fraction contained varying amounts of different RPA components, for comparisons of the amounts of RPA70 we combined equal parts of the cytoplasmic and nucleoplasmic fractions. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 SUMO2 pull-down U2OS cells that were stably transfected with a His-tagged SUMO2 were kindly provided by Dr. Michael Matunis at Johns Hopkins University, who obtained them from Dr. Mary Dasso’s lab at NIH. The cells were reverse-transfected with siRNAs using LifeTechnologies’ Lipofecta- mine RNAiMAX. Cells were treated with 2 mM HU for 16 hours. His-SUMO2 conjugates were purified as described by Tatham et al. [51]. Briefly, cells were washed with ice-cold phos- phate-buffered saline (PBS) and directly lysed in 4 ml lysis buffer (6 M guanidine-HCl, 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 3 mM imidazole and 2 mM β-mercaptoethanol) and soni- cated to reduce viscosity. Lysates were incubated with 50 ml Talon Metal Affinity Resin (Clon- tech Laboratories, Inc.) overnight at 4˚C with gentle mixing, and then washed 2 times with 4 ml guanidine wash buffer (6 M guanidine-HCl, 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100, 2 mM β-mercaptoethanol) followed by 3 washes in 4 ml urea wash buffer (8 M PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 15 / 25 Cytogenetic analysis For SCE analyses, cells were cultured with 10 μM BrdU (Sigma-Aldrich). After 60 hours, the cells were incubated with 0.02 μg per ml colcemid (Invitrogen) for up to 2 hours, harvested and processed as described earlier [14]. For the epistasis experiments using HEK293T cells, the cells were incubated in 0.6 μg per ml colcemid for 16 hours prior to harvest. The slides were examined under the microscope at 100×, and SCEs were counted from metaphases with an acceptable quality of sister-chromatid discrimination. For measurements of HU-induced SCEs, cells were cultured in 10 μM BrdU for 30 hours, washed one time with 1× PBS, and treated with 2 mM HU for 24 hours. Cells were then released into medium containing 10 μM BrdU for an additional 20 hours. Metaphases were collected in colcemid and processed as described above. For analysis of micronuclei, normal or NSCME2-depleted HeLa cells were seeded onto cov- erslips, treated or not with 2 mM HU for 24 hours, and fixed with 4% formaldehyde. The cells were stained with Nuc-Blue (Thermo Fisher) at 2 drops per ml in PBS for 30 min, washed briefly, mounted, and imaged using the GE DeltaVision Elite High Resolution Micro. For analysis of chromosomes at metaphase, normal and NSMCE2 null HEK293T cells were seeded into 60 mm dishes and incubated overnight. For analysis of γ-H2AX labeled chromo- somes, cells were treated with 2 mM HU for 24 hours, then washed and incubated in medium with 7.5 μM RO-3306 for 10 hours for HEK293T control or 20 hours for NSMCE2 null cells. Cells were then released into normal medium and harvested at the indicated time points. Cells were fixed with 4% formaldehyde and stained with anti-phospho-histone H3 (serine 10). For analysis of chromosome spreads, cells were treated with 0.02 μg per ml colcemid for 45 min or with 0.6 μg per ml colcemid for 16 hours prior to harvest. Cells from both experiments were harvested and metaphases prepared as described [14]. Metaphase chromosomes were stained with Nuc-Blue. Cells or chromosomes were imaged using the GE DeltaVision Elite High-Reso- lution Microscope. NSMCE2 and collapsed-fork rescue to visualize the distribution of foci. For analysis of EdU labeled forks and ultra-fine bridges (UFBs), z-stacks were created using 100x objective and deconvolved using the GE DeltaVision Elite High Resolution Microscope. Images were analyzed and 3D representations were created using the NIS Elements software. to visualize the distribution of foci. For analysis of EdU labeled forks and ultra-fine bridges (UFBs), z-stacks were created using 100x objective and deconvolved using the GE DeltaVision Elite High Resolution Microscope. Images were analyzed and 3D representations were created using the NIS Elements software. Flow cytometry Cells were harvested with trypsin/EDTA, resuspended in ice cold PBS, and fixed and stained using BioLegend True-Nuclear Transcription Factor Buffer. Cells were then stained for γ- H2AX using directly conjugated antibody and counterstained using 7AAD to monitor cell DNA content. For cell-cycle assays, analysis was performed using the BD Pharmingen FITC BrdU Flow Kit. A minimum of 30,000 events was recorded for each group using the BD FACS- Canto II or BD LSR II flow cytometer. Apoptosis analysis was carried out using the BioLegend FITC Annexin V Apoptosis Detection Kit with propidium iodide (PI). Immunofluorescence and image analysis The protocol was adapted from Dimitrova and Gilbert [53]. Cells were grown on coverslips overnight and washed with cold CSK buffer (10 mM HEPES pH 7.4, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2) and nucleoplasm was extracted for 90 seconds with cold extraction buffer (0.5% Triton X-100 in CSK buffer with 1 mM PMSF, 50 mM sodium fluoride, 0.1 mM sodium orthovanadate and 1x EDTA free Halt protease inhibitor) prior to 30-min fixation in 4% formaldehyde at room temperature. Cells were washed twice with cold PBS then treated with 0.5% Triton X-100 at room temperature before staining. Cells were blocked at room tem- perature for 1 hour using sterile filtered 3% BSA in PBS, then probed using primary antibodies for 1 hour at room temperature. Secondary antibodies (Alexa Fluor 488 and 546) were used at 1:1000 for 45 min and nuclei were stained using Molecular Probes NucBlue reagent (R37606). For 5-ethynyl-2´-deoxyuridine (EdU) labeling, cells were incubated in 10 μM EdU for 20 min. EdU labeling and detection was performed using Life technologies Click-iT EdU Alexa Fluor 647 imaging kit according to manufacturer’s instructions. Cells were mounted in Molecular Probes ProLong Gold Antifade Reagent. Fixed and stained cells were imaged using the Leica SP5-II spectral confocal microscope using the 63x/1.4 NA PL Apo objective. Using the nuclear signal to mask the region of interest enabled accurate measurement of the number and inten- sity of nuclear foci and the percent of nucleus occupied by signal for each antibody target. No less than 10,000 foci were analyzed per experimental group. Box and whisker plots were used PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 16 / 25 Statistics Statistical evaluations of experiments with continuous variables (e.g., quantitation of SUMO-BLM and DSBs) or discrete variables in which the data was normal were carried out by paired Students t-test. Evaluations of experiments with non-normal discrete variables (e.g., focal counts) were analyzed by Mann-Whitney test. All p-values were two-sided. NSMCE2 and collapsed-fork rescue lauroyl sarcosinate, 0.2% wt/vol sodium deoxycholate, and 1 mg/ml proteinase K) at 37˚C for 16 hours. Plugs were then washed four times in 20 mM Tris-HCl, pH 8.0, and 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 21 hours at 14˚C in 0.9% (wt/vol) agarose containing Tris-borate/EDTA buffer in a PFGE apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol: block I: 9 hours, 120˚ included angle, 5.5 V/cm, 30 to 18-s switch; block II: 6 hours, 117˚ included angle, 4.5 V/cm, 18 to 9-s switch; block III: 6 hours, 112˚ included angle, 4.0 V/cm, 9 to 5-s switch. The gel was then stained with SYBR Gold (1 part in 10,000 in water; Invitrogen) and analyzed by the AlphaIma- ger system (ProteinSimple). Relative DSB levels were assessed by comparing DSB signals for each treatment to the background levels observed in untreated conditions using Image J. Data were analyzed with GraphPad Prism software. Microfluidic-assisted replication track analysis maRTA was performed as previously described with some modifications [31]. Briefly, 36 hours after siRNA transfection, HeLa cells were pulse-labeled with 50 μM iododeoxyuridine (IdU) for 40 min. Cells were then treated or not with 2 mM HU for 5 hours or 16 hours. The cells were released in fresh medium containing 50 μM of chlorodeoxyuridine (CldU) for 40 min. Cells were then harvested and embedded into agarose plugs containing 20,000 cells/plug. After proteinase K digestion and agarose digestion by beta-agarase, DNA fibers were stretched on 3-aminopropyltriethoxysilane coated slides (LabScientific) using polydimethylsiloxane molds fashioned with micro-capillary channels prepared as described [31]. DNA fibers were then denatured in 2.5 M HCl, and probed with the following antibodies: mouse IgG1 anti- BrdU/IdU (clone BD44, Becton Dickinson), rat anti-BrdU/CldU (clone B1/75, Bio-Rad OBT0030), and mouse IgG2a anti-ssDNA (clone 16–19, Millipore). Secondary antibodies included Alexa Fluor 488 anti-mouse IgG1, Alexa Fluor 594 anti-rat, and Alexa Fluor 647 anti- mouse IgG2a, respectively (Life Technologies). Images were acquired on Leica DMI6000 epi- fluorescence microscope using Leica LAS-AF software. Signals were measured using NIH Ima- geJ software with custom-made modifications and the data analyzed with GraphPad Prism software. DSB detection by pulsed-field gel electrophoresis The procedure was performed as previously described [8, 14]. Sub-confluent cultures of HeLa cells or HEK293T cells untreated, treated with 2 mM HU for 24 hours, or treated with HU for 24 hours and released into normal medium for different times were harvested by trypsiniza- tion. Agarose plugs of 2.5 x 105 cells were prepared in disposable plug molds (Bio-Rad Labora- tories). In some experiments, cells were released into medium containing 10 μM aphidicolin for 24 hours. Plugs were then incubated in lysis buffer (100 mM EDTA, 1% wt/vol sodium PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 17 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 NSMCE2 and collapsed-fork rescue NSMCE2+/+ cells is approximately 20 hours per division and of NSMCE2-/- cells 40 hours per division. (D) Flow cytometric analysis of the cell cycle. Cells were treated or not with 2 mM HU and then released (Rel) into normal medium for 6 hours. Cells were pulsed with 10 μM EdU prior to harvest and processing for flow cytometry. The NSCME2 null cells exhibit a mild G1 delay. Normal cells were pulsed with EdU for 20 min and NSMCE2 null cells were pulsed for 40 min to account for the slower cell cycle. (TIF) NSMCE2+/+ cells is approximately 20 hours per division and of NSMCE2-/- cells 40 hours per division. (D) Flow cytometric analysis of the cell cycle. Cells were treated or not with 2 mM HU and then released (Rel) into normal medium for 6 hours. Cells were pulsed with 10 μM EdU prior to harvest and processing for flow cytometry. The NSCME2 null cells exhibit a mild G1 delay. Normal cells were pulsed with EdU for 20 min and NSMCE2 null cells were pulsed for 40 min to account for the slower cell cycle. (TIF) S2 Fig. (A) Representative Western blots of HeLa cells transfected with control or two differ- ent siRNAs against NSMCE2 and treated or not with 2 mM HU for 24 hours. Multiple loading controls (HSP90) are shown for separate gel runs and Westerns of the same cell lysate. (B) Western blot analysis of SMC5. For SMC5 experiments, β-actin was used as a loading control. (TIF) S3 Fig. (A) Complementation of accumulation of BLM foci by transfection of siRNA-resistant NSMCE2 cDNA construct. HeLa cells were exposed to control or NSMCE2 siRNAs and were treated with 2 mM HU for 24 hours. Box and whisker plots represent distributions of the num- ber of BLM foci per cell. The median values are shown in boxes. At least 10,000 BLM foci were analyzed in each experimental condition. Three independent experiments were performed. (B) A representative image of the colocalization of RPA (red) and RAD51 (green) in HeLa cells exposed to 2 mM HU for 24 hours prior to fixation (upper panel). Quantitation of the area of RAD51 foci (lower panel). Mean and standard error are shown. At least 10,000 RAD51 foci were analyzed in each experimental condition. Three independent experiments were per- formed. (C) Colocalization of RAD51 and EdU in HU-treated cells. Supporting information S1 Fig. (A) 80% reduction of the levels of NSMCE2 after depletion with siRNA in HeLa cells as measured by Western blot and qPCR analysis. (B-D) Analysis of the construction of NSMCE2 null cells in the HEK293T cell line. (B) (Upper panel) Western blot analysis of nor- mal, heterozygous, and two cell clones (clone 9 and clone 15) that are null for NSMCE2. HSC70 was used as a loading control. The homozygous null cell clones were both derived from the heterozygous mutant of NSCME2 that carried a 10-bp deletion in exon 2 of NSMCE2. (Lower panel) Analysis by PCR and sequencing showed that clone 9 and clone 15 each contain the 10-bp deletion and a second clone-specific frameshift mutation. The sequence of the PAM site is denoted in blue and the sequence of the guide RNA is denoted in red. (C) Analysis by hemocytometer-based cell counting of cell proliferation of normal, heterozygous, and null cell clones of NSMCE2. The cell counting experiments indicated that the rate of proliferation of PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 18 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 Merged images of untreated or HU-treated, control- and NSMCE2-depleted HeLa cells stained with antibod- ies to BLM (red) and PML (green) and counter stained with DAPI. Depletion of NSMCE2 is associated with increased numbers of PML nuclear bodies. In HU-treated, control-depleted cells, BLM moves to stalled replication forks. In HU-treated NSMCE2-depleted cells, BLM remains associated with PML. (B) Reduced accumulation of RPA foci in HU-treated, d f ll f ll d h h ll d S5 Fig. (A) DSBs that accumulate after release from HU block are replication-dependent. HeLa cells were treated or not with 2 mM HU or 10 μM aphidicolin for 24 hours. The HU- treated cells were released into normal medium or medium that contained 10 μM aphidicolin for an additional 24 hours. The cells were analyzed by PFGE. Means and SD are shown. Three independent experiments were performed. (B) Similar cell-cycle distributions in HeLa cells transfected with control or NSMCE2 siRNA after release from HU block. Flow cytometric analysis of the cell cycle after HU treatment and release. 24 hours after transfection, cells were treated or not with 2 mM HU for 24 hours prior to release into normal medium for 40 min, 6 hours, or 12 hours. The cells were then pulsed with 20 μM EdU for 20 min prior to harvest and staining with click reagents. A minimum of 10,000 events were recorded in each experimental condition. (C) No induction of DSBs in NSMCE2 null cells treated with HU. Quantitation of PFGE analysis of HEK293T cells treated or not with 2 mM HU for 24 hours prior to release into normal medium for 12 hours (upper panel). The bar graph shows the mean fold change values, normalized to the untreated normal HEK293T mean, and SEM values from three inde- pendent experiments. A representative gel image of one experiment is shown below the graph (lower panel). (D) Similar levels of apoptosis in HeLa cells exposed to control and NSMCE2 siRNAs after treatment with 2 mM HU for 24 hours and release into normal medium for 24 hours. Analysis of apoptosis staining with propidium iodide and antibodies against AnnexinV. A minimum of 10,000 events were recorded in each experimental condition. (E) Reduced lev- els of γ-H2AX in HU-treated NSMCE2 null cells. Shaded histograms represent the treated cell popula- tions. Three independent experiments were performed. (K) Complementation of accumula- tion of γ-H2AX foci by transfection of siRNA-resistant NSMCE2 cDNA construct. Quantitative analysis of γ-H2AX foci (upper panel). Box and whisker plots represent distribu- tions of the number of γ-H2AX foci per cell. The median values are shown in boxes. At least 10,000 γ-H2AX foci were analyzed in each experimental condition. Below the bar graph are representative immunofluorescence images. Three independent experiments were performed. (TIF) Quantitative analysis of γ-H2AX foci (upper panel). Box and whisker plots represent distribu- tions of the number of γ-H2AX foci per cell. The median values are shown in boxes. At least 10,000 γ-H2AX foci were analyzed in each experimental condition. Below the bar graph are representative immunofluorescence images. Three independent experiments were performed. (TIF) S4 Fig. Low magnification images of cells analyzed for the indirect immunofluorescence experiments. (A) BLM is retained in PML nuclear bodies in NSMCE2-deficient cells and the numbers of BLM foci induced by HU are reduced in NSMCE2-deficient cells. Merged images of untreated or HU-treated, control- and NSMCE2-depleted HeLa cells stained with antibod- ies to BLM (red) and PML (green) and counter stained with DAPI. Depletion of NSMCE2 is associated with increased numbers of PML nuclear bodies. In HU-treated, control-depleted cells, BLM moves to stalled replication forks. In HU-treated NSMCE2-depleted cells, BLM remains associated with PML. (B) Reduced accumulation of RPA foci in HU-treated, NSMCE2-deficient cells. Images of HeLa cells stained with phalloidin, DAPI, anti-RPA p32 antibodies. Actin staining also revealed striking morphological changes in NSMCE2-deficient HeLa cells. (C) Over-accumulation of RAD51 foci in HU-treated, NSMCE2-deficient cells. Images of HeLa cells stained with DAPI and anti-RAD51 antibodies. (D) Reduced accumula- tion of BrdU foci in HU-treated, NSMCE2-deficient cells. Images are of HeLa cells stained with DAPI and anti-BrdU antibodies. Cells were incubated with 10 μM BrdU for 48 hours, treated with HU for 24 hours, then processed for immunofluorescence. (E) Excess accumula- tion of 53BP1 foci in HU-treated NSMCE2-deficient cells. Images of HeLa cells treated with HU for 24 hours then released for 24 hours and stained with DAPI and anti-53BP1 antibodies. (TIF) S4 Fig. Low magnification images of cells analyzed for the indirect immunofluorescence experiments. (A) BLM is retained in PML nuclear bodies in NSMCE2-deficient cells and the numbers of BLM foci induced by HU are reduced in NSMCE2-deficient cells. Representative images of control and NSMCE2-depleted HeLa cells exposed to 2 mM HU for 24 hours. EdU was incor- porated for 12 min prior to HU treatment. After HU, cells were fixed and stained with RAD51. Images show the merge of EdU (green) and RAD51 (red) channels. (D) Reduced accumulation of RPA foci in HU-treated, NSMCE2-deficient U2OS cells. Box and whiskers plot represent distributions of the number of RPA foci in cells exposed to control or NSMCE2 siRNA and treated or not with 2 mM HU for 24 hours. The median values are shown in boxes. Three inde- pendent experiments were performed. (E) Complementation of accumulation of RPA foci by transfection of siRNA-resistant NSMCE2 cDNA construct. HeLa cells were exposed to control or NSMCE2 siRNAs and treated with 2 mM HU for 24 hours. Box and whiskers plot represent the distributions of the number of RPA foci per cell. The median values are shown in boxes. Three independent experiments were performed. (F) Reduced accumulation of chromatin- bound RPA in HU-treated NSMCE2 null cells compared to HU-treated normal HEK293T cells. Western blot analysis of levels of chromatin-bound RPA (RPA p70 subunit). Cells were treated or not with 2 mM HU for 16 hours. The M fraction contains equal parts of the cyto- plasmic and nucleoplasmic fractions. The C fraction contains the chromatin-bound material. The red carets point to the HU-induced chromatin-bound RPA. Four independent experi- ments were performed. (G) Quantitation of the experiment shown in F. (H) Reduced levels of ssDNA in HU-treated NSMCE2-deficient cells. Quantitation of immunofluorescence analysis of BrdU to measure exposed ssDNA in non-denaturing conditions (left panel). HeLa cells were exposed to control or NSMCE2 siRNAs and treated or not with 2 mM HU for 24 hours. The bar represents median values of the numbers of BrdU foci and the error bar represent the SEM values from three independent experiments. Representative images of BrdU foci are shown (right panel). (I) Similar levels of SCEs in normal HEK293T cells and NSMCE2 null cells. Box and whiskers plots represent the numbers of SCEs per metaphase. A minimum of 14 metaphases were scored in two independent experiments. (J) Reduced levels of γ-H2AX in HU-treated, NSMCE2-deficient cells. Flow cytometric analysis of γ-H2AX response in HeLa PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 19 / 25 (B) Similar cell-cycle distributions in HeLa c transfected with control or NSMCE2 siRNA after release from HU block. Flow cytomet analysis of the cell cycle after HU treatment and release. 24 hours after transfection, cell treated or not with 2 mM HU for 24 hours prior to release into normal medium for 40 m hours, or 12 hours. The cells were then pulsed with 20 μM EdU for 20 min prior to harv staining with click reagents. A minimum of 10,000 events were recorded in each experim condition. (C) No induction of DSBs in NSMCE2 null cells treated with HU. Quantitati PFGE analysis of HEK293T cells treated or not with 2 mM HU for 24 hours prior to rel into normal medium for 12 hours (upper panel). The bar graph shows the mean fold ch values, normalized to the untreated normal HEK293T mean, and SEM values from thre pendent experiments. A representative gel image of one experiment is shown below the (lower panel). (D) Similar levels of apoptosis in HeLa cells exposed to control and NSM siRNAs after treatment with 2 mM HU for 24 hours and release into normal medium fo hours. Analysis of apoptosis staining with propidium iodide and antibodies against Ann A minimum of 10,000 events were recorded in each experimental condition. (E) Reduc els of γ-H2AX in HU-treated NSMCE2 null cells. Graph showing fold change in median of HU-induced γ-H2AX in normal HEK293T cells, the heterozygous NSMCE2+/- cells, NSMCE2 and collapsed-for cells. Mean and standard deviation is shown. To the right of the bar graph are representative histograms showing γ-H2AX induction. Shaded histograms represent the treated cell popula- tions. Three independent experiments were performed. (K) Complementation of accumula- tion of γ-H2AX foci by transfection of siRNA-resistant NSMCE2 cDNA construct. cells. Mean and standard deviation is shown. To the right of the bar graph are representative histograms showing γ-H2AX induction. Shaded histograms represent the treated cell popula- tions. Three independent experiments were performed. (K) Complementation of accumula- tion of γ-H2AX foci by transfection of siRNA-resistant NSMCE2 cDNA construct. Quantitative analysis of γ-H2AX foci (upper panel). Box and whisker plots represent distribu- tions of the number of γ-H2AX foci per cell. The median values are shown in boxes. At least 10,000 γ-H2AX foci were analyzed in each experimental condition. Below the bar graph are representative immunofluorescence images. Three independent experiments were performed. (TIF) histograms showing γ-H2AX induction. PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 NSMCE2 and collapsed-fork rescue cells. Mean and standard deviation is shown. To the right of the bar graph are represent histograms showing γ-H2AX induction. Shaded histograms represent the treated cell po tions. Three independent experiments were performed. (K) Complementation of accum tion of γ-H2AX foci by transfection of siRNA-resistant NSMCE2 cDNA construct. Quantitative analysis of γ-H2AX foci (upper panel). Box and whisker plots represent dis tions of the number of γ-H2AX foci per cell. The median values are shown in boxes. At 10,000 γ-H2AX foci were analyzed in each experimental condition. Below the bar graph representative immunofluorescence images. Three independent experiments were perfo (TIF) S4 Fig. Low magnification images of cells analyzed for the indirect immunofluoresce experiments. (A) BLM is retained in PML nuclear bodies in NSMCE2-deficient cells an numbers of BLM foci induced by HU are reduced in NSMCE2-deficient cells. Merged i of untreated or HU-treated, control- and NSMCE2-depleted HeLa cells stained with an ies to BLM (red) and PML (green) and counter stained with DAPI. Depletion of NSMC associated with increased numbers of PML nuclear bodies. In HU-treated, control-depl cells, BLM moves to stalled replication forks. In HU-treated NSMCE2-depleted cells, BL remains associated with PML. (B) Reduced accumulation of RPA foci in HU-treated, NSMCE2-deficient cells. Images of HeLa cells stained with phalloidin, DAPI, anti-RPA antibodies. Actin staining also revealed striking morphological changes in NSMCE2-de HeLa cells. (C) Over-accumulation of RAD51 foci in HU-treated, NSMCE2-deficient ce Images of HeLa cells stained with DAPI and anti-RAD51 antibodies. (D) Reduced accu tion of BrdU foci in HU-treated, NSMCE2-deficient cells. Images are of HeLa cells stain with DAPI and anti-BrdU antibodies. Cells were incubated with 10 μM BrdU for 48 hou treated with HU for 24 hours, then processed for immunofluorescence. (E) Excess accu tion of 53BP1 foci in HU-treated NSMCE2-deficient cells. Images of HeLa cells treated HU for 24 hours then released for 24 hours and stained with DAPI and anti-53BP1 anti (TIF) S5 Fig. (A) DSBs that accumulate after release from HU block are replication-dependen HeLa cells were treated or not with 2 mM HU or 10 μM aphidicolin for 24 hours. The H treated cells were released into normal medium or medium that contained 10 μM aphid for an additional 24 hours. The cells were analyzed by PFGE. Means and SD are shown. independent experiments were performed. NSMCE2 and collapsed-fork rescue the two NSMCE2-/- clones 9 and 15. Data were normalized to HU-treated normal HEK293T cells. Two independent experiments were performed. (TIF) the two NSMCE2-/- clones 9 and 15. Data were normalized to HU-treated normal HEK293T cells. Two independent experiments were performed. S6 Fig. (A) Persistence of RAD51 at collapsed replication forks in NSMCE2 null cells. Immu- nofluorescence analysis of HEK293T cells treated or not 2 mM HU for 24 hours prior to release for 2 hours, 4 hours, or 6 hours before staining, imaging, and quantitation. Each point on the graph represents the median value and the error bar represent SEM values from ran- domly binned averages of 10 cells from at least 50 cells in each experimental condition. Two independent experiments were performed. (B) Representative images of data shown in (A) showing co-localization of γ-H2AX, RPA, and RAD51 in HEK293T cells after 24 hours treat- ment with HU and release into normal medium for 2 hours. (TIF) S7 Fig. Mitotic damage in HU-treated NSMCE2-deficient cells. (A) Quantitative analysis of G1 arrest after release of control and NSMCE2-depleted HeLa cells from HU block into normal medium for 24 hours. (B) Quantitative analysis of abnormal anaphases encountered after release from HU block (left panel). HeLa cells were exposed to control or NSMCE2 siRNAs and treated or not with 2 mM HU for 24 hours, then released into fresh media for 16 hours before fixation and staining. The graph plots percent values of normal mitoses (blue), mitoses with anaphase bridges (red), and mitoses with lagging chromosomes (green). The value shown above the graph is the percent of abnormal mitosis scored. Representative images of normal mitosis (B’), mitosis with an anaphase bridge (red caret in B”), and mitosis with a lagging chromosome (red caret in B”‘) are shown (right panel). At least 100 anaphase cells were analyzed in each experimental condition. Three independent experiments were performed. (C) Quantitative analysis of micronuclei formation after release from HU block (left panel). Experiment was performed three times and analyzed by chi square test. A repre- sentative image is shown to the right of the graph. Scale bars represent 10 microns. (D) Quantitation of 53BP1 nuclear bodies/cell using bins of 1, 2, and 3 or more bodies/cell. Graph showing fold change in median levels of HU-induced γ-H2AX in normal HEK293T cells, the heterozygous NSMCE2+/- cells, and PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 20 / 25 HeLa cells were exposed to control or NSMCE2 siRNAs and treated with 2 mM HU for 24 hours, then released into fresh media for 24 hours before fixation and staining. Each bar represents the mean percent of total cells observed in each class and the error bars represent the SEM values. Flow cytometric analysis showed that the majority of cells are in G1 (see panel A). Cells with large nuclei indicative of being in the G2 phase were excluded from the quantita- tion. Three independent experiments were performed. (E) Quantitative analysis of area of the nucleus inhabited by 53BP1 signal after release from HU block based on the same data in panel D. Box and whisker plot represents area distribution per cell in at least 250 cells in each experimental condition. (F) Representative images of cells stained for 53BP1 analyzed in panels D and E. (G) Flow cytometric analysis of the effect of the treatment schedule to enrich for mitotic cells used in the experiments shown in Fig 3. HeLa cells were transfected with control or NSMCE2 siRNAs. After 24 hours, they were treated or not with 2 mM HU for 24 hours followed by release into medium containing 7.5 μM RO-3306 for 15 hours, followed by release into normal media for 1 hour. Cells were harvested, fixed, and stained with 7-AAD. 10,000 events were analyzed in each experimental condition. This experiment was performed two times. S7 Fig. Mitotic damage in HU-treated NSMCE2-deficient cells. (A) Quantitative analysis of G1 arrest after release of control and NSMCE2-depleted HeLa cells from HU block into normal medium for 24 hours. (B) Quantitative analysis of abnormal anaphases encountered after release from HU block (left panel). HeLa cells were exposed to control or NSMCE2 siRNAs and treated or not with 2 mM HU for 24 hours, then released into fresh media for 16 hours before fixation and staining. The graph plots percent values of normal mitoses (blue), mitoses with anaphase bridges (red), and mitoses with lagging chromosomes (green). The value shown above the graph is the percent of abnormal mitosis scored. Representative images of normal mitosis (B’), mitosis with an anaphase bridge (red caret in B”), and mitosis with a lagging chromosome (red caret in B”‘) are shown (right panel). At least 100 anaphase cells were analyzed in each experimental condition. Three independent experiments were performed. The tabulated data in each cell is described in the legend in tab. The abbreviations for column and row headings follow the main text. See figure leg for more details. (XLSX) Acknowledgments medium containing 7.5 μM RO-3306 for 10 hours (normal HEK293T) or 20 hours (NSMCE2 null) to block cells at the G2/M boundary, and then released into normal medium and har- vested at the indicated times for analysis of metaphase chromosomes. Regions of interest were drawn using Image J based on the DAPI signal in phospho-histone H3-positive (serine 10) cells and γ-H2AX signal was quantified. Two independent experiments were performed. (B) Representative images of metaphase chromosomes stained with γ-H2AX, anti-phospho-his- tone H3 and DAPI. (TIF) S9 Fig. Increased chromosome aberrations in untreated and HU-treated NSMCE2 null cells. (A) Representative images of metaphases prepared from normal HEK293T and NSMCE null cells. (B) Quantitation of chromosome aberrations identified in untreated and HU-treated normal HEK293T and NSMCE2 null cells. Representative chromosome images are shown below the grid of counts of chromosome aberrations. Total indicates the number of chromo- somes scored. 1, chromatid break. 2, chromosome break. 3, chromosome gap. 4, telomere fusion. 5, tri-radial. 6, quadriradial. 7, secondary constriction. Approximately 25 metaphases were analyzed from each of two experiments. (TIF) S10 Fig. RAD51 and NSMCE2 are epistatic with respect to HU-induced phenotypes. Analy- sis of HU-induced phenotypes in normal HEK293T and NSMCE2 null cells in which RAD51 levels were reduced or not by siRNA-mediated depletion. (A) Analysis by flow cytometry of the fluorescence intensity of γ-H2AX after treatment with HU for 24 hours followed by release into normal medium for 6 hours (Rel). The error bars represent the SD of median fluorescence intensity from a minimum of 10,000 events in five independent experiments. (B) Analysis by PFGE of DSBs after treatment with HU for 24 hours followed by release into medium for 6 hours (Rel). The bar graph represents the mean fold change in DSBs detected by PFGE nor- malized to untreated normal HEK293T cells exposed to control siRNA. The error bars repre- sent the SD of three independent experiments. The inset gel shows the results from one experiment. (C) A representative histogram of the median fluorescence intensity of γ-H2AX from one of the experiments shown in A. (D) Western analysis of RAD51 levels from samples prepared for one of the PFGE experiments shown in (B). (TIF) S1 Data. The underlying numerical data for each figure in the article is tabulated in each separate tab of the excel file. The tabs are labeled F1A for Fig 1A, F1C for Fig 1C, and so on. Image files are not included. The tabulated data in each cell is described in the legend in each tab. The abbreviations for column and row headings follow the main text. See figure legends for more details. (XLSX) (C) Quantitative analysis of micronuclei formation after release from HU block (left panel). Experiment was performed three times and analyzed by chi square test. A repre- sentative image is shown to the right of the graph. Scale bars represent 10 microns. (D) S8 Fig. Increased γ-H2AX signal in metaphase chromosomes in HU-treated NSMCE2 null cells. (A) Quantitation of median fluorescence intensity of γ-H2AX on phospho-histone H3-positive chromosomes. Cells were treated with 2 mM HU for 24 hours, released into 21 / 25 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1007942 February 8, 2019 NSMCE2 and collapsed-fork rescue medium containing 7.5 μM RO-3306 for 10 hours (normal HEK293T) or 20 hours (NSM null) to block cells at the G2/M boundary, and then released into normal medium and h vested at the indicated times for analysis of metaphase chromosomes. Regions of interes drawn using Image J based on the DAPI signal in phospho-histone H3-positive (serine cells and γ-H2AX signal was quantified. Two independent experiments were performed Representative images of metaphase chromosomes stained with γ-H2AX, anti-phospho tone H3 and DAPI. (TIF) S9 Fig. Increased chromosome aberrations in untreated and HU-treated NSMCE2 n cells. (A) Representative images of metaphases prepared from normal HEK293T and N null cells. (B) Quantitation of chromosome aberrations identified in untreated and HU- normal HEK293T and NSMCE2 null cells. Representative chromosome images are show below the grid of counts of chromosome aberrations. Total indicates the number of chro somes scored. 1, chromatid break. 2, chromosome break. 3, chromosome gap. 4, telome fusion. 5, tri-radial. 6, quadriradial. 7, secondary constriction. Approximately 25 metaph were analyzed from each of two experiments. (TIF) S10 Fig. RAD51 and NSMCE2 are epistatic with respect to HU-induced phenotypes. sis of HU-induced phenotypes in normal HEK293T and NSMCE2 null cells in which RA levels were reduced or not by siRNA-mediated depletion. (A) Analysis by flow cytometr the fluorescence intensity of γ-H2AX after treatment with HU for 24 hours followed by into normal medium for 6 hours (Rel). The error bars represent the SD of median fluore intensity from a minimum of 10,000 events in five independent experiments. (B) Analys PFGE of DSBs after treatment with HU for 24 hours followed by release into medium fo hours (Rel). The bar graph represents the mean fold change in DSBs detected by PFGE n malized to untreated normal HEK293T cells exposed to control siRNA. The error bars r sent the SD of three independent experiments. The inset gel shows the results from one experiment. (C) A representative histogram of the median fluorescence intensity of γ-H from one of the experiments shown in A. (D) Western analysis of RAD51 levels from sa prepared for one of the PFGE experiments shown in (B). (TIF) S1 Data. The underlying numerical data for each figure in the article is tabulated in separate tab of the excel file. The tabs are labeled F1A for Fig 1A, F1C for Fig 1C, and s Image files are not included. Author Contributions Author Contributions Conceptualization: Kelvin W. Pond, Nathan A. Ellis. Formal analysis: Kelvin W. Pond. Funding acquisition: Nathan A. Ellis. Investigation: Kelvin W. Pond, Christelle de Renty, Mary K. Yagle, Nathan A. Ellis. Methodology: Kelvin W. Pond, Christelle de Renty, Mary K. Yagle. Project administration: Nathan A. Ellis. Resources: Nathan A. Ellis. Writing – original draft: Kelvin W. Pond, Nathan A. Ellis. Writing – review & editing: Kelvin W. Pond, Christelle de Renty, Mary K. Yagle, Nathan A. Ellis. Investigation: Kelvin W. Pond, Christelle de Renty, Mary K. Yagle, Nathan A. Ellis. Methodology: Kelvin W. Pond, Christelle de Renty, Mary K. Yagle. Project administration: Nathan A. Ellis. Resources: Nathan A. Ellis. Writing – original draft: Kelvin W. Pond, Nathan A. Ellis. Writing – original draft: Kelvin W. Pond, Nathan A. Ellis. Writing – review & editing: Kelvin W. Pond, Christelle de Renty, Mary K. Yagle, Nathan A. Ellis. References 1. Gaillard H, Garcia-Muse T, Aguilera A. Replication stress and cancer. Nat Rev Cancer. 2015; 15: 276– 289. https://doi.org/10.1038/nrc3916 PMID: 25907220 2. Stubbe J, van der Donk WA. Ribonucleotide reductases: radical enzymes with suicidal tendencies. Chem Biol. 1995; 2: 793–801. PMID: 8807812 3. Ying S, Minocherhomji S, Chan KL, Palmai-Pallag T, Chu WK, Wass T, et al. MUS81 promotes com- mon fragile site expression. Nat Cell Biol. 2013; 15: 1001–1007. https://doi.org/10.1038/ncb2773 PMID: 23811685 4. Leung JW, Ghosal G, Wang W, Shen X, Wang J, Li L, et al. 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https://openalex.org/W2965571378	https://www.mdpi.com/2571-9408/2/3/139/pdf?version=1564748113	English	null	Post-Processing of VIS, NIR, and SWIR Multispectral Images of Paintings. New Discovery on the The Drunkenness of Noah, Painted by Andrea Sacchi, Stored at Palazzo Chigi (Ariccia, Rome)	Heritage	2,019	cc-by	7,452	Received: 30 June 2019; Accepted: 30 July 2019; Published: 2 August 2019 Abstract: IR Reﬂectography applied to the identiﬁcation of hidden details of paintings is extremely useful for authentication purposes and for revealing technical hidden features. Recently, multispectral imaging has replaced traditional imaging techniques thanks to the possibility to select speciﬁc spectral ranges bringing out interesting details of the paintings. VIS–NIR–SWIR images of one of the The Drunkenness of Noah versions painted by Andrea Sacchi, acquired with a modiﬁed reﬂex and InGaAs cameras, are presented in this research. Starting from multispectral images we performed post-processing analysis, using visible and infrared false-color images and principal component analysis (PCA) in order to highlight pentimenti and underdrawings. Radiography was performed in some areas to better investigate the inner pictorial layers. This study represents the ﬁrst published scientiﬁc investigation of The Drunkenness of Noah’s artistic production, painted by Andrea Sacchi. Keywords: paintings; Andrea Sacchi; multispectral imaging; radiography; reﬂectography; false-color image; PCA Lucilla Pronti 1,,† , Martina Romani 1,,†, Gianluca Verona-Rinati 2,,†, Ombretta Tarquini 3,,†, Francesco Colao 4,†, Marcello Colapietro 3,†, Augusto Piﬀeri 3,† , Mariangela Cestelli-Guidi 1,† and Marco Marinelli 2,† Lucilla Pronti 1,,† , Martina Romani 1,,†, Gianluca Verona-Rinati 2,,†, Ombretta Tarquini 3,,†, Francesco Colao 4,†, Marcello Colapietro 3,†, Augusto Piﬀeri 3,† , Mariangela Cestelli-Guidi 1,† and Marco Marinelli 2,† 1 INFN-Laboratori Nazionali di Frascati, Via E. Fermi 40, 00044 Frascati, Italy y 2 INFN-Dipartimento di Ingegneria Industriale, Università degli Studi di Roma Tor Vergata, Department of Industrial Engineering, Via del Politecnico 1, 00133 Roma, Italy 3 C.N.R. Istituto di Cristallograﬁa - Area della Ricerca Roma 1, Via Salaria Km 29300, 00015 Monterotondo (RM), Italy y 4 ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Via E. Fermi 45, 00044 Frascati (RM), Italy 4 ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Via E. Fermi 45, 00044 Frascati (RM), Italy * Correspondence: lucilla.pronti@lnf.infn.it (L.P.); martina.romani@lnf.infn.it (M.R.); gianluca.verona.rinati@uniroma2.it (G.V.-R.); ombretta.tarquini@mlib.ic.cnr.it (O.T.) † These authors contributed equally to this work. † These authors contributed equally to this work. heritage heritage heritage heritage www.mdpi.com/journal/heritage 1. Introduction The early systems used PbO–PbS vidicon cameras with sensitivities up to 2200 nm; more suitable systems use InGaAs or PtSi detectors with a broader sensitivity. Usually, IR reﬂectography is carried out using a long-pass ﬁlter (in order to remove the UV-VIS portions of the spectrum) and capturing the IR image in a single large band. The acquired information is integrated over the broad spectral range instead of being obtained in a spectral narrow band. For this reason, over the last decade, multispectral imaging has gained importance in the cultural heritage ﬁeld, since it allows to select a speciﬁc IR portion in which the painted layers present low absorbance and granting a better visualization of the background layers [5,6]. Multispectral imaging was developed 40 years ago and its use was mainly restricted to astrophysics, remote sensing, and terrestrial military applications until the 1990s. This technology has undergone a rapid development during the last 20 years and has been applied in many research ﬁelds, such as the remote sensing of Earth’s atmosphere [7], the detection of contaminants on food [8], the analysis of plants and fruits [9], and the diagnostic of cancer tissue [10]. Multispectral imaging permits to acquire a set of two-dimensional images at diﬀerent wavelengths within a given spectral range. In this way, spatial and spectral information are simultaneously recorded: The light intensity is registered as a function of both location (pixel of the image) and wavelength. For the sake of the good order, another technique which is based on the same principles should be mentioned: ‘Hyperspectral imaging. This technique diﬀers from multispectral imaging because of the number of spectral images produced: Less than 12 in the multispectral imaging and much more in the hyperspectral system [11]. The post-processing represents an important step for a proper interpretation of multispectral images. Indeed, the development of digital acquisition procedures has permitted post-processing of raw images by the application of typical digital image processing algorithms, such as contrast stretching and luminance transformation ﬁlters (gamma, logarithmic, etc.). However, thanks to the modern image processing it is possible to achieve a more accurate radiometric calibration so that all images are corrected by the spectral sensitivity of the camera sensor and the spectral transmittance of optical lens and ﬁlters. 1. Introduction Infrared reﬂectography has been widely employed as preliminary investigation of paintings to evaluate the state of conservation, reveal underdrawings and provide similar and/or complementary information to the radiography [1,2]. The possibility to visualize the underlying drawing is related to the fact that many pigments result to be transparent to IR radiation in the infrared region of the electromagnetic spectrum. For this reason, a high contrast between the materials used for the drawing, like carbon black or black bone, and the preparation layer, consisting of chalk or white lead, is obtained. In some cases, infrared reﬂectography does not show underdrawings, but pictorial spreads that belong to a previous idea of the painting composition. These pictorial layers and changings in the drawing, which is concealed under the ﬁnal pictorial layer, are hidden to an external observer and could be Heritage 2019, 2, 2275–2286; doi:10.3390/heritage2030139 www.mdpi.com/journal/heritage www.mdpi.com/journal/heritage 2276 Heritage 2019, 2 deﬁned as pentimenti. Their visualization permits to acquire an important knowledge of the evolution of the artist’s compositional project, revealing the mental process behind the ﬁnal appearance of the painting [3]. Usually, copies do not show signiﬁcant pentimenti due to the fact that the painter replies a model. As a matter of fact, reﬂectograms could highlight the grids used to transpose the original painting to a diﬀerent scale [4]. This means that the studies of the underdrawings or pentimenti and the sketches, concerning a series of paintings and representing the same subject, are extremely important to determinate their temporal execution. Furthermore, the discovery of pentimenti can help to establish the originality of an artwork since a forgery often represents a faithful copy of the original or, at least, is realized without relevant changes. The infrared reﬂectography is performed by illuminating the painting with infrared radiation and collecting the infrared radiation backscattered from the painting with a dedicate detector. The less expensive acquisition system consists of two light sources, which uniformly illuminate the painting surface and a digital camera equipped with diﬀerent ﬁlters to take images in diﬀerent spectral range. The IR reﬂectography can be performed by using several types of cameras with diﬀerent image sensors. Cameras for NIR (Near Infrared Reﬂectography) imaging use silicon-based sensors, which can detect photons up to 1100 nm (even if the signal to noise ratio is very low at these wavelengths), while the other ones are properly projected for applications in the IR ranges. 1. Introduction Starting from calibrate images, it is possible to perform several post-processing procedures as false-color images and chemometric image analysis (PCA, PLS, K-means, etc.), in order to emphasize spectral features which are not evident in the raw data set [12–15]. The most traditional post-processing technique is the so called false-color image, which is a rendering method used to display colored images recorded in the visible and IR regions. Traditional false-color photography has been comprehensively used as a guide for identiﬁcation of pigments. The experimental procedure requires the combination of RGB color images and IR reﬂectogram, in which 2277 Heritage 2019, 2 the image taken in the green range of the electromagnetic spectrum (centered at about 500 nm) replaces the blue image (B), the image taken in the red range (centered at about 600 nm) replaces the green image (G), and the IR reﬂectogram substitutes the red image (R) [16–19]. This approach is very useful to recognize pigments which have large diﬀerences in the IR absorbance, although they appear similar to the eye (i.e., lapis lazuli shows a red false-color with respect to the much less expensive azurite that looks blue [20]). However, since any combination of three diﬀerent images (not mandatorily corresponding to the ones just mentioned) could be deﬁned as “False-color image”, new perspectives are open to image processing, not necessarily involving IR range but aiming to underline diﬀerent spectral features even in the visible range [21]. The second step is applying suitable chemometric approaches which have proven to be powerful tools to reduce data dimensionality, detecting hidden patterns also removing noise and redundant correlated data [22,23]. One of the most used chemometric methods in the analysis of paintings is the principal component analysis (PCA) that is a multivariate linear method which allows to represent data in a set of new loading, called principal components [24]. PCA is applied to the diagnostic imaging of paintings mainly for the recognition of pictorial materials [25–27] or to identify pictorial drafting and restoration works [28,29]. The new frontier for the analysis of multimodal images is the multisensor data fusion, that is a technology to enable combining information from several sources in order to form a uniﬁed data set to be processed [30]. 1. Introduction Paintings are good objects to apply data fusion technique due to the fact that their surface heterogeneities and complex stratigraphies required diﬀerent analytical methods, experimental set ups and devices to be analyzed [31–33]. In this research, false-color images and PCA of data fusion of visible and IR images of the painting The Drunkenness of Noah painted by Andrea Sacchi is presented. Some areas were compared with the radiography images which represent the complementary imaging technique par excellence. The importance of applying VIS–NIR–SWIR reﬂectrography, radiography, and post-processed imaging on this painting is related to the fact that it could be the ﬁrst version of the four oil paintings representing Drunkenness of Noah painted by Andrea Sacchi. This study represents the starting point for the scientiﬁc investigation of these paintings through the observations of pentimenti and the stylistic, artistic, and documental support of the conservators and humanistic researchers, with the aim to deﬁne which painting of them represents the ﬁrst version. D7100) equipped with a Si sensor, in the M Pi l Th i i d ith f 2.2. VIS–NIR–SWIR Multispectral Imaging MegaPixel. The camera is equipped with four Edmund Optics filters: One IR cut and three long pass filters above 780 nm, 830 nm, and 1000 nm. SWIR camera (XENICS “Xeva–1.7–640’’) has an InGaAs sensor with the spectral range at 900–1700 nm, a resolution of 640 × 512, a pixel pitch of 20 µm and an array cooling (TE1-cooled down to 263 K). All the multispectral images were properly radiometrically calibrated: For each acquisition, an image of the painting with a reflectance standard (spectralon white diffuse reflectance standard, Edmund Optics) was recorded Then each image was normalized to the spectral energy reflected in The VIS and NIR multispectral imaging were performed with a NIR converted camera (Nikon D7100) equipped with a Si sensor, in the spectral range of 370–1100 nm, with a resolution of 24.1 MegaPixel. The camera is equipped with four Edmund Optics ﬁlters: One IR cut and three long pass ﬁlters above 780 nm, 830 nm, and 1000 nm. SWIR camera (XENICS “Xeva–1.7–640”) has an InGaAs sensor with the spectral range at 900–1700 nm, a resolution of 640 × 512, a pixel pitch of 20 µm and an array cooling (TE1-cooled down to 263 K). Edmund Optics) was recorded. Then, each image was normalized to the spectral energy reflected in the acquisition band by the reflectance standard, in order to have images corrected by both the spectral sensitivity of the cameras and the spectral transmittance of the optical lens and the filters. To illuminate, we used two halogen bulbs (Uniflood 300 W, 3350 K, Cosmolight) placed at 45 degrees with respect to the normal of the painted surface. The images were registered by using ImageJ (an open source image processing software) and post-processed by using MATLAB 2017a integrated by Hypertools (Free Graphical User Interface for Hyperspectral Image Analysis) The VIS–NIR–SWIR images were also stacked in a multispectral cube All the multispectral images were properly radiometrically calibrated: For each acquisition, an image of the painting with a reﬂectance standard (spectralon white diﬀuse reﬂectance standard, Edmund Optics) was recorded. Then, each image was normalized to the spectral energy reﬂected in the acquisition band by the reﬂectance standard, in order to have images corrected by both the spectral sensitivity of the cameras and the spectral transmittance of the optical lens and the ﬁlters. 2.1. The Painting The Drunkenness of Noah represents a biblical scene (Genesi 9, 20–27) in which Noah is sleeping naked after getting drunk on his wine. His third son, Ham, saw his father unclothed and showed it to his two brothers (Shem and Japhet). They put a wrap on their backs and reclined it to cover his father with their faces turned away. The painting analyzed in this research is an oil on canvas (150.5 by 205.8 cm) and one of the four observable versions. This is stored in Palazzo Ghigi in Ariccia (Rome) and it is shown in Figure 1. It belongs to a British private collection and it was purchased from Sotherby’s (New York) in 2009. The other versions painted by the same artist are stored in: (1) Provincial Museum of Catanzaro (Italy), (2) Staatliche Museum of Berlin (Germany), (3) Kunsthistorisches Museum of Vienna (Austria) [34]. 2278 Heritage 2019, 2 (a) (b) Figure 1. The painting The Drunkenness of Noah placed at Palazzo Chigi (Ariccia) before (a) and after (b) restorations. Figure 1. The painting The Drunkenness of Noah placed at Palazzo Chigi (Ariccia) before (a) and after (b) restorations. (a) (b) (b) (a) Figure 1. The painting The Drunkenness of Noah placed at Palazzo Chigi (Ariccia) before (a) and after (b) restorations. Figure 1. The painting The Drunkenness of Noah placed at Palazzo Chigi (Ariccia) before (a) and after (b) restorations. 2.2. VIS–NIR–SWIR Multispectral Imaging The VIS and NIR multispectral imaging were performed with a NIR converted camera (Nikon The painting placed at Palazzo Chigi in Ariccia was restored in 2010 by removing ancient varnishes and repainting, which were realized to cover the Noah’s intimate parts, as showed in Figure 1a. 2.2. VIS–NIR–SWIR Multispectral Imaging The VIS and NIR multispectral imaging were performed with a NIR converted camera (Nikon The painting placed at Palazzo Chigi in Ariccia was restored in 2010 by removing ancient varnishes and repainting, which were realized to cover the Noah’s intimate parts, as showed in Figure 1a. D7100) equipped with a Si sensor, in the M Pi l Th i i d ith f 2.2. VIS–NIR–SWIR Multispectral Imaging To illuminate, we used two halogen bulbs (Uniﬂood 300 W, 3350 K, Cosmolight) placed at 45 degrees with respect to the normal of the painted surface. Hyperspectral Image Analysis). The VIS–NIR–SWIR images were also stacked in a multispectral cube and then combined in a false-color image (called also raw data). We performed PCA on the multispectral image cube, which is a chemometric processing method that carries out a linear transformation which decorrelates multivariate data by translating and/or rotating the axes of the original space. In this way, the data can be represented without any correlation in a new component space. Each component explains a certain percentage of the variance: The first component (PC1) explains the maximum variance, the second component (PC2) another consistent part. and so on, until reaching the 100% of the explained variance. 2.3. Radiography Th R di h f d i h X d I Pl d (IP) The images were registered by using ImageJ (an open source image processing software) and post-processed by using MATLAB 2017a integrated by Hypertools (Free Graphical User Interface for Hyperspectral Image Analysis). The VIS–NIR–SWIR images were also stacked in a multispectral cube and then combined in a false-color image (called also raw data). We performed PCA on the multispectral image cube, which is a chemometric processing method that carries out a linear transformation which decorrelates multivariate data by translating and/or rotating the axes of the original space. In this way, the data can be represented without any correlation in a new component space. Each component explains a certain percentage of the variance: The ﬁrst component (PC1) explains the maximum variance, the second component (PC2) another consistent part. and so on, until reaching the 100% of the explained variance. g with a reading sy 2.3. Radiography × 24 cm, 16 bit gray scale, and a resolution of 600 dpi. The operating conditions were 38 kV and 1 MAS. 3. Results and Discussion The Radiography was performed with a tungsten X-ray source and an Image Plate detector (IP) with a reading system (Kodak CR7400) that allows obtaining digital image with the dimension of 18 × 24 cm, 16 bit gray scale, and a resolution of 600 dpi. The operating conditions were 38 kV and 1 MAS. 2279 Heritage 2019, 2 3. Results and Discussion Several pentimenti were detected by capturing NIR and SWIR multispectral images, as shown in Figure 2. By acquiring images at diﬀerent spectral ranges, the readability of underdrawings is increased because of the fact that each pictorial layer is diﬀerently transparent in the IR range. Since it is impossible to know a priori of the whole chemical composition of the painting, multispectral imaging approach is always recommended. For each IR multispectral image, we can already recognize diﬀerences with the visible image by a point-to-point comparison. However, this approach results as a slow method and it depends on the capacity to ﬁnd changes in gray scale images. Thus, in order to better emphasize diﬀerences between the visible image and infrared ones, we generated a false RGB by replacing the red channel (R) with the image obtained from the InGaAs image, the green channel (G) with the image acquired by the Si sensor with the longpass ﬁlter at 1000 nm, and the blue channel (B) acquired by the Si sensor with the IR cut ﬁlter image. Heritage 2019, 2 FOR PEER REVIEW 5 imaging approach is always recommended. For each IR multispectral image, we can already recognize differences with the visible image by a point-to-point comparison. However, this approach results as a slow method and it depends on the capacity to find changes in gray scale images. Thus, in order to better emphasize differences between the visible image and infrared ones, we generated a false RGB by replacing the red channel (R) with the image obtained from the InGaAs image, the green channel (G) with the image acquired by the Si sensor with the longpass filter at 1000 nm, and the blue channel (B) acquired by the Si sensor with the IR cut filter image. (a) (b) (c) (d) Figure 2. Gray scale rendering of the reflectance images of the painting, taken with longpass filter at 780 nm (a), 830 nm (b), 1000 nm (c), and with the InGaAs camera (d). Figure 2. Gray scale rendering of the reﬂectance images of the painting, taken with longpass ﬁlter at 780 nm (a), 830 nm (b), 1000 nm (c), and with the InGaAs camera (d). (b) (a) (b) (a) ( ) (c) ( ) (d) (d) (c) Figure 2. 3. Results and Discussion Gray scale rendering of the reflectance images of the painting, taken with longpass filter at 780 nm (a), 830 nm (b), 1000 nm (c), and with the InGaAs camera (d). Figure 2. Gray scale rendering of the reﬂectance images of the painting, taken with longpass ﬁlter at 780 nm (a), 830 nm (b), 1000 nm (c), and with the InGaAs camera (d). The resulting image (Figure 3) shows some dark blue areas corresponding to the restoration integrations, see Figure 1b, appearing as lines in the bottom part of the painting. Other dark blue areas regard portions of the painting that were subjected to the removal of repainting. This means that the restauration materials used have low reflectivity under IR radiation. The resulting image (Figure 3) shows some dark blue areas corresponding to the restoration integrations, see Figure 1b, appearing as lines in the bottom part of the painting. Other dark blue areas regard portions of the painting that were subjected to the removal of repainting. This means that the restauration materials used have low reﬂectivity under IR radiation. Heritage 2019, 2 2280 Figure 3. False-color image obtained by the combination of the image taken with the IR cut filter (B), longpass filtered image (G), and the InGaAs image (R). Figure 3. False-color image obtained by the combination of the image taken with the IR cut ﬁlter (B), longpass ﬁltered image (G), and the InGaAs image (R). Figure 3. False-color image obtained by the combination of the image taken with the IR cut filter (B) longpass filtered image (G), and the InGaAs image (R). Figure 3. False-color image obtained by the combination of the image taken with the IR cut ﬁlter (B), longpass ﬁltered image (G), and the InGaAs image (R). Some pentimenti or not-visible aspects of the pictorial composition are colored in dark blue, too. Small pentimenti are related to the position of arms, legs, foot, and hands of all the ﬁgures represented. The false-color image highlights some details near the grape leaves and shadows of the rock on which Noah is lying down which are diﬃcult to interpret. Drapes and clothes present some light changings, except for Ham’s dress that shows another proﬁle on the left side. 3. Results and Discussion The images obtained by PCA are expected to better highlight small diﬀerences appearing in the images acquired in partially overlapped spectral windows, since the PCA components are obtained by simultaneously 2281 Heritage 2019, 2 considers all the acquired data, leaving out spectral correlations and highlighting the contrasts between spectral bands. Figure 3. False-color image obtained by the combination of the image taken with the IR cut filter (B), longpass filtered image (G), and the InGaAs image (R). Figure 4. The Drunkenness of Noah drawing by Andrea Sacchi, Metropolitan Museum of New York (Accession Number: 1977.168). Figure 4. The Drunkenness of Noah drawing by Andrea Sacchi, Metropolitan Museum of New York (Accession Number: 1977.168). eritage 2019, 2 FOR PEER REVIEW btained from single components coded as color grade are also shown in Figure 5b–d. It resulted th he PC1 explains approximately 94.5% of the variability, while PC2 and PC3 represent about 3.5 nd 1.5%. The images obtained by PCA are expected to better highlight small differences appeari n the images acquired in partially overlapped spectral windows, since the PCA componen re Figure 4. The Drunkenness of Noah drawing by Andrea Sacchi, Metropolitan Museum of New York (Accession Number: 1977.168). Figure 4. The Drunkenness of Noah drawing by Andrea Sacchi, Metropolitan Museum of New York (Accession Number: 1977.168). d 1.5%. The images obtained by PCA are expected to better highlight small differences appear the images acquired in partially overlapped spectral windows, since the PCA compone False-color with only three images is a reduc nalysis of post-processed images. A significant im ata is obtained by stacking multibands VIS, erforming additional processing through Hypert In order to improve the readability of the pe nalysis (PCA) of the entire set of multiple im ecomposition (SVD). A false-color image (called hree components of the entire set of VIS–NIR–SW (a) False-color with only three images is a reductive approach, as it cannot produce an exhaustive nalysis of post-processed images. A significant improvement in the ability to analyze multispectral ata is obtained by stacking multibands VIS, NIR, and SWIR images in a spectral cube and erforming additional processing through Hypertools (a free Matlab GUI). In order to improve the readability of the pentimenti, we computed the principal component nalysis (PCA) of the entire set of multiple images after mean centering and singular values ecomposition (SVD). 3. Results and Discussion Three important pentimenti were found in the ﬁgures representing the three sons of Noah: (1) The face of the son placed on the left side was shifted, in fact we, can recognize another proﬁle in the false-color image; (2) the son placed in the background right side shows short hair in the reﬂectograms. This is an important aspect that matches with the red chalk drawing placed at the Metropolitan Museum attribute to Andrea Sacchi, showed in Figure 4; (3) the son in the foreground shows two faces: The older version seems smaller than the last one. Furthermore, Ham’s dress shows another proﬁle on the left side. False-color with only three images is a reductive approach, as it cannot produce an exhaustive analysis of post-processed images. A signiﬁcant improvement in the ability to analyze multispectral data is obtained by stacking multibands VIS, NIR, and SWIR images in a spectral cube and performing additional processing through Hypertools (a free Matlab GUI). Figure 4. The Drunkenness of Noah drawing by Andrea Sacchi, Metropolitan Museum of New York (Accession Number: 1977.168). False-color with only three images is a reductive approach, as it cannot produce an exhaustive analysis of post-processed images. A significant improvement in the ability to analyze multispectral data is obtained by stacking multibands VIS, NIR, and SWIR images in a spectral cube and performing additional processing through Hypertools (a free Matlab GUI). In order to improve the readability of the pentimenti, we computed the principal component a aly i (PCA) of the e ti e et of ulti le i a e afte ea e te i a d i ula alue In order to improve the readability of the pentimenti, we computed the principal component analysis (PCA) of the entire set of multiple images after mean centering and singular values decomposition (SVD). A false-color image (called also raw data) was obtained by combining the ﬁrst three components of the entire set of VIS–NIR–SWIR in a RGB image (Figure 5a). The score images obtained from single components coded as color grade are also shown in Figure 5b–d. It resulted that the PC1 explains approximately 94.5% of the variability, while PC2 and PC3 represent about 3.5% and 1.5%. 3. Results and Discussion A false-color image (called also raw data) was obtained by combining the first hree components of the entire set of VIS–NIR–SWIR in a RGB image (Figure 5a). The score images (a) (b) (c) (d) Figure 5. False-color image obtained from the first three components of the processed multispectral data cube (a), intensity color coded images form principal component analysis (PCA) components are shown respectively as PC1 (b), PC2 (c), and PC3 (d). h l l f h h hl h d h f l l Figure 5. False-color image obtained from the ﬁrst three components of the processed multispectral data cube (a), intensity color coded images form principal component analysis (PCA) components are shown respectively as PC1 (b), PC2 (c), and PC3 (d). ve approach, as it cannot produce an exhaustive provement in the ability to analyze multispectral IR, and SWIR images in a spectral cube and ols (a free Matlab GUI). timenti, we computed the principal component ges after mean centering and singular values so raw data) was obtained by combining the first R in a RGB image (Figure 5a). The score images (b) (b) (a) (d) (c) (d) (c) Figure 5. False-color image obtained from the first three components of the processed multispectral data cube (a), intensity color coded images form principal component analysis (PCA) components are shown respectively as PC1 (b), PC2 (c), and PC3 (d). Figure 5. False-color image obtained from the ﬁrst three components of the processed multispectral data cube (a), intensity color coded images form principal component analysis (PCA) components are shown respectively as PC1 (b), PC2 (c), and PC3 (d). 2282 Heritage 2019, 2 Figure 5. F The analysis reveals most of the pentimenti highlighted in the previous false-color image (see Figure 3). However, since the processing is not supervised, hidden details well highlighted in the IR images are occasionally not so well evidenced. For example, the two faces’ sketches of the Noah’s son in the foreground are better identiﬁed in the original InGaAs image, as shown in the detail in Figure 6 rather than in the PCA images. Nevertheless, it is worth mentioning that none of the spectral information contained in the original data set is lost by the PCA processing. shown respectively as PC1 (b), PC2 (c), and PC3 (d). The analysis reveals most of the pentimenti highlighted in the previous false-color image (see Figure 3). 3. Results and Discussion However, since the processing is not supervised, hidden details well highlighted in the IR images are occasionally not so well evidenced. For example, the two faces’ sketches of the Noah’s son in the foreground are better identified in the original InGaAs image, as shown in the detail in Figure (a) (b) Heritage 2019, 2 FOR PEER REVIEW 8 (c) (d) Figure 6. Detail of the painting. InGaAs image (a), PC1 (b), PC2 (c), and PC3 (d). On the other hand, PC2 evidences few details not clearly identified in the InGaAs image: Here, Figure 6. Detail of the painting. InGaAs image (a), PC1 (b), PC2 (c), and PC3 (d). On the other hand, PC2 evidences few details not clearly identiﬁed in the InGaAs image: Here, h h f h l f ’ f h d h h (a) eritage 2019, 2 FOR PEER REVIEW (b) 8 (b) (a) (d) (c) (d) (c) Figure 6. Detail of the painting. InGaAs image (a), PC1 (b), PC2 (c), and PC3 (d). Figure 6. Detail of the painting. InGaAs image (a), PC1 (b), PC2 (c), and PC3 (d). On the other hand, PC2 evidences few details not clearly identified in the InGaAs image: Here, the chin of the earlier version of Ham’s face seems to have undergone changes, as shown in Figure 7. On the other hand, PC2 evidences few details not clearly identiﬁed in the InGaAs image: Here, the chin of the earlier version of Ham’s face seems to have undergone changes, as shown in Figure 7. To complete the investigation of the pentimenti, we performed radiographic images in the three most interesting parts of the paintings: Noah’s sons heads (Figure 8). The radiographic images showed the same pentimenti already seen in the post-processed VIS—NIR–SWIR images. Moreover, On the other hand, PC2 evidences few details not clearly identified in the InGaAs image: Here, the chin of the earlier version of Ham’s face seems to have undergone changes, as shown in Figure 7. On the other hand, PC2 evidences few details not clearly identiﬁed in the InGaAs image: Here, the chin of the earlier version of Ham’s face seems to have undergone changes, as shown in Figure 7. 3. Results and Discussion To complete the investigation of the pentimenti we performed radiographic images in the three On the other hand, PC2 evidences few details not clearly identified in the InGaAs image: Here, the chin of the earlier version of Ham’s face seems to have undergone changes, as shown in Figure 7. On the other hand, PC2 evidences few details not clearly identiﬁed in the InGaAs image: Here, the chin of the earlier version of Ham’s face seems to have undergone changes, as shown in Figure 7. the chin of the earlier version of Ham s face seems to have undergone changes, as shown in Figure 7. g g , g To complete the investigation of the pentimenti, we performed radiographic images in the three most interesting parts of the paintings: Noah’s sons heads (Figure 8). The radiographic images showed the same pentimenti already seen in the post-processed VIS—NIR–SWIR images. Moreover, the radiography performed on the double face of Noah’s son was evidence of the presence of the chin, reinforcing and conﬁrming the ﬁndings of the PC2 image. To complete the investigation of the pentimenti, we performed radiographic images in the three most interesting parts of the paintings: Noah’s sons heads (Figure 8). The radiographic images showed the same pentimenti already seen in the post-processed VIS—NIR–SWIR images. Moreover, the radiography performed on the double face of Noah’s son was evidence of the presence of the chin, reinforcing and conﬁrming the ﬁndings of the PC2 image. 2283 Heritage 2019, 2 On t (a) (b) Figure 7. Detail of the painting. False-color image obtained by not post-processed images (a) and PC2 restituted as intensity coded colors (b). Figure 7. Detail of the painting. False-color image obtained by not post-processed images (a) and PC2 restituted as intensity coded colors (b). H i 2019 2 FOR PEER REVIEW 9 (b) (a) (b) (a) Figure 7. Detail of the painting. False-color image obtained by not post-processed images (a) and PC2 restituted as intensity coded colors (b). Figure 7. Detail of the painting. False-color image obtained by not post-processed images (a) and PC2 restituted as intensity coded colors (b). To complete the investigation of the pentimenti, we performed radiographic images in the three most interesting parts of the paintings: Noah’s sons heads (Figure 8). The radiographic images showed the same pentimenti already seen in the post-processed VIS—NIR–SWIR images. 3. Results and Discussion Moreover, the radiography performed on the double face of Noah’s son was evidence of the presence of the chin, reinforcing and confirming the findings of the PC2 image. (a) (b) (c) (d) Figure 8. Detail of the painting (a). Radiographic images of Noah’s sons (b–d). l i Figure 8. Detail of the painting (a). Radiographic images of Noah’s sons (b–d). most interesting parts of the paintings: Noah’s sons heads (Figure 8). The radiographic images showed the same pentimenti already seen in the post-processed VIS—NIR–SWIR images. Moreover the radiography performed on the double face of Noah’s son was evidence of the presence of the chin, reinforcing and confirming the findings of the PC2 image. (a) (a) (d) (b) ( ) (c) (d) (b) (c) Figure 8. Detail of the painting (a). Radiographic images of Noah’s sons (b–d). Figure 8. Detail of the painting (a). Radiographic images of Noah’s sons (b–d). 2284 Heritage 2019, 2 Heritage 2019, 2 4. Conclusions Funding: This research was funded by ADAMO project (2.10.2018-1.01.2020). The ADAMO Project is fun within the Center-of-Excellence (CoE) of the District of Technologies for Culture (DTC) by Regione Lazio. Acknowledgments: The authors wish to acknowledge the Conservator of Palazzo Chigi (Ariccia, Rome): Arch. Francesco Petrucci for his kind availability. Thanks to Fernanda Benetti (INFN-LNF) for her collaboration for in situ measurements. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 4. Conclusions Infrared reﬂectography performed by selecting diﬀerent spectral regions permits to highlight several hidden details of the painting. We presented an almost complete diagnostic analysis for the study of Drunkenness of Noah’s underdrawings and pentimenti, exploiting the limitation of the univariate approach versus a more exhaustive multivariate approach. Multivariate approach was investigated by applying a “soft” post-processing procedure, modifying the consolidated false-color RGB combination by inserting the gray scaled visible image in one channel and two infrared images at diﬀerent spectral ranges in the other ones. This method emphasized the diﬀerences between visible and underdrawing features, permitting an easier visualization of the meaningful areas. The use of PCA was very important to reveal other spectral variances that were conﬁrmed by radiographic images. We conﬁrmed that the multispectral image acquisition is required and recommended to visualize hidden details of a painting. In addition, post-processing reveals important spectral features which were not visible otherwise, starting from non-conventional false-color images to chemometric methods (such as PCA). The occurrence of the pentimenti found here shows that the composition originally conceived diﬀers from its ﬁnal realization. Except for the pentimenti, which regard the small changes in the position of the represented ﬁgures, the short hair of the Noah’s son in the background, realized in the ﬁrst instance, remains the main aspect to investigate further. As a matter of fact, these features are similar to the drawing conserved at the Metropolitan Museum. Although it is not possible to make deﬁnitive conclusions without carefully studying the paintings exhibited in the other museums, the occurrence of the pentimenti suggests that this painting could be one of the ﬁrst versions realized by the original artist. g The next step will be the analysis of the other Drunkenness of Noah versions painted by Andrea Sacchi in order to add diagnostic information to be further integrated with documental, stylistic, and artistic expertise. Author Contributions: Investigation, L.P., M.R., G.V.R., O.T., M.C., and A.P.; writing—original draft, L.P.; writing—review and editing, M.C.G., M.M., and F.C. Author Contributions: Investigation, L.P., M.R., G.V.R., O.T., M.C., and A.P.; writing—original draft, L.P.; writing—review and editing, M.C.G., M.M., and F.C. Author Contributions: Investigation, L.P., M.R., G.V.R., O.T., M.C., and A.P.; writing—original draft, L.P.; writing—review and editing, M.C.G., M.M., and F.C. 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https://openalex.org/W2611769032	https://infoscience.epfl.ch/record/274662/files/arXiv_1509.09029.pdf	English	null	Measurement of transverse momentum relative to dijet systems in PbPb and pp collisions at s N N = 2.76 $$ \sqrt{s_{\mathrm{NN}}}=2.76 $$ TeV	The Journal of high energy physics/The journal of high energy physics	2,016	cc-by	30,968	Measurement of transverse momentum relative to dijet systems in PbPb and pp collisions at √sNN = 2.76 TeV v:1509.09029v2 [nucl-ex] 12 Feb 2016 The CMS Collaboration∗ arXiv:1509.09029v2 [nucl-ex] 12 Feb 2 UROPEAN ORGANIZATION FOR NUCLEAR RESEARCH (CERN CMS-HIN-14-010 Published in the Journal of High Energy Physics as doi:10.1007/JHEP01(2016)006. Abstract arXiv:1509.09029v2 [nucl-ex] An analysis of dijet events in PbPb and pp collisions is performed to explore the prop- erties of energy loss by partons traveling in a quark-gluon plasma. Data are collected at a nucleon-nucleon center-of-mass energy of 2.76 TeV at the LHC. The distribution of transverse momentum (pT) surrounding dijet systems is measured by selecting charged particles in different ranges of pT and at different angular cones of pseudo- rapidity and azimuth. The measurement is performed as a function of centrality of the PbPb collisions, the pT asymmetry of the jets in the dijet pair, and the distance parameter R used in the anti-kT jet clustering algorithm. In events with unbalanced dijets, PbPb collisions show an enhanced multiplicity in the hemisphere of the sub- leading jet, with the pT imbalance compensated by an excess of low-pT particles at large angles from the jet axes. Published in the Journal of High Energy Physics as doi:10.1007/JHEP01(2016)006. c⃝2016 CERN for the beneﬁt of the CMS Collaboration. CC-BY-3.0 license 1 Introduction Partons produced at large transverse momenta (pT) through hard-scattering processes in heavy- ion collisions are expected to lose energy as they travel through the quark-gluon plasma (QGP) created in these interactions [1]. Experiments at RHIC and the LHC have observed a sup- pression in the yield of high-pT particles relative to suitably scaled pp collision data, and a signiﬁcant reduction in back-to-back high-pT hadron correlations [2–10] that have been inter- preted as evidence for strong partonic interactions within the dense medium that causes the quenching of jets. A direct observation of this effect using jets was provided by ATLAS [11] and CMS [12, 13] through a comparison of the pT balance of dijets in PbPb and pp collisions. In head-on PbPb collisions, a large increase in asymmetric dijet events was observed relative to the pp reference. This reﬂects the difference in energy lost by the two scattered partons in the medium, an effect that becomes more pronounced as the path lengths travelled by the partons and the energy density of the medium increase. In pPb collisions, no excess in unbalanced dijets was observed [14], leading to the conclusion that the dijet imbalance does not originate from initial-state effects. A wide range of models was proposed to accommodate the depen- dence of dijet data on the jet pT and the centrality of the collision, i.e. on the degree of overlap of the two colliding nuclei [15–20]. Further evidence for parton energy loss was found in studies of correlations between isolated photons and jets in PbPb events [21], where the unmodiﬁed isolated photon provides a measure of the initial parton momentum [22]. As energy is conserved in all interactions in the medium, parton energy loss does not imply the disappearance of energy, but its redistribution in phase space such that it is not recovered with standard jet ﬁnding clustering methods. The observed jet quenching naturally leads to questions of how the angular and pT distributions of charged particles are modiﬁed by the energy loss of partons as they traverse the medium. A measurement of these spectra can pro- vide information about the physical processes underlying parton energy loss, which can yield insights into the properties of the strongly interacting medium [23]. c⃝2016 CERN for the beneﬁt of the CMS Collaboration. CC-BY-3.0 license 1 considered. The original CMS analysis used a PbPb data sample corresponding to an integrated luminosity of 10 µb−1 [12], which was insufﬁcient for a detailed study of the angular pattern. In addition, no pp data at the same collision energy was available at the time. In this paper, PbPb data cor- responding to an integrated luminosity of 166 µb−1 from a heavy-ion run at a nucleon-nucleon center-of-mass energy of 2.76 TeV, and pp data corresponding to an integrated luminosity of 5.3 pb−1 taken at the same center-of-mass energy are used in a more comprehensive study. The new data provide an opportunity for detailed characterization of the multiplicity, momentum, and angular distribution of particles associated with the ﬂow of pT in dijet events in PbPb and pp collisions, as a function of collision centrality and dijet pT asymmetry. Collision centrality refers to conﬁgurations with different impact parameters of the lead nuclei. By changing cen- trality, the dependence of jet quenching can be studied as a function of the size and density of the medium. To study the pT ﬂow relative to the dijet system, two complementary approaches are pursued, both relying on the cancellation of contributions from the uncorrelated UE. First, the pT of individual tracks are projected onto the dijet axis, deﬁned as the bisector of the leading (highest pT) jet axis and the subleading (next highest pT) jet axis, with the latter ﬂipped by π in φ. These projections are then summed to investigate the overall pT ﬂow in dijet events. This “missing pT” analysis is used to study how the lost momentum is distributed as a function of the separation of the track from the jet axis, ∆. The second approach involves the study of the difference in the total number of particles emitted in the leading and subleading jet hemispheres. The measurements are carried out as a function of the collision centrality in PbPb collisions, and as a function of the dijet pT imbalance in pp and PbPb collisions. To investigate how differences in jet fragmentation affect energy loss mechanisms, jets are clustered using several anti-kT R parameters (0.2, 0.3, 0.4 and 0.5) [30, 31]. 1 Introduction Particle distributions in- side the jet cone (within ∆= √ (ηtrk −ηjet)2 + (φtrk −φjet)2 = 0.2–0.4, where φ is the azimuthal angle in radians and η is the pseudorapidity) were studied in terms of jet fragmentation func- tions and jet shapes [24–27]. These distributions show a moderate softening and broadening of the in-cone fragmentation products in PbPb collisions compared to pp data. However, the ob- served changes account for only a small fraction of the dijet momentum imbalance, indicating that a large amount of energy is transported outside of the jet cone through interactions in the medium. Identifying the distribution of particle pT surrounding the jets (i.e. the pattern of pT “ﬂow” relative to the dijet system) is challenging, as the “lost” pT is only of the order of 10 GeV, while the total pT from soft processes forming the underlying event (UE) in a head-on (central) PbPb collision is about three orders of magnitude larger [28, 29]. The angular distribution of the radiated energy is a priori unknown. To overcome these difﬁculties, CMS previously used the “missing pT ” method that exploits momentum conservation and azimuthal symmetry in dijet events. This method makes it possible to distinguish the correlated particles carrying the energy lost by jets from the uncorrelated particles, the directions of which are not related to the axes of the jets [12]. The momenta of all charged-particle tracks were therefore projected onto the jet direction, leading to a balancing of the uncorrelated particles, and thereby revealed the pT ﬂow relative to the dijet system. In pp events, imbalance in the pT of leading and subleading jets is accommodated through three-jet and multijet ﬁnal states. In PbPb collisions, quenching effects modify the spectrum and angular distribution of particles that recover the pT balance within the dijet system. These studies showed that the overall energy balance is restored only when low-momentum particles (pT ≈0.5–2 GeV) at large angles to the jet axis (∆> 0.8) are 2 CMS detector 2 considered. 3 Monte Carlo simulation To study the performance of jet reconstruction in PbPb and pp collisions, dijet events in nucleon- nucleon collisions are simulated with the PYTHIA Monte Carlo (MC) event generator [40] (ver- sion 6.423, tune Z2 [41, 42]). To account for isospin effects present in PbPb collisions, the un- derlying pp, pn, and nn subcollisions are weighted by cross sections using the model from Ref. [43]. For the simulation of dijet signals, a minimum hard-interaction scale of 30 GeV is used to increase the number of dijet events. To model the PbPb UE, minimum bias PbPb events are simulated with the HYDJET event gener- ator, version 1.8 [43]. The parameters of this version are tuned to reproduce total particle mul- tiplicities, improve agreement with the observed charged-hadron spectra, and to approximate the ﬂuctuations in UE seen in data. Proton-proton collisions are generated using leading-order (LO) PYTHIA (without HYDJET simulation). Full detector simulation using the GEANT4 pack- age [44] and the standard CMS analysis chain are used to process both PYTHIA dijet events and PYTHIA dijet events embedded into HYDJET events (denoted PYTHIA+HYDJET in this paper). Jet reconstruction is studied using the jet information in the PYTHIA generator in comparison to the same fully reconstructed jet in PYTHIA+HYDJET, after matching the generator-level and reconstructed jets in angular regions of ∆reco,gen = √ (ηgen jet −ηreco jet )2 + (φgen jet −φreco jet )2 < R. 2 CMS detector The central feature of the CMS apparatus is a superconducting solenoid with a 6 m internal diameter. Within the superconducting solenoid volume are a silicon pixel and strip tracker, a lead tungstate crystal electromagnetic calorimeter (ECAL), and a brass and scintillator hadron calorimeter (HCAL), each composed of a barrel and two endcap sections. Forward calorimeters extend the pseudorapidity [32] coverage provided by the barrel and endcap detectors. Muons are measured in gas-ionization detectors embedded in the steel ﬂux-return yoke outside the solenoid. The silicon tracker measures charged particles within the pseudorapidity range \|η\| < 2.5. It consists of 1440 silicon pixel and 15 148 silicon strip detector modules and is located in the 3.8 T ﬁeld of the superconducting solenoid. For nonisolated particles of 1 < pT < 10 GeV and \|η\| < 1.4, the track resolutions are typically 1.5% in pT and 25–90 (45–150) µm in the transverse (longitudinal) impact parameter [33]. The ECAL has coverage up to \|η\| = 1.48, and the HCAL up to \|η\| = 3. Steel and quartz ﬁbre hadron forward (HF) calorimeters extend the acceptance to \|η\| = 5. For central η, the calorimeter cells are grouped in projective towers of granularity ∆η × ∆φ = 0.087 × 0.087. The ECAL was initially calibrated using test beam electrons, and then with photons from π0 and η meson decays and electrons from Z boson decays [34–36]. The energy scale in data agrees with that in the simulation to better than 1 (3)% in the barrel (endcap) region, \|η\| < 1.5 (1.3 < \|η\| < 3.0) [37]. Hadron calorimeter cells in the \|η\| < 3 region are calibrated primarily with test beam data and radioactive sources [38, 39]. A more 3 detailed description of the CMS detector, together with a deﬁnition of the coordinate system and kinematic variables, can be found in Ref. [32]. detailed description of the CMS detector, together with a deﬁnition of the coordinate system and kinematic variables, can be found in Ref. [32]. 4 Jet reconstruction Jet reconstruction in heavy-ion collisions at CMS is performed using the anti-kT algorithm and distance parameters R = 0.2 through 0.5, encoded in the FastJet framework [30]. Jets are re- constructed based on energies deposited in the CMS calorimeters. The probability of having a pileup collision is 23%, and the average transverse energy (ET) associated with the UE is less than 1 GeV. For pp collisions, no subtraction is employed for the underlying event (UE) nor for pileup from overlapping pp interactions. Whereas, for PbPb collisions, a new “HF/Voronoi” algorithm is used to subtract the heavy-ion background [45]. The transverse energy is deﬁned by ET = ∑Ei sin (θi), where Ei is the energy of the ith particle in the calorimeter, θi is the polar angle of particle i measured from the beam axis, and the sum is over all particles emitted into a ﬁxed ∆in an event. The HF/Voronoi algorithm removes the UE contribution by estimating the ET contribution from the UE at central η, and its azimuthal dependence, from deposition in the HF detector. The estimation is performed using a polynomial model that is trained using a singular-value decomposition method [46], separately on minimum bias data and MC simulation. After an average ET is subtracted from each calorimeter tower, based on its location in η and φ, the calorimeter towers containing non-physical negative ET are evened out by redistributing the energy in neighboring positive ET towers in a circular region of the parameter R + 0.1. The redistribution is implemented by minimizing a metric that describes the total energy difference before and after the process, given that after the redistribution all towers have positive energy. The initial calorimetric ET values are corrected as a function of pT and η to match the jets clustered using all particles, except neutrinos, at the generator level of PYTHIA. The consistency of the corrected jet energy scale (JES), deﬁned as ⟨preco T /pgen T ⟩, is checked as a function of pT and 5 Track reconstruction 4 5 η using PYTHIA+HYDJET events in bins of event centrality. The deviations are within 2% for all centrality, pT, and η bins, and less than 1% for jet pT greater than 60 GeV. η using PYTHIA+HYDJET events in bins of event centrality. The deviations are within 2% for all centrality, pT, and η bins, and less than 1% for jet pT greater than 60 GeV. 4 Jet reconstruction The nonlinear response of the calorimeter as a function of particle energy gives jets that frag- ment into many particles with smaller energies a smaller response relative to the jets of same energy but with fewer fragments. To account for the dependence of JES on the fragmentation of jets, an additional correction is applied as a function of reconstructed jet pT, and as a func- tion of the number of charged particles with pT > 2 GeV in a cone of R around the jet axis. The number of charged particles in PYTHIA+HYDJET is calculated using the pT values obtained after the “HF/Voronoi” subtraction. For PYTHIA, pT values without any UE subtraction are used to calculate the number of charged particles. The fragmentation-dependent correction applied in PbPb collisions is calculated using PYTHIA+HYDJET events with matching UE activity. This cor- rection results in a reduction in separation of the JES for quark and gluon jets, and also lessens the impact of jet reconstruction on fragmentation of the leading and subleading jets. The residual JES that accounts for the difference in calorimeter response in data and MC events is calculated using dijet balance in pp and peripheral (50–100% centrality) PbPb collisions [47], based on data. This difference is found to be less than 2% for \|η\| < 2. 6 Analysis Events are selected using an inclusive single-jet trigger with jet pT > 80 GeV. To suppress elec- tronic noise, cosmic rays, and beam backgrounds, events are required to satisfy selection crite- ria documented in refs [12, 21]. Events passing selections are subject to ofﬂine jet reconstruction. To select samples containing high-pT dijets, events are required to have a leading (subleading) jet in the range of \|η\| < 2 with a corrected jet pT > 120 (50) GeV. The single-jet trigger is fully efﬁcient for events with the requirement on the leading jet pT for all the R parameters in the analysis. To select a dijet topology, the azimuth between the leading and subleading jets is required to be ∆φ1,2 = \|φ1 −φ2\| > 5π/6. Once leading and subleading jets are identiﬁed within the initial range of \|η\| < 2, both jets are then restricted to be within a tighter \|η\|. For measurements that offer comparison to a previous analysis [12], we use the previous selection of \|η\| < 1.6. For those that extend up to large angular distances ∆, a tighter requirement of \|η\| < 0.6 is applied, such that leading and subleading jets are far from the edge of the tracker and all ranges in ∆fall within the acceptance. This analysis aims to provide information that would aid the characterization of the energy loss mechanisms responsible for the increase in the fraction of unbalanced dijet pairs in central PbPb relative to pp collisions. As hard-scattered partons travel and shower in the QGP, they can both trigger a coherent medium response and undergo interactions in the medium that modify the showers of both partons. However, the enhancement in unbalanced dijet pairs suggests that, on average, the subleading jet loses more energy than the leading jet. The modiﬁcation in jet balance must be compensated by the remaining, unclustered constituents of the event, as each interaction conserves overall momentum. The particles that provide the pT balance are correlated with the jet axes, but the particles that are not affected by the interaction of the partons with the medium are evenly distributed in azimuth relative to the individual directions of the leading and subleading jets. The total pT of these particles is uncorrelated with the dijet pair. To differentiate the uncorrelated and cor- related particles, we compare differences in multiplicity in leading and subleading jet hemi- spheres. 5 Track reconstruction For studies of pp data and PYTHIA MC events, charged particles are reconstructed using the same iterative method [33] as in previous CMS analyses of pp collisions. However, for PbPb data and PYTHIA+HYDJET events, a different iterative reconstruction [8, 25] is employed after extending the global tracking information down to pT = 0.4 GeV. To minimize the impact of inefﬁciencies in track reconstruction caused by the pT resolution in track seeds near the 0.4 GeV threshold, only tracks with pT > 0.5 GeV are used in this analysis. Reconstructed tracks in PYTHIA and PYTHIA+HYDJET simulations are matched to primary par- ticles using the associated hits, i.e., charged particles that are produced in the interaction or are remnants of particles with a mean proper lifetime of less than 5 × 1013 GeV−1. The misiden- tiﬁcation rate is deﬁned as the fraction of reconstructed tracks that do not match any charged particle (primary or otherwise). The multiple reconstruction rate is given by the fraction of primary particles that are matched to more than one reconstructed track. Tight track quality criteria are applied to reduce the rate of misidentiﬁed or secondary particles [33]. Requirements are less restrictive for pp than for PbPb collisions. Heavy-ion tracking requires a larger number of hits in the tracker and a smaller normalized ﬁt χ2 value for ﬁts to reconstructed tracks. For both systems, tracks are required to be compatible with the vertex with the largest value in the sum of their pT. In pp collisions, the track reconstruction efﬁciency is ≈90% at pT = 10 GeV and 80% at 0.5 GeV. The misidentiﬁcation rate for tracks is <2% for pT > 1 GeV and slightly higher below this value. The contribution from secondary particles is subtracted, as the secondary-particle rate is as high as 2%. The multiple reconstruction rate is smaller than 1%. The efﬁciency and misidentiﬁcation corrections are calculated as a function of η, φ, pT, and the distance to the nearest jet axis, while simpler secondary-particle and multiple reconstruction corrections are applied that depend only on the η and pT values of charged particles. As the track reconstruction efﬁciency in pp collisions is larger than in PbPb collisions, the momentum ﬂow can be measured with higher precision, while in the high-multiplicity en- vironment of heavy-ion collisions track reconstruction remains a challenge. 5 Track reconstruction In PbPb collisions, 5 the reconstruction efﬁciency for primary charged particles, after implementing the above track quality criteria, is approximately 70% at pT ≈10 GeV. Efﬁciency starts to drop for pT be- low 5 GeV and at 0.5 GeV the efﬁciency is 30%. The misidentiﬁcation rate for tracks with pT = 0.5 GeV is ≈35%, but decreases to values smaller than 2% and for pT > 1 GeV. The secondary-particle rate and multiple-reconstruction rate are, respectively, less than 0.5% and 0.3% over the whole pT range in the analyis. No corrections are applied for these in PbPb colli- sions. Using PYTHIA+HYDJET simulations, track reconstruction efﬁciency and misidentiﬁcation rates are evaluated as a function of the η, φ, and pT of the track, as well as the centrality of the collision, and the smallest distance in ∆between the track and a jet with pT > 50 GeV. Tracks used in the analysis are weighted with a factor to correct for the effects described above. The value of this correction is ctrk = (1 −misreconstruction) (1 −secondary-particle) (efﬁciency) (1 + multiple-reconstruction) , (1) (1) where secondary-particle and multiple-reconstruction rates are set to zero for PbPb collisions. where secondary-particle and multiple-reconstruction rates are set to zero for PbPb collisions. 6 Analysis In addition, we measure modiﬁcations in the pT spectrum of charged particles that contribute to the overall pT balance in the event, as well as their angular distribution with respect to the dijet system. Using the azimuthal symmetry of the jet axes relative to the UE makes it possible to perform precise measurements for particles down to pT = 0.5 GeV, and 6 Analysis 6 angles as large as ∆= 1.8. This provides constraints on energy loss mechanisms despite the small signal-to-background ratio. angles as large as ∆= 1.8. This provides constraints on energy loss mechanisms despite the small signal-to-background ratio. The cancellation of the uncorrelated UE depends on azimuthal symmetry of the areas selected around the leading and subleading jets relative to the axis of projection. As mentioned above, to ensure this requirement, the dijet azimuthal angle (φdijet) is deﬁned as the average φ of the leading and subleading jets after the subleading jet is reﬂected around the origin. In contrast with previous publications [12], φdijet is preferred over φ1 (the φ of the leading jet) for the pro- jection axis, because the latter choice breaks azimuthal symmetry, by generating particles near the leading jet that have larger projections at small angles relative to particles produced at the same distance to the subleading jet. The perfect cancellation of contributions from particles to pT ﬂow, and to differences in hemi- sphere multiplicities from UE, take place only when there is no interaction between UE and the jets. This is the case in PYTHIA+HYDJET simulations. In data, due to the variations in path length in medium traversed by jets there are complicated correlations between particles from different interactions and jet directions. These correlations comprise a part of the signal probed in this analysis. The observables used in this analysis are measured in bins of centrality and dijet imbalance. The dependence on centrality in PbPb collisions is investigated in terms of the emergence and enhancement of jet quenching effects as the size of the medium and energy density increase, and the dijet imbalance enriches events with subleading jets that lose more energy than the leading jet. To deﬁne centrality classes, collisions with inelastic hadronic interactions are di- vided into percentages according to the ET of calorimeter towers summed in the HF, and events are assigned into classes of centrality based on these total sums in the HF. 6 Analysis The distribution in this ET is used to divide the event sample into bins, each representing 0.5% of the total nucleus- nucleus interaction cross section. Following Refs. [12, 13], we quantify pT imbalance through the asymmetry ratio AJ = (pT,1 −pT,2)/(pT,1 + pT,2), where pT,1 and pT,2 are the pT of the lead- ing and subleading jets within η < 2.0, respectively. The AJ boundaries used in the analysis are 0.11, 0.22, 0.33 and 0.44, which correspond to pT,2/pT,1 values of 0.8, 0.64, 0.50 and 0.42, respectively. 6.1 Difference in multiplicities The events are bisected with a plane perpendicular to φdijet into two hemispheres associated with the leading and subleading jets. The multiplicity difference is deﬁned as the difference between the corrected number of tracks with pT > 0.5 GeV (NCorrected trk = ∑ctrk) in these two hemispheres: ∆mult = NCorrected trk \|\|φtrk−φdijet\|>π/2 −N Corrected trk \|\|φtrk−φdijet\|<π/2. (2) (2) Positive ∆mult means that an excess of particles is found in the hemisphere of the subleading jet, relative to the number of particles in the leading jet hemisphere. This quantity is measured event-by-event and then averaged in bins of the observables of interest. It is sensitive to the number of jets in a given hemisphere and their fragmentation, as well as to the additional particles produced in jet quenching or through some speciﬁc response of the QGP medium in one of the two hemispheres. Positive ∆mult means that an excess of particles is found in the hemisphere of the subleading jet, relative to the number of particles in the leading jet hemisphere. This quantity is measured event-by-event and then averaged in bins of the observables of interest. It is sensitive to the number of jets in a given hemisphere and their fragmentation, as well as to the additional particles produced in jet quenching or through some speciﬁc response of the QGP medium in one of the two hemispheres. To select events that show consequences of jet quenching, the measurement is carried out as a function of AJ and collision centrality. The AJ-dependent measurement is performed for jets with a distance parameter of R = 0.3. To see modiﬁcations in the pT spectrum associated with the difference in multiplicities in two Transverse momentum balance 7 6.2 hemispheres, ∆mult is measured for track pT ranges of 0.5–1, 1–2, 2–4, 4–8, and 8–300 GeV, and divided by the bin width. The measurement is repeated for different R parameters. To be consistent with the measurement of pT balance, the leading and subleading jets used in the AJ-dependent ∆mult measurement are required to fall in the pseudorapidity region of \|η\| < 1.6. The leading and subleading jets used in the R-dependent measurement are required to be within \|η\| < 0.6. 6.2 Transverse momentum balance Detailed information about the pT ﬂow relative to the dijet system can be obtained by studying the contribution of tracks to the overall pT balance in the event, as characterized by individual track pT and angle relative to the jets. To calculate the pT balance, the pT of tracks are projected onto the dijet axis. For each track, this projection is deﬁned as p∥ T = −ctrk ptrk T cos (φtrk −φdijet), (3) (3) where, as mentioned in Section 5, the correction for reconstruction effects accounts for the misreconstruction rate and reconstruction efﬁciency for PbPb collisions, with values speciﬁed by Eq. 1. In addition, secondary particle and multiple reconstruction rates are corrected in pp collisions. Particles that make a positive contribution in ∆mult also have positive p∥ T, as the cosine function changes sign at π/2. These two observables therefore map onto each other with a weight in track pT and cos (φtrk −φdijet). To study the angular recovery rate (rate at which imbalance is restored, as momentum contri- butions are included further from the jet cone) and the associated spectra of pT balance, tracks that fall in annular regions around the jet axes are grouped together according to their pT. In each event, p∥ T values of these group of tracks are summed to obtain pT / ∥. For each region, pT / ∥ is calculated in track pT ranges of 0.5–1, 1–2, 2–4, 4–8, and 8–300 GeV. Annular regions are de- ﬁned in ∆= √ (φtrk −φjet)2 + (ηtrk −ηjet)2 and binned between ∆= 0.0–1.8 in steps of 0.2. In addition, the contribution from charged particles that fall outside of this range are all collected in an extra overﬂow bin. These particles lie in the range of 1.8 < ∆< 3.6, depending on the η of the dijet pair. No anti-kT clustering is employed in the calculation of ∆, and tracks are deﬁned to lie within circular regions in pseudorapidity and azimuth. The axes used to deﬁne the annuli differ from the projection axis, φdijet. For large ∆, the annuli around the leading and subleading jets can overlap, in which case, the track used in the overlap region when calculating pT / ∥, is the one in the annulus at smaller radius. The overlaps do not occur before ∆= 5π/12. 6.1 Difference in multiplicities Although in both cases jets with \|η\| > 2 are excluded, it is important to note that starting jet reconstruction with a cutoff \|η\| < 1.6, (or < 0.6) is different than using the \|η\| < 2 selection for determining the highest-pT jets and then applying a tighter requirement, since events in which the leading or subleading jets are found in the range between \|η\| = 1.6 (or 0.6) and \|η\| = 2.0 are also excluded. 6.2 Transverse momentum balance Instead of summing all ∆ bins, to calculate the recovery of balance as radius gets larger, the annuli can be summed from ∆= 0 up to the angle of interest, and a cumulative balance inside a cone calculated, as ⟨pT / ∥⟩[0,∆] = ∆′=∆ ∑ ∆′=0 ⟨pT / ∥⟩∆′. (7) (7) As mentioned previously, for consistency with the analysis in Ref. [12], in calculations that in- tegrate over ∆, e.g. for, ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ, only events in which both leading and subleading jets fall within \|η\| < 1.6 are included in the measurement of pT balance. For measurements where contributions of different annuli are studied, to ensure full tracker coverage around jets over ∆< 1.8 for ⟨pT / ∥⟩ptrk T ,∆, ⟨pT / ∥⟩∆, and ⟨pT / ∥⟩[0,∆], tighter restrictions are required on the pseu- dorapidity of leading and subleading jets (\|η\| < 0.6) after they are found within \|η\| < 2. 6.2 Transverse momentum balance The pT / ∥is averaged over events with a speciﬁc AJ value separately for pp and PbPb collisions, and for PbPb collisions they are divided into classes of collision centrality. This average is denoted as ⟨pT / ∥⟩ptrk T ,∆, to indicate that within each event the balance is calculated using a subset of tracks with speciﬁc ∆and pT. Using the track pT and ∆parameters limits the selections on collision centrality and AJ because of the statistical imprecision of the data. For more detailed analysis of the dependance of track pT on event properties, ∆binning can be removed by adding up the ⟨pT / ∥⟩ptrk T ,∆values for each 8 7 Systematic uncertainties ∆bin, which is identical to not having annular requirements in the ﬁrst place, to obtain ⟨pT / ∥⟩ptrk T = ∑ ∆ ⟨pT / ∥⟩ptrk T ,∆. (4) (4) The pT balance, as in Eq. 4, calculated for tracks in a given pT range usually yields nonzero values, because of the differences in pT spectra of particles in subleading jet hemisphere relative to the spectra in the leading jet hemisphere. Summing the signed ⟨pT / ∥⟩ptrk T values for each track pT bin provides an overall pT balance in the event for tracks with 0.5 < pT < 300 GeV, that takes values close to zero, because of momentum conservation. There can still be a deviation from zero because of the particles with pT < 0.5 GeV, as well as for those particles that fall out of the tracker coverage in pseudorapidity that are not included in the measurement. This sum corresponds to ⟨pT / ∥⟩Σ = ∑ ptrk T ⟨pT / ∥⟩ptrk T . (5) (5) The angular distribution of pT balance is studied differentially in bins of track pT by ⟨pT / ∥⟩ptrk T ,∆, as described above, and adding up the contribution from different track pT bins gives The angular distribution of pT balance is studied differentially in bins of track pT by ⟨pT / ∥⟩ptrk T ,∆, as described above, and adding up the contribution from different track pT bins gives ⟨pT / ∥⟩∆= ∑ ptrk T ⟨pT / ∥⟩ptrk T ,∆, (6) (6) which deﬁnes the contribution of all tracks with 0.5 < pT < 300 GeV in a given annulus to total pT balance. This ⟨pT / ∥⟩∆, summed over all ∆intervals, yields ⟨pT / ∥⟩Σ. 7 Systematic uncertainties The sources of major systematic uncertainty can be categorized into two groups; biases related to jet reconstruction and those related to track reconstruction. Effects associated with event selection and beam background rejection are found to be negligible. The biases related to jet reconstruction are caused by smearing of jet pT due to energy resolu- tion and uncertainties in the JES. These factors can change the pT-ordering of jets in the event, resulting in the interchanging of leading and subleading jets, or causing third jet to replace the subleading jet. The uncertainties are estimated as a function of centrality and AJ in each charged-particle pT range, using PYTHIA and PYTHIA+HYDJET simulations to compare observ- ables calculated with reconstructed jets to generator-level jets. A bin-by-bin correction is ap- plied to data to account for the observed jet reconstruction bias. This uncertainty includes the 9 Table 1: Systematic uncertainties in ⟨pT / ∥⟩∆for jets clustered with distance parameter of 0.3 in pp, and in central and peripheral PbPb collisions, for different AJ selections. Uncertainties are shown as shifts in the values in units of GeV (rather than as fractions) for two ∆selections. shown as shifts in the values in units of GeV (rather than as fractions) for two ∆selections. 7 Systematic uncertainties Values integrated over AJ pp PbPb, 30–100% PbPb, 0–30% ∆ <0.2 0.2–2.0 <0.2 0.2–2.0 <0.2 0.2–2.0 Jet reconstruction <1 0.0–0.2 1 0.1–0.2 1 0.1–0.4 Data/MC differences for JES 1 0.1–0.2 2 0.1–0.3 2 0.1–0.3 Fragmentation dependent JES <1 0.1–0.2 2 0.1–0.2 1 0.1–0.4 Track corrections <1 <0.1 1 0.0–0.2 2 0.2–0.9 Data/MC differences for tracking 1 0.0–0.1 1 0.1–0.2 1 0.1–0.2 Total 1 0.1–0.3 2 0.2–0.3 3 0.2–1.0 AJ < 0.22 pp PbPb, 30–100% PbPb, 0–30% ∆ <0.2 0.2–2.0 <0.2 0.2–2.0 <0.2 0.2–2.0 Jet reconstruction <1 0.1–0.2 1 0.1–0.2 1 0.1–0.4 Data/MC differences for JES 1 0.1–0.2 2 0.1–0.4 2 0.2–0.4 Fragmentation dependent JES <1 0.1 2 0.1–0.4 1 0.1–0.5 Track corrections <1 <0.1 1 0.1 2 0.1–0.6 Data/MC differences for tracking <1 0.0–0.1 1 0.1 1 0.1 Total 1 0.1–0.3 2 0.2–0.4 3 0.2–0.6 AJ > 0.22 pp PbPb, 30–100% PbPb, 0–30% ∆ <0.2 0.2–2.0 <0.2 0.2–2.0 <0.2 0.2–2.0 Jet reconstruction 2 0.1–0.5 1 0.1–0.6 2 0.2–0.6 Data/MC differences for JES 2 0.1–0.3 3 0.2–0.5 3 0.3–0.6 Fragmentation dependent JES 1 0.1–0.5 1 0.1–0.7 1 0.2–0.6 Track corrections <1 0.1 1 0.1–0.3 3 0.2–1.1 Data/MC differences for tracking 2 0.1–0.2 2 0.1–0.2 2 0.1–0.3 Total 3 0.3–0.8 3 0.3–0.9 4 0.4–1.4 effect of jet-angular resolution. However, the size of the bins in the ∆-dependent measurement is signiﬁcantly larger than a typical angular resolution, which therefore has a negligible effect on the observables. Going from R = 0.2 to 0.5, the angular resolution, deﬁned by the standard deviation of the ∆reco,gen distribution, increases from 0.020 to 0.025 for leading jets, and from 0.025 to 0.035 for subleading jets in pp. The same holds in 30–100% centrality PbPb collisions. In the most central 0–30% of events, the corresponding ranges are 0.020–0.035 and 0.025–0.045, respectively. After implementing the fragmentation-dependent jet energy corrections there is up to 5% dif- f b h JES f k d l j 50 G V d h diff di Values integrated over AJ pp PbPb, 30–100% PbPb, 0–30% ∆ <0.2 0.2–2.0 <0.2 0.2–2.0 <0.2 0.2–2.0 Jet reconstruction <1 0.0–0.2 1 0.1–0.2 1 0.1–0.4 Data/MC differences for JES 1 0.1–0.2 2 0.1–0.3 2 0.1–0.3 Fragmentation dependent JES <1 0.1–0.2 2 0.1–0.2 1 0.1–0.4 Track corrections <1 <0.1 1 0.0–0.2 2 0.2–0.9 Data/MC differences for tracking 1 0.0–0.1 1 0.1–0.2 1 0.1–0.2 Total 1 0.1–0.3 2 0.2–0.3 3 0.2–1.0 effect of jet-angular resolution. 7 Systematic uncertainties Because the number of charged parti- cles is a parameter in these corrections, and can make the fragmentation-dependent jet energy corrections sensitive to quenching effects, the difference in the observables before and after cor- rections in MC events is compared to the corresponding change in data, and the discrepancy between data and simulation is quoted as an additional source of uncertainty. ance in a control sample of peripheral PbPb events [47]. The jet pT is changed up and down for leading and subleading jets in an asymmetric manner (leading JES is increased while sub- leading JES is decreased) as a function of jet pT, to account for the differences in JES between quark and gluon jets and the data-based JES uncertainty. Because the number of charged parti- cles is a parameter in these corrections, and can make the fragmentation-dependent jet energy corrections sensitive to quenching effects, the difference in the observables before and after cor- rections in MC events is compared to the corresponding change in data, and the discrepancy between data and simulation is quoted as an additional source of uncertainty. Uncertainties related to track reconstruction are calculated in PYTHIA and PYTHIA+HYDJET by comparing the results with generator-level charged particles to those with reconstructed tracks, after applying the track corrections discussed in Section 5. The small uninstrumented regions in the detector, and the correlation between track reconstruction efﬁciency and JES are the main causes of discrepancies observed between results with generator-level particles and reconstructed tracks. The track corrections account for the inefﬁciencies due to uninstrumented regions. However, the bins used in η and φ to calculate the reconstruction efﬁciency are larger than the size of the uninstrumented regions, and as a result cannot completely correct the effect of these. An additional uncertainty is therefore added to account for the effect of differences in detector conditions and simulation of track reconstruction. This is achieved using the ratio of corrected to initial track pT and η spectra in data and simulations that are compared as track- quality selections are changed. The difference is found to be less than 5%, which is included in the systematic uncertainty. To calculate the total uncertainty, the uncertainties from sources mentioned above are summed in quadrature. The contribution of each item is summarized in Tables 1–3 for the ⟨pT / ∥⟩∆mea- surement. 7 Systematic uncertainties However, the size of the bins in the ∆-dependent measurement is signiﬁcantly larger than a typical angular resolution, which therefore has a negligible effect on the observables. Going from R = 0.2 to 0.5, the angular resolution, deﬁned by the standard deviation of the ∆reco,gen distribution, increases from 0.020 to 0.025 for leading jets, and from 0.025 to 0.035 for subleading jets in pp. The same holds in 30–100% centrality PbPb collisions. In the most central 0–30% of events, the corresponding ranges are 0.020–0.035 and 0.025–0.045, respectively. effect of jet-angular resolution. However, the size of the bins in the ∆-dependent measurement is signiﬁcantly larger than a typical angular resolution, which therefore has a negligible effect on the observables. Going from R = 0.2 to 0.5, the angular resolution, deﬁned by the standard deviation of the ∆reco,gen distribution, increases from 0.020 to 0.025 for leading jets, and from 0.025 to 0.035 for subleading jets in pp. The same holds in 30–100% centrality PbPb collisions. In the most central 0–30% of events, the corresponding ranges are 0.020–0.035 and 0.025–0.045, respectively. After implementing the fragmentation-dependent jet energy corrections there is up to 5% dif- ference between the JES for quark and gluon jets at pT < 50 GeV, and the difference disappears for high-pT jets. Additional checks are therefore pursued to account for possible discrepancies in the performance of jet energy corrections in data and in MC simulations. A modiﬁcation in ﬂavor content of jets due to quenching can lead to an under- or over-correction of the jet energy in data. Also, the uncertainty in the JES from differences in simulation and detector conditions is calculated to be 2% using a data-based “tag-and-probe” technique that depends on dijet bal- 10 7 Systematic uncertainties ance in a control sample of peripheral PbPb events [47]. The jet pT is changed up and down for leading and subleading jets in an asymmetric manner (leading JES is increased while sub- leading JES is decreased) as a function of jet pT, to account for the differences in JES between quark and gluon jets and the data-based JES uncertainty. 7 Systematic uncertainties The systematic sources are given in terms of shifts in the value of each observable in a given bin in units of GeV instead of % changes, as the ⟨pT / ∥⟩∆can vanish and can take values arbitrarily close to zero. Typically, ⟨pT / ∥⟩∆is between 15–40 GeV near the jet axes (∆< 0.2), and less than 10 GeV at larger angles. The dependence of uncertainties in dijet asymmetry and centrality is summarized in Table 1 for jets with a distance parameter R = 0.3. The jet energy resolution, can cause events to move across the AJ boundaries. Moreover, it is more likely for the leading jet in a highly imbalanced dijet event to be located in a region of an upward UE ﬂuctuation in PbPb collisions. For these reasons, uncertainties related to jet reconstruction are larger in imbalanced dijet events. For well-balanced events, the uncertainty is comparable to that in the inclusive AJ selection, be- cause the increase in effects from jet energy resolution balances the reduction of effects related to UE ﬂuctuations. Uncertainties in track reconstruction are larger in imbalanced than in bal- anced events, because of the correlation of track reconstruction efﬁciency and reconstructed jet energy. When a high-pT track that carries a signiﬁcant fraction of jet pT is not reconstructed, the jet energy is under-corrected, and vice versa, the energy is over-corrected in events where the high-pT track is found, because jet energy corrections are obtained for the average case where the high-pT track might not be reconstructed. Events with highly imbalanced dijets can result from miscalculated jet energies caused by inefﬁciencies in track reconstruction. Centrality of PbPb collisions does not affect the uncertainties within the jet cone as much as at larger angles, where the signal-to-background ratio gets smaller. Track and jet reconstruction uncertainties, caused by over-correction of the leading jet pT because of upward UE ﬂuctuations, in particu- lar, tend to increase in central collisions. Uncertainties are smaller in pp than in PbPb collisions because of the absence of a heavy-ion UE, and differences in jet and track reconstruction that provide better measurement of jet pT, larger track reconstruction efﬁciency, and lower track misidentiﬁcation rates. 7 Systematic uncertainties 11 Uncertainties for small ∆are dominated by charged particles with pT > 8 GeV, while at larger ∆, low-pT particles make up a larger fraction of the total uncertainty in events when there is no selection made on charged-particle pT. The contribution from each range of track pT to the uncertainty in ⟨pT / ∥⟩∆, in other words the uncertainty in ⟨pT / ∥⟩ptrk T ,∆, is shown in Table 2 for R = 0.3, in events with 0–30% central PbPb collisions. Finally, as shown in Table 3, uncertainties in jet reconstruction and track reconstruction in MC events increase together with increasing R, as the UE inside the jet cone gets larger. However, JES difference between quark and gluon jets is smaller for large R parameters, and uncertainties that account for JES differences in data and in MC events therefore decrease. Table 2: Systematic uncertainties in ⟨pT / ∥⟩ptrk T ,∆in 0–30% PbPb collisions, for jets clustered with a distance parameter of 0.3, as a function of charged-particle pT. Uncertainties are shown as shifts in the values in units of GeV (rather than as fractions) for two ∆selections. 0.5 < pT < 2 GeV 2 < pT < 8 GeV pT > 8 GeV ∆ <0.2 0.2–2.0 <0.2 0.2–2.0 <0.2 0.2–2.0 Jet reconstruction 0.04 0.06–0.25 0.13 0.04–0.14 0.85 0.01–0.07 Data/MC differences for JES 0.14 0.07–0.24 0.42 0.03–0.11 0.97 0.01–0.12 Fragmentation dependent JES 0.03 0.10–0.14 1.1 0.05–0.23 0.19 0.02–0.06 Track corrections 0.09 0.08–0.64 0.27 0.06–0.13 1.78 0.01–0.07 Data/MC differences for tracking 0.04 0.03–0.08 1.2 0.01–0.05 1.16 0.00–0.02 Total 0.17 0.20–0.69 1.1 0.11–0.29 2.3 0.04–0.10 Table 3: Systematic uncertainties in ⟨pT / ∥⟩ptrk T ,∆in 0–30% PbPb collisions are shown for jets clus- tered with distance parameters of 0.2, 0.4 and 0.5. Uncertainties are shown as shifts in the values in units of GeV (rather than as fractions) for two ∆selections. 7 Systematic uncertainties R = 0.2 R = 0.4 R = 0.5 ∆ <0.2 0.2–2.0 <0.2 0.2–2.0 <0.2 0.2–2.0 Jet reconstruction 1 0.1–0.4 1 0.1–0.5 1 0.1–0.7 Data/MC differences for JES 2 0.1–0.5 2 0.1–0.4 2 0.1–0.3 Fragmentation dependent JES 1 0.1–0.4 1 0.1–0.3 1 0.1–0.3 Track corrections 2 0.2–0.7 2 0.1–1.1 2 0.1–1.1 Data/MC differences for tracking 1 0.1–0.2 1 0.1 1 0.1 Total 3 0.2–0.9 3 0.3–1.1 3 0.2–1.1 Although uncertainties in differences in multiplicities are calculated separately, their values are not listed in a table, because they can be approximated from the uncertainties in ⟨pT / ∥⟩divided by the average charged particle pT in that range. In 0–10% central events, for R = 0.3, the dom- inant source is jet reconstruction, with an uncertainty caused by an upward ﬂuctuation in the background under the leading jet, which is followed by the uncertainty in track reconstruction, and residual track reconstruction in data and in MC events that change by 0.5–1.5 particles, as a function of AJ. The uncertainties increase with R and with centrality from peripheral to central collisions. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets Angular distribution of the pT relative to the axis deﬁned by the parton direction is a key for studying QCD processes responsible for parton energy loss. In models, large-angle modiﬁca- 12 8 Results 8 Results tions in the event due to jet quenching have been accommodated qualitatively through a re- sponse triggered in the hydrodynamic medium by the deposited energy [48] and through the cascade of gluons created in medium-induced radiation processes [49–52]. Moreover, some MC implementations of jet quenching that modify partonic showers in PYTHIA, such as Q-PYTHIA, can generate soft particles at angles ∆> 0.8, but this treatment modiﬁes the fragmentation functions more severely than found in data [53, 54]. Angular scales for different jet quench- ing mechanisms in perturbative QCD are related to momentum scales through time evolu- tion of partonic interactions [23]. Especially for QCD cascades in a sufﬁciently large medium, angular broadening is independent of the path length, and this mechanism might therefore produce a cumulative effect even after taking averages over different events where jets travel different path lengths in the QGP. The medium response may not have the same correlation be- tween angular and momentum scales. The relative importance of each mechanism is unknown. Measuring the pT spectra of ⟨pT / ∥⟩as a function of ∆from the jet axis, denoted as ⟨pT / ∥⟩ptrk T ,∆, as discussed in Section 6, can provide information on the momentum scales at which certain quenching mechanisms become dominant. The analysis is performed for pp collisions, and two PbPb centrality selections of 30–100% and 0–30%. The resulting differential distributions in ⟨pT / ∥⟩ptrk T ,∆are shown for different regions of track pT (in terms of the colored boxes) as a function of ∆in the upper row of Fig. 1. The sum of ⟨pT / ∥⟩ptrk T ,∆for different ptrk T ranges as a function of ∆, ⟨pT / ∥⟩∆, are given by the open markers, and follow the leading jet at small ∆and subleading jet at large ∆. The cumulative values, ⟨pT / ∥⟩[0,∆] (i.e. from summing and smoothing the ⟨pT / ∥⟩∆over bins in ∆, starting at ∆= 0 and ending at the point of interest) are shown as dashed lines for pp and solid lines for PbPb. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets These lines demonstrate the evolution of the overall pT balance from small to large distances relative to the jet axis, reaching an overall balance close to zero only at large radii. The cumulative curve in PbPb collisions for 0–30% centrality is slightly narrower than for pp collisions. The distributions in pp collisions have characteristic features, and understanding these is im- portant for interpreting the PbPb results. The magnitude of the ⟨pT / ∥⟩∆in the ﬁrst bin, with ∆< 0.2, is related to the average dijet imbalance, and takes a negative value indicating that the momentum projection points along the direction of the leading jet. In the rest of the ∆bins, ⟨pT / ∥⟩∆takes a positive value, and ⟨pT / ∥⟩ptrk T ,∆for lower track pT make up larger fractions of ⟨pT / ∥⟩∆. We refer to the ⟨pT / ∥⟩ptrk T ,∆and ⟨pT / ∥⟩∆for bins with ∆> 0.2 as the “balancing distri- bution” of the corresponding quantity, because they reduce the large pT imbalance observed in the ﬁrst bin in ∆. The balancing distribution has a peak in the range 0.4 < ∆< 0.6, which is at the most likely ∆position for a third jet relative to the subleading jet. In PbPb collisions, the peak of the balancing ⟨pT / ∥⟩∆distribution shifts towards smaller angles (0.2 < ∆< 0.4). This can be due to the modiﬁcation in the fragmentation of the leading and subleading jets after quenching, as it occurs at angles close to their axes, where the low- pT particles make largest contributions. It is therefore not possible to claim a direct relation between the peak position of the balancing ⟨pT / ∥⟩∆distribution and the location of other jets in the event, unless only the highest-pT particles are considered, i.e. not likely to be related to the leading and subleading jets at large ∆values. The peak position of the balancing ⟨pT / ∥⟩ptrk T ,∆ distribution of the highest-pT particles is located at the same place as in pp collisions (0.4 < ∆< 0.6), but with smaller magnitude. This suggests that the position of a third jet relative to the subleading jet is not modiﬁed signiﬁcantly. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets However, the magnitude of ⟨pT / ∥⟩ptrk T ,∆for tracks with 8 < pT < 300 GeV associated with the third jet can be reduced for several reasons, such as quenching of the third jet, which makes its fragmentation softer, or a change in the ordering of the jets relative to original partonic conditions, i.e. leading parton losing more energy compared In PbPb collisions, the peak of the balancing ⟨pT / ∥⟩∆distribution shifts towards smaller angles (0.2 < ∆< 0.4). This can be due to the modiﬁcation in the fragmentation of the leading and subleading jets after quenching, as it occurs at angles close to their axes, where the low- pT particles make largest contributions. It is therefore not possible to claim a direct relation between the peak position of the balancing ⟨pT / ∥⟩∆distribution and the location of other jets in the event, unless only the highest-pT particles are considered, i.e. not likely to be related to the leading and subleading jets at large ∆values. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -20 -10 0 pp CMS (2.76 TeV) -1 5.3 pb Inclusive J A ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp [GeV] ∆ , T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 ∆ 0.5 1 1.5 -20 -10 0 PbPb 30-100% (2.76 TeV) -1 b µ 166 R = 0.3 t anti-k ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb ∆ 0.5 1 1.5 -5 0 5 (PbPb 30-100%) - pp ∆〉 \|\| T p 〈 PbPb - pp ∆ 0.5 1 1.5 -20 -10 0 > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η \| PbPb 0-30% ∆ 0.5 1 1.5 -5 0 5 (PbPb 0-30%) - pp ∥ , PbPb 0-30% ∆ Figure 1: (Color online) Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0– Figure 1: (Color online) Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0– 30% PbPb data for ﬁve track-pT ranges (colored boxes), for momentum ranges from 0.5 < pT < 1 GeV (light blue) to 8 < pT < 300 GeV (red), as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp (open squares) and PbPb data (open plus symbols). Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference between the PbPb and pp ⟨pT / ∥⟩ptrk T ,∆distributions according to the range in pT, as a function of ∆(colored boxes), and difference of ⟨pT / ∥⟩∆as a function of ∆(open circles), error bars and brackets represent statistical and systematic uncertainties, respectively. pT , 30% PbPb data for ﬁve track-pT ranges (colored boxes), for momentum ranges from 0.5 < pT < 1 GeV (light blue) to 8 < pT < 300 GeV (red), as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp (open squares) and PbPb data (open plus symbols). Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets The peak position of the balancing ⟨pT / ∥⟩ptrk T ,∆ 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets 13 ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -20 -10 0 pp CMS (2.76 TeV) -1 5.3 pb Inclusive J A ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp [GeV] ∆ , T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 ∆ 0.5 1 1.5 -20 -10 0 PbPb 30-100% (2.76 TeV) -1 b µ 166 R = 0.3 t anti-k ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb ∆ 0.5 1 1.5 -5 0 5 (PbPb 30-100%) - pp ∆〉 \|\| T p 〈 PbPb - pp ∆ 0.5 1 1.5 -20 -10 0 > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η \| PbPb 0-30% ∆ 0.5 1 1.5 -5 0 5 (PbPb 0-30%) - pp Figure 1: (Color online) Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0– 30% PbPb data for ﬁve track-pT ranges (colored boxes), for momentum ranges from 0.5 < pT < 1 GeV (light blue) to 8 < pT < 300 GeV (red), as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp (open squares) and PbPb data (open plus symbols). Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference between the PbPb and pp ⟨pT / ∥⟩ptrk T ,∆distributions according to the range in pT, as a function of ∆(colored boxes), and difference of ⟨pT / ∥⟩∆as a function of ∆(open circles), error bars and brackets represent statistical and systematic uncertainties, respectively. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference between the PbPb and pp ⟨pT / ∥⟩ptrk T ,∆distributions according to the range in pT, as a function of ∆(colored boxes), and difference of ⟨pT / ∥⟩∆as a function of ∆(open circles), error bars and brackets represent statistical and systematic uncertainties, respectively. to the subleading parton, which causes the third jet to be found in the leading jet hemisphere, instead of the subleading jet hemisphere. A comparison of pp and PbPb collisions is provided in the lower row of Fig. 1, showing the difference in PbPb and pp for ⟨pT / ∥⟩ptrk T ,∆, and ⟨pT / ∥⟩∆as a function of ∆. For central events, the ﬁrst bin with ∆< 0.2 ⟨pT / ∥⟩ptrk T ,∆for high-pT tracks and ⟨pT / ∥⟩∆point in the leading jet direction, although the excess is not signiﬁcant. While in the second bin with 0.2 < ∆< 0.4, there is a signiﬁcant positive excess in ⟨pT / ∥⟩∆. The excess towards the subleading jet in this bin may either be because the leading jet is narrower, or the subleading jet wider in PbPb collisions compared to pp collisions. The excess in ⟨pT / ∥⟩∆along the subleading jet direction extends up to larger angles (∆≈1–1.2), with decreasing signiﬁcance. In this angular range, there is an excess in ⟨pT / ∥⟩ptrk T ,∆for tracks with pT that fall in the ranges of 0–0.5, 0.5–1, and 1–2 GeV, and a depletion for particles with pT > 4 GeV. This is consistent with results shown in the previous section and earlier CMS studies that demonstrate that the small-angle imbalance towards the leading jet is compensated by particles of small pT emitted at large angles to the jet axes [12]. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -15 -10 -5 0 5 pp CMS (2.76 TeV) -1 5.3 pb < 0.22 J A ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp [GeV] ∆ , T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 ∆ 0.5 1 1.5 -15 -10 -5 0 5 PbPb 30-100% (2.76 TeV) -1 b µ 166 R = 0.3 t anti-k ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb ∆ 0.5 1 1.5 -5 0 5 (PbPb 30-100%) - pp ∆〉 \|\| T p 〈 PbPb - pp ∆ 0.5 1 1.5 -15 -10 -5 0 5 > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η \| PbPb 0-30% ∆ 0.5 1 1.5 -5 0 5 (PbPb 0-30%) - pp ∆ Figure 2: (Color online) Same as Fig. 1, but with a balanced dijet selection (AJ < 0.22). Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0–30% PbPb data for ﬁve track pT ranges (colored boxes), as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp (open squares) and for PbPb data (open plus symbols). Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference in the ⟨pT / ∥⟩ptrk T ,∆distributions for the PbPb and pp according to the range in pT, as a function of ∆(colored boxes), and difference of ⟨pT / ∥⟩∆as a function of ∆(open circles). Error bars and brackets represent statistical and systematic uncertainties, respectively. The y-axis range on the top panels are smaller than in Fig. 1. 8.1 Dependence of the pT balance in pp and PbPb on opening angles around jets 14 8 Results ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -15 -10 -5 0 5 pp CMS (2.76 TeV) -1 5.3 pb < 0.22 J A ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp [GeV] ∆ , T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 ∆ 0.5 1 1.5 -15 -10 -5 0 5 PbPb 30-100% (2.76 TeV) -1 b µ 166 R = 0.3 t anti-k ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb ∆ 0.5 1 1.5 -5 0 5 (PbPb 30-100%) - pp ∆〉 \|\| T p 〈 PbPb - pp ∆ 0.5 1 1.5 -15 -10 -5 0 5 > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η \| PbPb 0-30% ∆ 0.5 1 1.5 -5 0 5 (PbPb 0-30%) - pp Figure 2: (Color online) Same as Fig. 1, but with a balanced dijet selection (AJ < 0.22). Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0–30% PbPb data for ﬁve track pT ranges (colored boxes), as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp (open squares) and for PbPb data (open plus symbols). Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference in the ⟨pT / ∥⟩ptrk T ,∆distributions for the PbPb and pp according to the range in pT, as a function of ∆(colored boxes), and difference of ⟨pT / ∥⟩∆as a function of ∆(open circles). Error bars and brackets represent statistical and systematic uncertainties, respectively. The y-axis range on the top panels are smaller than in Fig. 1. 8.2 Study of the pT balance in pp and PbPb collisions, as a function of opening angles around jets in bins of AJ More information can be obtained by repeating the previous study as a function of dijet asym- metry AJ. The results for a sample containing more balanced dijets (AJ < 0.22) is shown in Fig. 2, again comparing pp data with two PbPb centrality bins. As expected, ⟨pT / ∥⟩∆and ⟨pT / ∥⟩ptrk T ,∆for all track pT take smaller values compared to inclusive AJ selection, meaning that events with a more balanced dijet selection show an overall better pT balance in both small ∆< 0.2, as well as larger ∆. This is also seen in the difference in ⟨pT / ∥⟩∆for PbPb and pp collisions, although, as before, an preference of ⟨pT / ∥⟩ptrk T ,∆for low-pT tracks to point along the subleading side can be seen for central PbPb events. Complementary to the selection of more balanced dijets, Fig. 3 shows a selection for unbalanced dijets with AJ > 0.22. The AJ selection is reﬂected in the overall larger contributions in the small- and large-angle regions relative to the jet axes. This large AJ selection, which enhances 8.2 Study of the pT balance in pp and PbPb collisions, as a function of opening angles around jets in bins of AJ 15 15 J ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -40 -20 0 pp CMS (2.76 TeV) -1 5.3 pb > 0.22 J A ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp [GeV] ∆ , T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 ∆ 0.5 1 1.5 -40 -20 0 PbPb 30-100% (2.76 TeV) -1 b µ 166 R = 0.3 t anti-k ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb ∆ 0.5 1 1.5 -5 0 5 (PbPb 30-100%) - pp ∆〉 \|\| T p 〈 PbPb - pp ∆ 0.5 1 1.5 -40 -20 0 > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η \| PbPb 0-30% ∆ 0.5 1 1.5 -5 0 5 (PbPb 0-30%) - pp Figure 3: (Color online) Same as Fig. 1, but with an unbalanced dijet selection (AJ > 0.22). 8.2 Study of the pT balance in pp and PbPb collisions, as a function of opening angles around jets in bins of AJ Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0–30% PbPb data for ﬁve track pT ranges, as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp and for PbPb data. Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference in the ⟨pT / ∥⟩ptrk T ,∆distributions for the PbPb and pp. Error bars and brackets represent statistical and systematic uncertainties, respectively. The y-axis range on the top panels are larger than in Fig. 1. J ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -40 -20 0 pp CMS (2.76 TeV) -1 5.3 pb > 0.22 J A ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp [GeV] ∆ , T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 ∆ 0.5 1 1.5 -40 -20 0 PbPb 30-100% (2.76 TeV) -1 b µ 166 R = 0.3 t anti-k ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb ∆ 0.5 1 1.5 -5 0 5 (PbPb 30-100%) - pp ∆〉 \|\| T p 〈 PbPb - pp ∆ 0.5 1 1.5 -40 -20 0 > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η \| PbPb 0-30% ∆ 0.5 1 1.5 -5 0 5 (PbPb 0-30%) - pp Fi 3 (C l li ) S Fi 1 b i h b l d dij l i (A 0 22) ∆ Figure 3: (Color online) Same as Fig. 1, but with an unbalanced dijet selection (AJ > 0.22). Upper row: ⟨pT / ∥⟩ptrk T ,∆distributions for pp, and for 30–100% and 0–30% PbPb data for ﬁve track pT ranges, as a function of ∆. Also shown is ⟨pT / ∥⟩∆as a function of ∆for pp and for PbPb data. Dashed lines (pp) and solid lines (PbPb) show ⟨pT / ∥⟩[0,∆] (i.e. integrating the ⟨pT / ∥⟩∆over ∆from ∆= 0 up to the point of interest). Lower row: Difference in the ⟨pT / ∥⟩ptrk T ,∆distributions for the PbPb and pp. Error bars and brackets represent statistical and systematic uncertainties, respectively. 8.3 Dependence of dijet asymmetry on pT balance and multiplicity difference in jet hemispheres To study the pT ﬂow relative to the dijet system as a function of event properties, such as centrality and AJ, in more detail, the⟨pT / ∥⟩ptrk T ,∆is summed over all annuli to obtain ⟨pT / ∥⟩ptrk T , i.e. the average pT balance in the event calculated for a given range of track pT. In Fig. 4, we display ⟨pT / ∥⟩ptrk T for different ranges of track pT (displayed in terms of the colored boxes) as a function of AJ, ranging from almost balanced to very unbalanced dijets in pp collisions, and in four selections of PbPb centrality from most peripheral to most central. The balance in the event for all tracks with pT > 0.5 GeV, denoted as ⟨pT / ∥⟩Σ, which is obtained by adding up the ⟨pT / ∥⟩ptrk T for different pT ranges, is also included, and shown as open markers, with associated systematic uncertainties as brackets around the points. In PbPb events, overall pT is balanced to better than 10 GeV, i.e. \|⟨pT / ∥⟩Σ\| < 10 GeV for all AJ selections. The small negative trend in ⟨pT / ∥⟩Σ as a function of AJ is observed also in pp events, and in generator-level PYTHIA events, once the pT threshold set on charged particles and the acceptance of the tracker are imposed. When selecting events containing dijets with AJ > 0.11, an expected excess of high-pT particles in the direction of the leading jet (indicated by the red areas in Fig. 4) is seen for all selections in pp and PbPb collisions. For pp and peripheral PbPb collisions, this excess is mostly balanced by particles with intermediate pT of 2–8 GeV. Going to more central collisions, ⟨pT / ∥⟩ptrk T on the subleading jet side is modiﬁed from the intermediate pT range towards low pT (0.5–2 GeV). This effect is most pronounced for events with large AJ in central PbPb collisions. The lower row of Fig. 4 shows the difference between ⟨pT / ∥⟩ptrk T in PbPb and pp collisions, after requiring the speciﬁc PbPb collision centralities and dijet imbalance. While the contributions from different pT ranges are similar for pp and peripheral PbPb collisions, a difference can be seen for central collisions, where a signiﬁcant excess of low-pT charged particles is observed for asymmetric jets in PbPb collisions. 8.2 Study of the pT balance in pp and PbPb collisions, as a function of opening angles around jets in bins of AJ The y-axis range on the top panels are larger than in Fig. 1. the fraction of jets having undergone signiﬁcant energy loss in PbPb collisions, also enhances the differences between PbPb and pp, as shown in the lower row of Fig. 3. It is important to note that in pp collisions, only 30% of selected dijet events have AJ > 0.22, but this number increases to 42% for central PbPb selections. This again suggests the presence of an additional mechanism creating asymmetric dijets in PbPb, i.e. parton energy loss in the medium. Consistent with this picture, the AJ dependence of the ⟨pT / ∥⟩ptrk T ,∆distributions in PbPb and pp collisions and their difference suggests that asymmetric dijet systems in pp and PbPb collisions are created through different mechanisms, with semi-hard radiation (e.g., three- jet events) dominating pp collisions. In contrast, a large fraction of asymmetric dijet events in PbPb is created through a differential energy loss mechanism as the partons traverse the medium, which leads to the observed excess in ⟨pT / ∥⟩ptrk T ,∆for the low-pT bins. The depletion of high-pT particle contributions at large angles in PbPb is more dominant with AJ > 0.22 relative to an inclusive AJ selection, because of the difference in relative fractions of three-jet events among all selected events. 16 8 Results 8.3 Dependence of dijet asymmetry on pT balance and multiplicity difference in jet hemispheres Systematic uncertainties are shown only for ⟨pT / ∥⟩Σ, and not for ⟨pT / ∥⟩ptrk T . Uncertainties in ⟨pT / ∥⟩Σ provide an upper bound on systematic uncertainties for individual pT ranges, as uncertainties in low-pT particles are, in fact, signiﬁcantly smaller. The excess observed in low-pT particles in the range of 0.5–2 GeV has therefore a signiﬁcance of 3–4 standard deviations for AJ > 0.11 for most central events. The difference in ⟨pT / ∥⟩ between PbPb and pp collisions for all tracks with pT > 0.5 GeV is consistent with zero across all centrality and AJ selections. The overall pT balance observed through ⟨pT / ∥⟩Σ in PbPb events agrees with pp events, within systematic and statistical uncertainties, over all ranges of AJ and centrality, while the ⟨pT / ∥⟩ptrk T distributions show excess of low-pT particles. This implies that there are more particles in the subleading jet hemispheres compared to the leading jet hemispheres, because more particles are required to obtain the same pT sum. Figure 5 shows the mean difference in multiplicities between leading and subleading jet hemi- spheres, denoted as ⟨∆mult⟩, as a function of AJ and collision centrality. The ⟨∆mult⟩is pre- sented for both PbPb and pp collisions. Measurements in pp collisions are in good agreement with PYTHIA and PYTHIA+HYDJET simulations. In general, the ⟨∆mult⟩increases as a function of AJ in pp, PbPb, PYTHIA, and PYTHIA+HYDJET events. The events in pp collisions with large AJ contain a larger fraction of three-jet or multijet events, where more particles are produced in the direction of the subleading jet. The observed increase in ⟨∆mult⟩for pp collisions with increasing AJ is therefore expected. Going from peripheral (50–100%) to central (0–10%) PbPb events, for a given AJ selection an excess in ⟨∆mult⟩is visible compared to pp collisions. 8.3 Dependence of dijet asymmetry on pT balance and multiplicity difference in jet hemispheres The difference in ⟨∆mult⟩between pp and PbPb collisions increases monotonically as a function 8.4 Dependence of transverse momentum balance on jet distance parameter R 17 [GeV] T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 PbPb 50-100% (2.76 TeV) -1 b µ 166 Σ〉 \|\| T p 〈 PbPb J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 50-100%) - pp Σ〉 \|\| T p 〈 PbPb - pp J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 R = 0.3 t anti-k PbPb 30-50% J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 30-50%) - pp J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 PbPb 10-30% > 120 GeV T,1 p > 50 GeV T,2 p J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 10-30%) - pp J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 PbPb 0-10% \| < 1.6 2 η \|,\| 1 η\| /6 π > 5 1,2 φ ∆ J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 0-10%) - pp J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -60 -40 -20 0 20 40 CMS pp (2.76 TeV) -1 5.3 pb Σ〉 \|\| T p 〈 pp Gen. PYTHIA Figure 4: (Color online) Upper row has ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ in pp collisions (leftmost) and in four selections of PbPb for collision centralities from 50–100% to 0–10%. The open markers show ⟨pT / ∥⟩Σ, pT balance for tracks with 0.5 < pT < 300 GeV, while the colored boxes show the ⟨pT / ∥⟩ptrk T contributions for different track pT ranges. For each panel, ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ values are shown as a function of dijet asymmetry. The lower row shows the difference between ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ for PbPb and pp data. Error bars and brackets represent statistical and systematic uncertainties, respectively. 8.3 Dependence of dijet asymmetry on pT balance and multiplicity difference in jet hemispheres [GeV] T trk p〉 \|\| T p 〈 \| < 2.4 trk η \| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 8.0-300.0 J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 PbPb 50-100% (2.76 TeV) -1 b µ 166 Σ〉 \|\| T p 〈 PbPb J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 50-100%) - pp Σ〉 \|\| T p 〈 PbPb - pp J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 R = 0.3 t anti-k PbPb 30-50% J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 30-50%) - pp J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 PbPb 10-30% > 120 GeV T,1 p > 50 GeV T,2 p J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 10-30%) - pp J A 0.1 0.2 0.3 0.4 -60 -40 -20 0 20 40 PbPb 0-10% \| < 1.6 2 η \|,\| 1 η\| /6 π > 5 1,2 φ ∆ J A 0.1 0.2 0.3 0.4 -20 -10 0 10 20 (PbPb 0-10%) - pp J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -60 -40 -20 0 20 40 CMS pp (2.76 TeV) -1 5.3 pb Σ〉 \|\| T p 〈 pp Gen. PYTHIA J A 2 0 J A Figure 4: (Color online) Upper row has ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ in pp collisions (leftmost) and in four selections of PbPb for collision centralities from 50–100% to 0–10%. The open markers show ⟨pT / ∥⟩Σ, pT balance for tracks with 0.5 < pT < 300 GeV, while the colored boxes show the ⟨pT / ∥⟩ptrk T contributions for different track pT ranges. For each panel, ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ values are shown as a function of dijet asymmetry. The lower row shows the difference between ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ for PbPb and pp data. Error bars and brackets represent statistical and systematic uncertainties, respectively. of AJ at all collision centralities, with the biggest effect seen for most central PbPb collisions. This is consistent with the expected dependence of medium-induced energy loss on collision centrality, where systems of the largest size (i.e. smallest centrality) should show the largest medium-related effects. The multiplicity difference is up to ≈15 particles in the most central 0–10 % collisions. 8.4 Dependence of transverse momentum balance on jet distance parameter R In pp collisions, jets clustered with small R are narrower and fragment into components with higher pT than jets clustered with large R. In addition, using small R tends to bias the clustered jets to contain a larger fraction of quark jets [55, 56]. Changing the R parameter can provide a handle on the size and shower proﬁles of individual jets. In heavy ion collisions, studying the R dependence of momentum ﬂow in dijet events makes it possible to investigate whether jet quenching mechanisms act differently on jets with different fragmentation patterns on a jet-by-jet basis. It is important to note that there is an overlap in the ﬁnal set of dijet events obtained for different R parameters, and therefore it is not possible to interpret the dependence of the pT-balance distributions on R as simply a dependence on jet size. A change in R can induce a modiﬁcation in pT / ∥in two ways: events that satisfy the dijet requirements for one R can fail for another R value, or events that satisfy the dijet requirements for both R parameters, but for which the ordering of jets change, can impact φdijet, as well as the value of parameters used in the binning of the measurements, such as AJ and ∆. The requirements on the pT of leading and subleading jets are the main sources of variations in the ﬁnal set of dijet events for different R parameters. For each R, a jet pT selection translates into a different requirement on initial parton pT. 8.4 Dependence of transverse momentum balance on jet distance parameter R 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 CMS 50-100% (2.76 TeV) -1 b µ PbPb 166 (2.76 TeV) -1 pp 5.3 pb PYTHIA+HYDJET PYTHIA J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 R = 0.3 t anti-k > 120 GeV T,1 p > 50 GeV T,2 p /6 π > 5 1,2 φ ∆ \| < 1.6 2 η \|, \| 1 η\| 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 30-50% J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 > 0.5 GeV trk T p \| < 2.4 trk η\| 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 10-30% J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 0-10% J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 J A J A Figure 5: (Color online) Upper panels show the comparison of the mean difference in multiplic- ity ⟨∆mult⟩between the subleading jet hemisphere and leading jet hemisphere, as a function of dijet asymmetry AJ for pp (blue squares), PbPb (red ﬁled circles), PYTHIA (dashed histogram), and PYTHIA+HYDJET events (black histogram). The centralities of PbPb collisions are 50–100%, 30–50%, 10–30 %, and 0–10%, respectively, from leftmost to rightmost panel. Lower panels provide the difference in ⟨∆mult⟩between PbPb and pp collisions. Statistical and systematic uncertainties are shown as error bars and brackets, respectively. parton is recovered using jets of smaller size. Although fewer events pass the dijet requirement for R = 0.2 jets, strictly speaking, such events do not form a subset of dijet events with larger R parameters. A small fraction of R = 0.2 dijet events (4–7% in PbPb collisions and 2–4% in pp collisions) does not satisfy the dijet requirements for other R values, mainly because jets fall outside of the η range or the ∆φ requirement for the dijet pair. This can happen because of the merging of the subleading and third jets, and because of the resolution in jet angular direction. Such events make up a statistically negligible contribution to the results and are therefore not the focus of the discussion. 8.4 Dependence of transverse momentum balance on jet distance parameter R A smaller fraction of the initial energy of the 18 8 Results 8 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 CMS 50-100% (2.76 TeV) -1 b µ PbPb 166 (2.76 TeV) -1 pp 5.3 pb PYTHIA+HYDJET PYTHIA J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 R = 0.3 t anti-k > 120 GeV T,1 p > 50 GeV T,2 p /6 π > 5 1,2 φ ∆ \| < 1.6 2 η \|, \| 1 η\| 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 30-50% J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 > 0.5 GeV trk T p \| < 2.4 trk η\| 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 10-30% J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 0.1 0.2 0.3 0.4 > mult ∆ Hemisphere < 10 20 30 0-10% J A 0.1 0.2 0.3 0.4 PbPb - pp -5 0 5 10 15 20 25 Figure 5: (Color online) Upper panels show the comparison of the mean difference in multiplic- ity ⟨∆mult⟩between the subleading jet hemisphere and leading jet hemisphere, as a function of dijet asymmetry AJ for pp (blue squares), PbPb (red ﬁled circles), PYTHIA (dashed histogram), and PYTHIA+HYDJET events (black histogram). The centralities of PbPb collisions are 50–100%, 30–50%, 10–30 %, and 0–10%, respectively, from leftmost to rightmost panel. Lower panels provide the difference in ⟨∆mult⟩between PbPb and pp collisions. Statistical and systematic uncertainties are shown as error bars and brackets, respectively. 8.4 Dependence of transverse momentum balance on jet distance parameter R The fraction of events that pass the dijet selection both for the largest R = 0.5 and for other values are shown in the second column of Table 4, without matching the directions of the jets. Compared to pp collisions, the fraction of events that pass both cutoffs on jets is reduced in PbPb collisions more rapidly as R decreases. This observation is qualitatively consistent with the measurement showing that inclusive jet suppression is smaller in PbPb collisions for large R values [57], which can be interpreted as due to the recovery of part of the energy lost in the initial hard scatter of partons. Additional information can therefore be extracted by requiring the leading and subleading jets with a given R to be in the same direction as the corresponding jets found using R = 0.5. As shown in the third column of Table 4, the fraction of such events is similar for pp and PbPb collisions. These events produce almost no change in φdijet and the jet axes, which change only slightly due to jet angular resolution, and therefore yield approximately the same pT / ∥. However, these events can accommodate the change in the pT of jets that originate from the 8.4 Dependence of transverse momentum balance on jet distance parameter R 19 8.4 Table 4: Overlap in event selections for 0–100% PbPb and pp collisions. The second column gives the percentage of events that pass dijet selections and a tight pseudorapidity requirement ( \|η\| < 0.6 ) for R = 0.5, and an additional dijet selection also required for a smaller R value. In columns 3–6 the leading and subleading jets with R = 0.5 are matched to the leading and subleading jets with smaller R values, requiring only R = 0.5 selection on jets. The third column shows the percentage of these events where both leading and subleading jets point in the same direction (∆i = √ (ηR i −ηR=0.5 i )2 + (φR i −φR=0.5 i )2 < 0.5 for i = 1 and 2). The average value of the ratio of pT of the leading and subleading jets at jet for a given R, to their pT for R = 0.5 are shown in the fourth and ﬁfth columns, respectively. 8.4 Dependence of transverse momentum balance on jet distance parameter R The sixth column shows percentage of events in which subleading jets with the given R parameter match the R = 0.5 leading jet, and the leading jet matches the R = 0.5 subleading jet. Additional Matched Swapped R dijet selection [%] jet directions [%] ⟨pR T,1/pR=0.5 T,1 ⟩ ⟨pR T,2/pR=0.5 T,2 ⟩ jet directions [%] PbPb 0.2 48 ± 2 83 ± 5 0.89 ± 0.001 0.79 ± 0.002 10 ± 3 0.3 62 ± 2 90 ± 4 0.93 ± 0.002 0.88 ± 0.004 7 ± 3 0.4 77 ± 1 94 ± 3 0.96 ± 0.002 0.94 ± 0.005 3 ± 2 pp 0.2 58 ± 2 83 ± 5 0.91 ± 0.001 0.83 ± 0.002 14 ± 3 0.3 73 ± 2 90 ± 4 0.95 ± 0.001 0.90 ± 0.001 8 ± 3 0.4 86 ± 1 95 ± 3 0.98 ± 0.001 0.96 ± 0.001 4 ± 2 same initial hard-scattered parton for different R parameters. For jets matched to each other spatially, the ratio of the pT of the leading or subleading jet at some given R to respective jets with R = 0.5, ⟨pR T,1(2)/pR=0.5 T,1(2) ⟩, is calculated and the values are shown in columns 4 and 5 in Table 4. As expected, in both PbPb and pp collisions, ⟨pR T,1/pR=0.5 T,1 ⟩and ⟨pR T,2/pR=0.5 T,2 ⟩are reduced as R gets smaller. In PbPb collisions, a smaller fraction of jet pT is recovered at small R for both the leading and subleading jets, which may be due to the broadening of quenched jets. This effect is larger for the subleading than for the leading jet. same initial hard-scattered parton for different R parameters. For jets matched to each other spatially, the ratio of the pT of the leading or subleading jet at some given R to respective jets with R = 0.5, ⟨pR T,1(2)/pR=0.5 T,1(2) ⟩, is calculated and the values are shown in columns 4 and 5 in Table 4. As expected, in both PbPb and pp collisions, ⟨pR T,1/pR=0.5 T,1 ⟩and ⟨pR T,2/pR=0.5 T,2 ⟩are reduced as R gets smaller. In PbPb collisions, a smaller fraction of jet pT is recovered at small R for both the leading and subleading jets, which may be due to the broadening of quenched jets. This effect is larger for the subleading than for the leading jet. 8.4 Dependence of transverse momentum balance on jet distance parameter R same initial hard-scattered parton for different R parameters. For jets matched to each other spatially, the ratio of the pT of the leading or subleading jet at some given R to respective jets with R = 0.5, ⟨pR T,1(2)/pR=0.5 T,1(2) ⟩, is calculated and the values are shown in columns 4 and 5 in Table 4. As expected, in both PbPb and pp collisions, ⟨pR T,1/pR=0.5 T,1 ⟩and ⟨pR T,2/pR=0.5 T,2 ⟩are reduced as R gets smaller. In PbPb collisions, a smaller fraction of jet pT is recovered at small R for both the leading and subleading jets, which may be due to the broadening of quenched jets. This effect is larger for the subleading than for the leading jet. As R parameters become smaller, leading and subleading jets fall below the pT requirements. Most of the time, the leading jet satisﬁes the pT selection for R = 0.5, but falls below the threshold for smaller R, because the subleading jet pT is already biased towards values above the 50 GeV threshold by the leading jet with pT > 120 GeV in the event. However, as shown in Figs. 2 and 3, for R = 0.3 jets the ⟨pT / ∥⟩ptrk T ,∆signal is dominated by dijet events with large imbalance, which is true for all other R parameters as well. For events with AJ > 0.22, ⟨pT,2⟩≈ 70–80 GeV is sufﬁciently close to the 50 GeV threshold for subleading jets falling below the threshold to create sizable effects on the results. The last column of Table 4 gives the fraction of events with swapped leading and subleading jets compared to those with R = 0.5. For these events, the pT / ∥has an opposite sign relative to the value for R = 0.5, as φdijet points in the opposite hemisphere. Especially in pp colli- sions, swapping of the leading and subleading jet is the main source of events in which the jet directions are not matched. In PbPb collisions, swapping is slightly less frequent than in pp collisions, suggesting that the third jet may be replacing the subleading jet. For events that satisfy dijet requirements for different R parameters, the pT / ∥in each event can still change as a function of R because of the swapping of jets in the dijet pairs, and the replacement of the subleading jet by the third jet. The dependence of ⟨pT / ∥⟩ptrk T ,∆on ∆and R is shown in Fig. 6, without any AJ requirement, for 8.4 Dependence of transverse momentum balance on jet distance parameter R pendence of ⟨pT / ∥⟩ptrk T ,∆on ∆and R is shown in Fig. 6, without any AJ requirement, for The dependence of ⟨pT / ∥⟩ptrk T ,∆on ∆and R is shown in Fig. 6, without any AJ requirement, for 20 8 Results ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -20 -10 0 pp R = 0.2 (2.76 TeV) -1 5.3 pb ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -20 -10 0 PbPb R = 0.2 (2.76 TeV) -1 b µ 166 ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -5 0 PbPb - pp R = 0.2 ∆ 0.5 1 1.5 -20 -10 0 pp R = 0.3 ∆ 0.5 1 1.5 -20 -10 0 PbPb R = 0.3 ∆ 0.5 1 1.5 -5 0 PbPb - pp R = 0.3 ∆ 0.5 1 1.5 -20 -10 0 pp R = 0.4 ∆ 0.5 1 1.5 -20 -10 0 PbPb R = 0.4 ∆ 0.5 1 1.5 -5 0 PbPb - pp R = 0.4 ∆ 0.5 1 1.5 -20 -10 0 pp R = 0.5 ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp ∆ 0.5 1 1.5 -20 -10 0 ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb PbPb R = 0.5 ∆ 0.5 1 1.5 -5 0 PbPb - pp R = 0.5 ∆〉 \|\| T p 〈 PbPb - pp CMS Inclusive J A Jet; 0-30% t anti-k > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η\| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 [GeV] ∆ , T trk p〉 \|\| T p 〈 8.0-300.0 \| < 2.4 trk η\| Figure 6: (Color online) Upper row shows ⟨pT / ∥⟩ptrk T ,∆in pp collisions as a function of ∆, for a distance parameter R = 0.2, 0.3, 0.4, and 0.5, from left to right for different ranges of track pT, and ⟨pT / ∥⟩∆(i.e. ⟨pT / ∥⟩ptrk T ,∆summed over all pT for a given ∆bin). Dashed lines indicate cumu- lative results for ⟨pT / ∥⟩[0,∆] in pp, for each distance parameter (i.e. integrating ⟨pT / ∥⟩∆over the ∆ range from ∆= 0 to the point of interest). 8.4 Dependence of transverse momentum balance on jet distance parameter R Middle row provides ⟨pT / ∥⟩ptrk T ,∆and ⟨pT / ∥⟩∆in PbPb collisions of centrality range 0–30% as a function of ∆, for distance parameters R = 0.2, 0.3, 0.4, and 0.5 from left to right. Solid line indicates ⟨pT / ∥⟩[0,∆] in PbPb for each distance parameter. Lower row has the difference between PbPb and pp. Error bars and brackets represent statisti- cal and systematic uncertainties, respectively. The results are inclusive in the dijet asymmetry parameter AJ. ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -20 -10 0 pp R = 0.2 (2.76 TeV) -1 5.3 pb ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -20 -10 0 PbPb R = 0.2 (2.76 TeV) -1 b µ 166 ∆ 0.5 1 1.5 [GeV] 〉 \|\| T p 〈 -5 0 PbPb - pp R = 0.2 ∆ 0.5 1 1.5 -20 -10 0 pp R = 0.3 ∆ 0.5 1 1.5 -20 -10 0 PbPb R = 0.3 ∆ 0.5 1 1.5 -5 0 PbPb - pp R = 0.3 ∆ 0.5 1 1.5 -20 -10 0 pp R = 0.4 ∆ 0.5 1 1.5 -20 -10 0 PbPb R = 0.4 ∆ 0.5 1 1.5 -5 0 PbPb - pp R = 0.4 ∆ 0.5 1 1.5 -20 -10 0 pp R = 0.5 ∆〉 \|\| T p 〈 pp ∆ 0, 〉 \|\| T p 〈 pp ∆ 0.5 1 1.5 -20 -10 0 ∆〉 \|\| T p 〈 PbPb ∆ 0, 〉 \|\| T p 〈 PbPb PbPb R = 0.5 ∆ 0.5 1 1.5 -5 0 PbPb - pp R = 0.5 ∆〉 \|\| T p 〈 PbPb - pp CMS Inclusive J A Jet; 0-30% t anti-k > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 0.6; 2 η \|,\| 1 η\| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 [GeV] ∆ , T trk p〉 \|\| T p 〈 8.0-300.0 \| < 2.4 trk η\| ∥ Inclusive J A ∆ Figure 6: (Color online) Upper row shows ⟨pT / ∥⟩ptrk T ,∆in pp collisions as a function of ∆, for a distance parameter R = 0.2, 0.3, 0.4, and 0.5, from left to right for different ranges of track pT, and ⟨pT / ∥⟩∆(i.e. ⟨pT / ∥⟩ptrk T ,∆summed over all pT for a given ∆bin). PbPb system, the peak also shifts towards greater ∆, but less than in pp collisions due 8.4 Dependence of transverse momentum balance on jet distance parameter R 6, is narrower than in pp collisions, shown by the dashed black curves, meaning that the slope is larger in PbPb relative to pp collisions. This becomes slightly more signiﬁcant at R = 0.5, where bias in gluon or quark jets that have large angular width becomes smaller. This is also reﬂected in the increase in the magnitude of ⟨pT / ∥⟩∆in the leading jet direction in the ﬁrst bin, and in the subleading jet direction in the second bin. This modiﬁcation is dominated by particles with pT > 2 GeV, and may arise from quenching effects, causing leading jets to narrow or subleading jets to widen in central PbPb relative to pp collisions. To summarize the dependence of differences in pT balance among different R bins on AJ, and to investigate the observed changes in the associated track pT spectrum in more central events, our measurement of the dependence of the pT balance on R and AJ, is shown in Fig. 7 for pp and 0–10% central PbPb events, respectively, in the top and middle rows. The leftmost panels correspond to a selection of R = 0.2 jets, while the rightmost panels correspond to R = 0.5. For pp collisions, there is a slight decrease in the magnitude of signal in each pT range as R increases. This behavior is consistent with the observed reduction in the incone ⟨pT / ∥⟩ptrk T ,∆for high-pT tracks with ∆< 0.2 shown in the top panels of Fig. 6 as a function of R, which was discussed above, and is also observed in generator-level PYTHIA. This kind of behavior is not observed in central PbPb events. The bottom row of Fig. 7 displays the difference between PbPb and pp results. The R parameter is correlated with a small change in the magnitude of the ⟨pT / ∥⟩ptrk T excess of low-pT particles, as jets of larger R give a greater excess. When pT ranges 0.5–2.0 GeV are combined, the increase in the low-pT excess becomes more signiﬁcant. The systematic uncertainties shown in the plot are dominated primarily by the pT range 8.0–300.0 GeV, and as such cannot be used to charac- terize the signiﬁcance of ⟨pT / ∥⟩ptrk T in the low track-pT ranges, nor the slight dependence on the distance parameter in the low-pT excess. 8.4 Dependence of transverse momentum balance on jet distance parameter R Dashed lines indicate cumu- lative results for ⟨pT / ∥⟩[0,∆] in pp, for each distance parameter (i.e. integrating ⟨pT / ∥⟩∆over the ∆ range from ∆= 0 to the point of interest). Middle row provides ⟨pT / ∥⟩ptrk T ,∆and ⟨pT / ∥⟩∆in PbPb collisions of centrality range 0–30% as a function of ∆, for distance parameters R = 0.2, 0.3, 0.4, and 0.5 from left to right. Solid line indicates ⟨pT / ∥⟩[0,∆] in PbPb for each distance parameter. Lower row has the difference between PbPb and pp. Error bars and brackets represent statisti- cal and systematic uncertainties, respectively. The results are inclusive in the dijet asymmetry parameter AJ. pp and for PbPb events with 0–30% centralities. The R-dependent evolution in pp collisions, which is attributed to the softening and broadening of jets, can be seen as a shift in the position of the sign change of ⟨pT / ∥⟩ptrk T ,∆and as a decrease in the total imbalance within the jet cones ∆≲0.2–0.4 . Moreover, the peaking point of the balancing distribution shifts towards larger ∆, as jet distance parameter R increases (from ∆= 0.2–0.4 for R = 0.2 jets, to ∆= 0.6–1.0 for R = 0.5 jets). As stated for R = 0.3 jets in Section 8.1, the peak position is correlated with the most likely position of the third jet relative to the subleading jet, which also moves to larger angles by increasing R. 8.4 Dependence of transverse momentum balance on jet distance parameter R 21 8.4 to the additional soft particles at small angles associated to the quenching of the dijet pair and reduction in the number of high-pT particles associated with the third jet. In the PbPb−pp bottom panels, this manifests in the depletion of higher ranges at pT, 4–8 and 8–300 GeV, which shift to greater angular distance with increasing R. There is a modest increase observed in the excess in the pT ranges of 0.5–1 and 1–2 GeV with increasing R. The overall distribution in the low-pT excess in PbPb relative to pp does not change signiﬁcantly with the distance parameter, and especially not at larger angular distance ∆. There is a hint that the ⟨pT / ∥⟩[0,∆] distribution in central PbPb collisions, shown by the black curves in Fig. 8.4 Dependence of transverse momentum balance on jet distance parameter R The sum of track pT ranges ⟨pT / ∥⟩Σ is insensitive to the distance parameter, and the difference between PbPb and pp collisions is consistent with zero for all R values. Finally, the multiplicity associated with excess of low-pT particles shown in Figs. 6 and 7, and the charged-particle spectrum for ⟨∆mult⟩are given in Fig. 8 for events with 0–30% centrality, without any AJ requirement, for several distance parameters in pp and PbPb collisions, and for their difference. In pp collisions the fragmentation of leading jets with high pT provides more high-pT and fewer low-pT particles in the hemisphere of the leading jet relative to the subleading-jet hemispheres. As a result, ⟨d∆mult/dpT⟩has a positive value for charged particles with pT < 8 GeV and a negative value for charged particles with pT > 8 GeV. Also, in PbPb collisions, ⟨d∆mult/dpT⟩ is positive for particles with pT < 8 GeV and becomes negative in the last bin, although the spectrum is much steeper, and has a large excess of soft particles. 8.4 Dependence of transverse momentum balance on jet distance parameter R By taking the difference in ⟨d∆mult/dpT⟩between PbPb and pp collisions, a signiﬁcant excess (>5 standard deviations) is observed at pT < 2 GeV, and a depletion at pT > 4 GeV, while there is only a slight excess in 22 9 Summary and conclusions J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -40 -20 0 20 40 pp R = 0.2 (2.76 TeV) -1 5.3 pb J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -40 -20 0 20 40 PbPb R = 0.2 (2.76 TeV) -1 b µ 166 J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -30 -20 -10 0 10 20 PbPb - pp R = 0.2 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 pp R = 0.3 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 PbPb R = 0.3 J A 0.1 0.2 0.3 0.4 -30 -20 -10 0 10 20 PbPb - pp R = 0.3 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 pp R = 0.4 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 PbPb R = 0.4 J A 0.1 0.2 0.3 0.4 -30 -20 -10 0 10 20 PbPb - pp R = 0.4 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 pp R = 0.5 Σ〉 \|\| T p 〈 pp J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 Σ〉 \|\| T p 〈 PbPb PbPb R = 0.5 J A 0.1 0.2 0.3 0.4 -30 -20 -10 0 10 20 PbPb - pp R = 0.5 Σ〉 \|\| T p 〈 PbPb - pp CMS Jet; 0-10% t anti-k > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 1.6; 2 η \|,\| 1 η\| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 [GeV] T trk p〉 \|\| T p 〈 8.0-300.0 \| < 2.4 trk η\| Figure 7: (Color online) Upper row shows ⟨pT / ∥⟩ptrk T (the individual track pT) and ⟨pT / ∥⟩Σ (sum over all ranges of track pT) as a function of AJ in pp collisions for distance parameters R = 0.2, 0.3, 0.4, and 0.5, from left to right. The dijet asymmetry ranges from almost balanced (AJ < 0.11) to unbalanced (AJ > 0.33) dijets. 8.4 Dependence of transverse momentum balance on jet distance parameter R Middle row provides ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ as a function of AJ in PbPb collisions of centrality range 0–10%, for distance parameter R = 0.2, 0.3, 0.4, and 0.5, from left to right. Lower row has the difference PbPb −pp of the ⟨pT / ∥⟩ptrk T , and ⟨pT / ∥⟩Σ, which are shown in the upper panels. Error bars and brackets represent statistical and systematic uncertainties, respectively. the range 2 < pT < 4 GeV. Changing R does not have an effect on the results in pp collisions, while in PbPb collisions there is a small enhancement in the excess for low-pT charged particles as R is increased from 0.2 to 0.5. the range 2 < pT < 4 GeV. Changing R does not have an effect on the results in pp collisions, while in PbPb collisions there is a small enhancement in the excess for low-pT charged particles as R is increased from 0.2 to 0.5. 8.4 Dependence of transverse momentum balance on jet distance parameter R Middle row provides ⟨pT / ∥⟩ptrk T and ⟨pT / ∥⟩Σ as a function of AJ in PbPb collisions of centrality range 0–10%, for distance parameter R = 0.2, 0.3, 0.4, and 0.5, from left to right. Lower row has the difference PbPb −pp of the ⟨pT / ∥⟩ptrk T , and ⟨pT / ∥⟩Σ, which are shown in the upper panels. Error bars and brackets represent statistical and systematic uncertainties, respectively. 8.4 Dependence of transverse momentum balance on jet distance parameter R J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -40 -20 0 20 40 pp R = 0.2 (2.76 TeV) -1 5.3 pb J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -40 -20 0 20 40 PbPb R = 0.2 (2.76 TeV) -1 b µ 166 J A 0.1 0.2 0.3 0.4 [GeV] 〉 \|\| T p 〈 -30 -20 -10 0 10 20 PbPb - pp R = 0.2 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 pp R = 0.3 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 PbPb R = 0.3 J A 0.1 0.2 0.3 0.4 -30 -20 -10 0 10 20 PbPb - pp R = 0.3 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 pp R = 0.4 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 PbPb R = 0.4 J A 0.1 0.2 0.3 0.4 -30 -20 -10 0 10 20 PbPb - pp R = 0.4 J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 pp R = 0.5 Σ〉 \|\| T p 〈 pp J A 0.1 0.2 0.3 0.4 -40 -20 0 20 40 Σ〉 \|\| T p 〈 PbPb PbPb R = 0.5 J A 0.1 0.2 0.3 0.4 -30 -20 -10 0 10 20 PbPb - pp R = 0.5 Σ〉 \|\| T p 〈 PbPb - pp CMS Jet; 0-10% t anti-k > 50 GeV T,2 > 120; p T,1 p /6 π > 5 1,2 φ ∆ \| < 1.6; 2 η \|,\| 1 η\| 0.5-1.0 1.0-2.0 2.0-4.0 4.0-8.0 [GeV] T trk p〉 \|\| T p 〈 8.0-300.0 \| < 2.4 trk η\| J A 2 0 J A 0 J A 0 J A Figure 7: (Color online) Upper row shows ⟨pT / ∥⟩ptrk T (the individual track pT) and ⟨pT / ∥⟩Σ (sum over all ranges of track pT) as a function of AJ in pp collisions for distance parameters R = 0.2, 0.3, 0.4, and 0.5, from left to right. The dijet asymmetry ranges from almost balanced (AJ < 0.11) to unbalanced (AJ > 0.33) dijets. 9 Summary and conclusions The transverse momentum ﬂow relative to the dijet axis in PbPb and pp collisions contain- ing jets with large pT has been studied using data corresponding to integrated luminosities of 166 µb−1 and 5.3 pb−1, respectively, collected at a nucleon-nucleon center-of-mass energy of 2.76 TeV. Dijet events were selected containing a leading jet with transverse momentum pT,1 > 120 GeV and a subleading jet with pT,2 > 50 GeV, reconstructed using the anti-kT algo- rithm, with distance parameters of R = 0.2, 0.3, 0.4 and 0.5. For PbPb collisions, the dijet events 23 [GeV] trk T p 1 10 2 10 -1 [GeV] 〉 trk T dp mult ∆ d 〈 0 10 20 CMS (2.76 TeV) -1 5.3 pb pp R=0.2 R=0.3 R=0.4 R=0.5 [GeV] trk T p 1 10 2 10 -1 [GeV] 〉 trk T dp mult ∆ d 〈 0 10 20 (2.76 TeV) -1 b µ 166 PbPb 0-30% > 120 GeV T,1 p > 50 GeV T,2 p [GeV] trk T p 1 10 2 10 -1 [GeV] 〉 trk T dp mult ∆ d 〈 0 10 20 PbPb - pp Inc. J A \| < 0.6 2 η \|, \| 1 η \| /6 π > 5 1,2 φ ∆ Figure 8: (Color online) Difference in differential multiplicity ⟨d∆mult/dpTtrk⟩between the away-side and leading-jet hemispheres as a function of track pT, using an inclusive dijet asym- metry selection. Left panel has measurements in pp for jet radii R = 0.2, 0.3, 0.4, and 0.5, and the middle panel displays similar measurements in PbPb. Right panel provides the differ- ence in ⟨d∆mult/dptrk T ⟩between PbPb and pp collisions for each momentum range. Systematic uncertainties are shown as boxes. Error bars represent statistical uncertainties. [GeV] trk T p 1 10 2 10 -1 [GeV] 〉 trk T dp mult ∆ d 〈 0 10 20 CMS (2.76 TeV) -1 5.3 pb pp R=0.2 R=0.3 R=0.4 R=0.5 [GeV] trk T p 1 10 2 10 -1 [GeV] 〉 trk T dp mult ∆ d 〈 0 10 20 (2.76 TeV) -1 b µ 166 PbPb 0-30% > 120 GeV T,1 p > 50 GeV T,2 p [GeV] trk T p 1 10 2 10 -1 [GeV] 〉 trk T dp mult ∆ d 〈 0 10 20 PbPb - pp Inc. 9 Summary and conclusions These results constrain the redistribution of transverse momen- tum in the modelling of QCD energy loss processes of partons traversing the hot and dense medium created in heavy-ion collisions. show a larger asymmetry in pT between the leading and subleading jets than in pp collisions. The multiplicity, angular, and pT spectra of the radiation balancing this asymmetry are charac- terized using several techniques as a function of PbPb collision centrality and pT asymmetry. For a given dijet asymmetry, the imbalance in pT in PbPb collisions is found to be compen- sated by particles at pT = 0.5–2 GeV, whereas in pp collisions most of the momentum balance is found in the pT range of 2–8 GeV, reﬂecting a softening of the radiation responsible for the imbalance in pT of the asymmetric dijet system in PbPb interactions. Correspondingly, a larger multiplicity of associated particles is seen in PbPb than in pp collisions. Both measurements show larger differences between PbPb and pp for more central PbPb collisions. The current data provide the ﬁrst detailed study of the angular dependence of charged particle contribu- tions to the asymmetry up to large angles from the jet axis (∆= 1.8). Despite the large shift in the pT spectrum of particles, the angular pattern of energy ﬂow in PbPb events as a func- tion of ∆matches that seen in pp collisions, especially for small R parameters. The results suggest that either the leading jet is getting narrower, or the subleading jet is getting broader after quenching. In pp collisions, the balancing distribution shifts to larger ∆with increasing distance parameter R, likely because of the presence of a third jet further away from the dijet axis. The shift is more pronounced than in PbPb collisions, where there is an excess of low pT particles close to the jet axes. These results constrain the redistribution of transverse momen- tum in the modelling of QCD energy loss processes of partons traversing the hot and dense medium created in heavy-ion collisions. 9 Summary and conclusions J A \| < 0.6 2 η \|, \| 1 η \| /6 π > 5 1,2 φ ∆ Figure 8: (Color online) Difference in differential multiplicity ⟨d∆mult/dpTtrk⟩between the away-side and leading-jet hemispheres as a function of track pT, using an inclusive dijet asym- metry selection. Left panel has measurements in pp for jet radii R = 0.2, 0.3, 0.4, and 0.5, and the middle panel displays similar measurements in PbPb. Right panel provides the differ- ence in ⟨d∆mult/dptrk T ⟩between PbPb and pp collisions for each momentum range. Systematic uncertainties are shown as boxes. Error bars represent statistical uncertainties. show a larger asymmetry in pT between the leading and subleading jets than in pp collisions. The multiplicity, angular, and pT spectra of the radiation balancing this asymmetry are charac- terized using several techniques as a function of PbPb collision centrality and pT asymmetry. For a given dijet asymmetry, the imbalance in pT in PbPb collisions is found to be compen- sated by particles at pT = 0.5–2 GeV, whereas in pp collisions most of the momentum balance is found in the pT range of 2–8 GeV, reﬂecting a softening of the radiation responsible for the imbalance in pT of the asymmetric dijet system in PbPb interactions. Correspondingly, a larger multiplicity of associated particles is seen in PbPb than in pp collisions. Both measurements show larger differences between PbPb and pp for more central PbPb collisions. The current data provide the ﬁrst detailed study of the angular dependence of charged particle contribu- tions to the asymmetry up to large angles from the jet axis (∆= 1.8). Despite the large shift in the pT spectrum of particles, the angular pattern of energy ﬂow in PbPb events as a func- tion of ∆matches that seen in pp collisions, especially for small R parameters. The results suggest that either the leading jet is getting narrower, or the subleading jet is getting broader after quenching. In pp collisions, the balancing distribution shifts to larger ∆with increasing distance parameter R, likely because of the presence of a third jet further away from the dijet axis. The shift is more pronounced than in PbPb collisions, where there is an excess of low pT particles close to the jet axes. Acknowledgments We congratulate our colleagues in the CERN accelerator departments for the excellent perfor- mance of the LHC and thank the technical and administrative staffs at CERN and at other CMS institutes for their contributions to the success of the CMS effort. In addition, we gratefully acknowledge the computing centres and personnel of the Worldwide LHC Computing Grid for delivering so effectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: BMWFW and FWF (Austria); FNRS and 9 Summary and conclusions 24 FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COLCIENCIAS (Colombia); MSES and CSF (Croatia); RPF (Cyprus); MoER, ERC IUT and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NIH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Re- public of Korea); LAS (Lithuania); MOE and UM (Malaysia); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Dubna); MON, RosAtom, RAS and RFBR (Russia); MESTD (Serbia); SEIDI and CPAN (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEPCenter, IPST, STAR and NSTDA (Thailand); TUBITAK and TAEK (Turkey); NASU and SFFR (Ukraine); STFC (United Kingdom); DOE and NSF (USA). We congratulate our colleagues in the CERN accelerator departments for the excellent performance of the LHC and thank the technical and administrative staffs at CERN and at other CMS institutes for their contributions to the suc- cess of the CMS effort. In addition, we gratefully acknowledge the computing centres and personnel of the Worldwide LHC Computing Grid for delivering so effectively the comput- ing infrastructure essential to our analyses. Acknowledgments Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the fol- lowing funding agencies: the Austrian Federal Ministry of Science, Research and Economy and the Austrian Science Fund; the Belgian Fonds de la Recherche Scientiﬁque, and Fonds voor Wetenschappelijk Onderzoek; the Brazilian Funding Agencies (CNPq, CAPES, FAPERJ, and FAPESP); the Bulgarian Ministry of Education and Science; CERN; the Chinese Academy of Sciences, Ministry of Science and Technology, and National Natural Science Foundation of China; the Colombian Funding Agency (COLCIENCIAS); the Croatian Ministry of Science, Ed- ucation and Sport, and the Croatian Science Foundation; the Research Promotion Foundation, Cyprus; the Ministry of Education and Research, Estonian Research Council via IUT23-4 and IUT23-6 and European Regional Development Fund, Estonia; the Academy of Finland, Finnish Ministry of Education and Culture, and Helsinki Institute of Physics; the Institut National de Physique Nucl´eaire et de Physique des Particules / CNRS, and Commissariat `a l’´Energie Atomique et aux ´Energies Alternatives / CEA, France; the Bundesministerium f¨ur Bildung und Forschung, Deutsche Forschungsgemeinschaft, and Helmholtz-Gemeinschaft Deutscher Forschungszentren, Germany; the General Secretariat for Research and Technology, Greece; the National Scientiﬁc Research Foundation, and National Innovation Ofﬁce, Hungary; the Depart- ment of Atomic Energy and the Department of Science and Technology, India; the Institute for Studies in Theoretical Physics and Mathematics, Iran; the Science Foundation, Ireland; the Isti- tuto Nazionale di Fisica Nucleare, Italy; the Ministry of Science, ICT and Future Planning, and National Research Foundation (NRF), Republic of Korea; the Lithuanian Academy of Sciences; the Ministry of Education, and University of Malaya (Malaysia); the Mexican Funding Agen- cies (CINVESTAV, CONACYT, SEP, and UASLP-FAI); the Ministry of Business, Innovation and Employment, New Zealand; the Pakistan Atomic Energy Commission; the Ministry of Science and Higher Education and the National Science Centre, Poland; the Fundac¸˜ao para a Ciˆencia e a Tecnologia, Portugal; JINR, Dubna; the Ministry of Education and Science of the Russian Federation, the Federal Agency of Atomic Energy of the Russian Federation, Russian Academy of Sciences, and the Russian Foundation for Basic Research; the Ministry of Education, Science and Technological Development of Serbia; the Secretar´ıa de Estado de Investigaci´on, Desar- rollo e Innovaci´on and Programa Consolider-Ingenio 2010, Spain; the Swiss Funding Agencies (ETH Board, ETH Zurich, PSI, SNF, UniZH, Canton Zurich, and SER); the Ministry of Sci- ence and Technology, Taipei; the Thailand Center of Excellence in Physics, the Institute for the Promotion of Teaching Science and Technology of Thailand Special Task Force for Activat- 25 References Scientiﬁc and Technical Research Council of Turkey, and Turkish Atomic Energy Authority; the National Academy of Sciences of Ukraine, and State Fund for Fundamental Researches, Ukraine; the Science and Technology Facilities Council, UK; the US Department of Energy, and the US National Science Foundation. Acknowledgments Individuals have received support from the Marie-Curie programme and the European Re- search Council and EPLANET (European Union); the Leventis Foundation; the A. P. Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Of- ﬁce; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA- Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT-Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOMING PLUS programme of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund; the OPUS programme of the National Science Center (Poland); the Compagnia di San Paolo (Torino); the Consorzio per la Fisica (Trieste); MIUR project 20108T4XTM (Italy); the Thalis and Aristeia programmes coﬁnanced by EU-ESF and the Greek NSRF; the National Priorities Research Program by Qatar National Research Fund; the Rachadapisek Sompot Fund for Postdoctoral Fellowship, Chula- longkorn University (Thailand); and the Welch Foundation, contract C-1845. 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De Souza Santosb, S. Dograa, T.R. Fernandez Perez Tomeia, Universidade Estadual Paulista a, Universidade Federal do ABC b, S˜ao Paulo, Brazil S. Ahujaa, C.A. Bernardesb, A. De Souza Santosb, S. Dograa, T.R. Fernandez Perez Tomeia, 32 A The CMS Collaboration E.M. Gregoresb, P.G. Mercadanteb, C.S. Moona,8, S.F. Novaesa, Sandra S. Padulaa, D. Romero Abad, J.C. Ruiz Vargas E.M. Gregoresb, P.G. Mercadanteb, C.S. Moona,8, S.F. Novaesa, Sandra S. Padulaa, D. Romero Abad, J.C. Ruiz Vargas Institute for Nuclear Research and Nuclear Energy, Soﬁa, Bulgaria A. Aleksandrov, R. Hadjiiska, P. Iaydjiev, M. Rodozov, S. Stoykova, G. Sultanov, M. Vutova A. Aleksandrov, R. Hadjiiska, P. Iaydjiev, M. Rodozov, S. Stoykova, G. Sultanov, M. Vut University of Soﬁa, Soﬁa, Bulgaria A. Dimitrov, I. Glushkov, L. Litov, B. Pavlov, P. Petkov University of Soﬁa, Soﬁa, Bulgaria A. Dimitrov, I. Glushkov, L. Litov, B. Pavlov, P. Petkov University of Soﬁa, Soﬁa, Bulgaria Institute of High Energy Physics, Beijing, China Institute of High Energy Physics, Beijing, China M. Ahmad, J.G. Bian, G.M. Chen, H.S. Chen, M. Chen, T. Cheng, R. Du, C.H. Jiang, R. Plestina9, F. Romeo, S.M. Shaheen, A. Spiezia, J. Tao, C. Wang, Z. Wang, H. Zhang State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China C. Asawatangtrakuldee, Y. Ban, Q. Li, S. Liu, Y. Mao, S.J. Qian, D. Wang, Z. Xu C. Avila, A. Cabrera, L.F. Chaparro Sierra, C. Florez, J.P. Gomez, B. Gomez Moreno, J.C. Sanabria University of Split, Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, Split, Croatia N. Godinovic, D. Lelas, I. Puljak, P.M. Ribeiro Cipriano University of Split, Faculty of Science, Split, Croatia Z. Antunovic, M. Kovac University of Split, Faculty of Science, Split, Croatia Z. Antunovic, M. Kovac Institute Rudjer Boskovic, Zagreb, Croatia V. Brigljevic, K. Kadija, J. Luetic, S. Micanovic, L. Sudic Institute Rudjer Boskovic, Zagreb, Croatia V. Brigljevic, K. Kadija, J. Luetic, S. Micanovic, L. Sudic University of Cyprus, Nicosia, Cyprus A. Attikis, G. Mavromanolakis, J. Mousa, C. Nicolaou, F. Ptochos, P.A. Razis, H. Rykaczewski Charles University, Prague, Czech Republic M. Bodlak, M. Finger10, M. Finger Jr.10 Charles University, Prague, Czech Republic M. Bodlak, M. Finger10, M. Finger Jr.10 Academy of Scientiﬁc Research and Technology of the Arab Republic of Egypt, Egyptian Network of High Energy Physics, Cairo, Egypt A.A. Abdelalim11,12, A. Awad, M. El Sawy13,14, A. Mahrous11, A. Radi14,15 National Institute of Chemical Physics and Biophysics, Tallinn, Estonia B. Calpas, M. Kadastik, M. Murumaa, M. Raidal, A. Tiko, C. Veelken Department of Physics, University of Helsinki, Helsinki, Finland P. Eerola, J. Pekkanen, M. Voutilainen Helsinki Institute of Physics, Helsinki, Finland J. H¨ark¨onen, V. Karim¨aki, R. Kinnunen, T. Lamp´en, K. Lassila-Perini, S. Lehti, T. Lind´en, P. Luukka, T. M¨aenp¨a¨a, T. Peltola, E. Tuominen, J. Tuominiemi, E. Tuovinen, L. Wendland Lappeenranta University of Technology, Lappeenranta, Finland J. Talvitie, T. Tuuva DSM/IRFU, CEA/Saclay, Gif-sur-Yvette, France M. Besancon, F. Couderc, M. Dejardin, D. Denegri, B. Fabbro, J.L. Faure, C. Favaro, F. Ferri, S. Ganjour, A. Givernaud, P. Gras, G. Hamel de Monchenault, P. Jarry, E. Locci, M. Machet, J. Malcles, J. Rander, A. Rosowsky, M. Titov, A. Zghiche J. Malcles, J. Rander, A. Rosowsky, M. Titov, A. Zghiche 33 Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau, France I. Antropov, S. Bafﬁoni, F. Beaudette, P. Busson, L. Cadamuro, E. Chapon, C. Charlot, T. Dahms, O. Davignon, N. Filipovic, A. Florent, R. Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S Gadrat Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France d Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S G d Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, C. Bernet, G. Boudoul, E. Bouvier, C.A. Carrillo Montoya, R. Chierici, D. Contardo, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S. Perries, J.D. Ruiz Alvarez, D. Sabes, L. Sgandurra, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret Institute of High Energy Physics, Beijing, China Granier de Cassagnac, S. Lisniak, L. Mastrolorenzo, P. Min´e, I.N. Naranjo, M. Nguyen, C. Ochando, G. Ortona, P. Paganini, P. Pigard, S. Regnard, R. Salerno, J.B. Sauvan, Y. Sirois, T. Strebler, Y. Yilmaz, A. Zabi Institut Pluridisciplinaire Hubert Curien, Universit´e de Strasbourg, Universit´e de Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France J.-L. Agram16, J. Andrea, A. Aubin, D. Bloch, J.-M. Brom, M. Buttignol, E.C. Chabert, N. Chanon, C. Collard, E. Conte16, X. Coubez, J.-C. Fontaine16, D. Gel´e, U. Goerlach, C. Goetzmann, A.-C. Le Bihan, J.A. Merlin2, K. Skovpen, P. Van Hove Georgian Technical University, Tbilisi, Georgia T. Toriashvili17 Georgian Technical University, Tbilisi, Georgia T. Toriashvili17 Tbilisi State University, Tbilisi, Georgia Z. Tsamalaidze10 RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, M. Edelhoff, L. Feld, A. Heister, M.K. Kiesel, K. Klein, M RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C Autermann S Beranek M Edelhoff L Feld A Heister M K Kiesel K K RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, M. Edelhoff, L. Feld, A. Heister, M.K. Kiesel, K. Klein, M. Lipinski, A. Ostapchuk, M. Preuten, F. Raupach, S. Schael, J.F. Schulte, T. Verlage, H. Weber, B. Wittmer, V. Zhukov6 p A. Ostapchuk, M. Preuten, F. Raupach, S. Schael, J.F. Schulte, T. Verlage, H. Weber, B. Wittmer, V. Zhukov6 RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany M. Ata, M. Brodski, E. Dietz-Laursonn, D. Duchardt, M. Endres, M. Erdmann, S. Erdweg, T. Esch, R. Fischer, A. G¨uth, T. Hebbeker, C. Heidemann, K. Hoepfner, S. Knutzen, P. Kreuzer, M. Merschmeyer, A. Meyer, P. Millet, M. Olschewski, K. Padeken, P. Papacz, T. Pook, M. Radziej, H. Reithler, M. Rieger, F. Scheuch, L. Sonnenschein, D. Teyssier, S. Th¨uer y y y V. Cherepanov, Y. Erdogan, G. Fl¨ugge, H. Geenen, M. Geisler, F. Hoehle, B. Kargoll, T. Kress, Y. Kuessel, A. K¨unsken, J. Lingemann, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl Deutsches Elektronen-Synchrotron, Hamburg, Germany M. Aldaya Martin, I. Asin, N. Bartosik, O. Behnke, U. Behrens, A.J. Bell, K. Borras18, A. Burgmeier, A. Campbell, S. Choudhury19, F. Costanza, C. Diez Pardos, G. Dolinska, S. Dooling, T. Dorland, G. Eckerlin, D. Eckstein, T. Eichhorn, G. Flucke, E. Gallo20, J. Garay Garcia, A. Geiser, A. Gizhko, P. Gunnellini, J. Hauk, M. Hempel21, H. Jung, A. Kalogeropoulos, O. Karacheban21, M. Kasemann, P. Katsas, J. Kieseler, C. Kleinwort, I. Korol, W. Lange, J. Leonard, K. Lipka, A. Lobanov, W. Lohmann21, R. Mankel, I. Marﬁn21, I.-A. Melzer-Pellmann, A.B. Meyer, G. Mittag, J. Mnich, A. Mussgiller, S. Naumann-Emme, A. Nayak, E. Ntomari, A The CMS Collaboration 34 H. Perrey, D. Pitzl, R. Placakyte, A. Raspereza, B. Roland, M. ¨O. Sahin, P. Saxena, T. Schoerner- Sadenius, M. Schr¨oder, C. Seitz, S. Spannagel, K.D. Trippkewitz, R. Walsh, C. Wissing University of Hamburg, Hamburg, Germany V. Blobel, M. Centis Vignali, A.R. Draeger, J. Erﬂe, E. Garutti, K. Goebel, D. Gonzalez, M. G¨orner, J. Haller, M. Hoffmann, R.S. H¨oing, A. Junkes, R. Klanner, R. Kogler, N. Kovalchuk, T. Lapsien, T. Lenz, I. Marchesini, D. Marconi, M. Meyer, D. Nowatschin, J. Ott, F. Pantaleo2, T. Peiffer, A. Perieanu, N. Pietsch, J. Poehlsen, D. Rathjens, C. Sander, C. Scharf, H. Schettler, P. Schleper, E. Schlieckau, A. Schmidt, J. Schwandt, V. Sola, H. Stadie, G. Steinbr¨uck, H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald T. Peiffer, A. Perieanu, N. Pietsch, J. Poehlsen, D. Rathjens, C. Sander, C. Scharf, H. Schettler, P. Schleper, E. Schlieckau, A. Schmidt, J. Schwandt, V. Sola, H. Stadie, G. Steinbr¨uck, H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald j P. Schleper, E. Schlieckau, A. Schmidt, J. Schwandt, V. Sola, H. S D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany M. Akbiyik, C. Barth, C. Baus, J. Berger, C. B¨oser, E. Butz, T. Chwalek, F. Colombo, W. De Boer, A. Descroix, A. Dierlamm, S. Fink, F. Frensch, R. Friese, M. Giffels, A. Gilbert, D. Haitz, F. Hartmann2, S.M. Heindl, U. Husemann, I. Katkov6, A. Kornmayer2, P. Lobelle Pardo, B. Maier, H. Mildner, M.U. Mozer, T. M¨uller, Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, S. R¨ocker, F. Roscher, G. Sieber, H.J. Simonis, F.M. Stober, R. Ulrich, J. Wagner-Kuhr, S. Wayand, M. Weber, T. Weiler, C. W¨ohrmann, R. Wolf Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia Paraskevi, Greece G. Anagnostou, G. Daskalakis, T. Geralis, V.A. Giakoumopoulou, A. Kyriakis, D. Loukas, A. Psallidas, I. Topsis-Giotis University of Athens, Athens, Greece A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi University of Athens, Athens, Greece A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi y , , A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi University of Io´annina, Io´annina, Greece I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos, E. Paradas, J. Strologas E. Paradas, J. Strologas Wigner Research Centre for Physics, Budapest, Hungary G. Bencze, C. Hajdu, A. Hazi, P. Hidas, D. Horvath22, F. Sikler, V. Veszpremi, G. Vesztergombi23, A.J. Zsigmond Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi24, J. Molnar, Z. Szillasi2 University of Debrecen, Debrecen, Hungary M. Bart´ok25, A. Makovec, P. Raics, Z.L. Trocsanyi, B. Ujvari University of Debrecen, Debrecen, Hungary y g y M. Bart´ok25, A. Makovec, P. Raics, Z.L. Trocsanyi, B. Ujvari National Institute of Science Education and Research, Bhubaneswar, India P. Mal, K. Mandal, D.K. Sahoo, N. Sahoo, S.K. Swain Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, J.B. Singh, G. Walia University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, A. Kumar, S. Malhotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma Saha Institute of Nuclear Physics, Kolkata, India Saha Institute of Nuclear Physics, Kolkata, India S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutta, Sa. Jain, N. Majumdar, A. Modak, K. Mondal, S Mukherjee S Mukhopadhyay A Roy D Roy S Roy Chowdhury S Sarkar M Sharan S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutta, Sa. Jain, N. Majumdar, A. Modak, K. Mondal, S. Mukherjee, S. Mukhopadhyay, A. Roy, D. Roy, S. Roy Chowdhury, S. Sarkar, M. Shar 35 Bhabha Atomic Research Centre, Mumbai, India Bhabha Atomic Research Centre, Mumbai, India A. Abdulsalam, R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty2, L.M. Pant, P. Shukla, A. Topkar , , A. Abdulsalam, R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty2, L.M. Pant, P. Shukla, A. Topkar Tata Institute of Fundamental Research, Mumbai, India T. Aziz, S. Banerjee, S. Bhowmik26, R.M. Chatterjee, R.K. Dewanjee, S. Dugad, S. Ganguly, S. Ghosh, M. Guchait, A. Gurtu27, G. Kole, S. Kumar, B. Mahakud, M. Maity26, G. Majumder, K. Mazumdar, S. Mitra, G.B. Mohanty, B. Parida, T. Sarkar26, N. Sur, B. Sutar, N. Wickramage28 Indian Institute of Science Education and Research (IISER), Pune, India S. Chauhan, S. Dube, K. Kothekar, S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran Institute for Research in Fundamental Sciences (IPM), Tehran, Iran H. Bakhshiansohi, H. Behnamian, S.M. Etesami29, A. Fahim30, R. Goldouzian, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi, F. Rezaei Hosseinabadi, B. Safarzadeh31, M. Zeinali ( ), , H. Bakhshiansohi, H. Behnamian, S.M. Etesami29, A. Fahim30, R. Goldouzian, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi, F. Rezaei Hosseinabadi, B S f d h31 M Z i li H. Bakhshiansohi, H. Behnamian, S.M. Etesami29, A. Fahim30, R. Goldouzian, M. Khakzad, M M h di N j f b di M N i S P kti t M hdi b di F R i H i b di H. Bakhshiansohi, H. Behnamian, S.M. Etesami29, A. Fahim30, R. Goldouzian, M M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi, F. Rezaei Hosseinabadi, B. Safarzadeh31, M. Zeinali University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald INFN Sezione di Bari a, Universit`a di Bari b, Politecnico di Bari c, Bari, Italy M. Abbresciaa,b, C. Calabriaa,b, C. Caputoa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b, N. De Filippisa,c, M. De Palmaa,b, L. Fiorea, G. Iasellia,c, G. Maggia,c, M. Maggia, G. Minielloa,b, S. Mya,c, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa,b, A. Ranieria, G. Selvaggia,b, L. Silvestrisa,2, R. Vendittia,b, P. Verwilligena INFN Sezione di Bologna a, Universit`a di Bologna b, Bologna, Italy G. Abbiendia, C. Battilana2, A.C. Benvenutia, D. Bonacorsia,b, S. Braibant-Giacomellia,b, L. Brigliadoria,b, R. Campaninia,b, P. Capiluppia,b, A. Castroa,b, F.R. Cavalloa, S.S. Chhibraa,b, G. Codispotia,b, M. Cufﬁania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b, P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,2, R. Travaglinia,b F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,2, R. Travaglinia,b INFN Sezione di Catania a, Universit`a di Catania b, Catania, Italy G. Cappelloa, M. Chiorbolia,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b, A. Tricomia,b, C. Tuvea,b INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, S. Gonzia,b, V. Goria,b, P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, A. Tropianoa,b, L. Viliania,b,2 INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera2 INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera2 INFN Sezione di Genova a, Universit`a di Genova b, Genova, Italy V. Calvellia,b, F. Ferroa, M. Lo Veterea,b, M.R. Mongea,b, E. Robuttia, S. Tosia,b INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy L. Brianza, M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, R. Gerosaa,b, A. Ghezzia,b, P. Govonia,b, S. Malvezzia, R.A. Manzonia,b,2, B. Marzocchia,b,2, D. Menascea, L. Moronia, M. Paganonia,b, D. Pedrinia, S. Ragazzia,b, N. Redaellia, T. Tabarelli de Fatisa,b INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy L. Brianza, M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, R. Gerosaa,b, A. Ghezzia,b, P. Govonia,b, S M l ia R A M ia b 2 B M hia b 2 D M a L M ia M P ia b L. Brianza, M.E. Dinardo , S. Fiorendi , S. Gennai , R. Gerosa , A. Ghezzi , P. Govoni , S. Malvezzia, R.A. Manzonia,b,2, B. Marzocchia,b,2, D. Menascea, L. Moronia, M. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Paganonia,b, D Pedrinia S Ragazzia,b N Redaellia T Tabarelli de Fatisa,b D. Pedrinia, S. Ragazzia,b, N. Redaellia, T. Tabarelli de Fatisa,b A The CMS Collaboration 36 INFN Sezione di Napoli a, Universit`a di Napoli ’Federico II’ b, Napoli, Italy, Universit`a della Basilicata c, Potenza, Italy, Universit`a G. Marconi d, Roma, Italy y y S. Buontempoa, N. Cavalloa,c, S. Di Guidaa,d,2, M. Espositoa,b, F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,2, M. Merolaa, P. Paoluccia,2, C. Sciaccaa,b, F. Thyssen INFN Sezione di Padova a, Universit`a di Padova b, Padova, Italy, Universit`a di Trento c, Trento, Italy 2 b b b b 2 P. Azzia,2, N. Bacchettaa, L. Benatoa,b, A. Bolettia,b, A. Brancaa,b, M. Dall’Ossoa,b,2, T. Dorigoa, F. Fanzagoa, F. Gonellaa, A. Gozzelinoa, K. Kanishcheva,c, M. Margonia,b, G. Marona,32, A.T. Meneguzzoa,b, M. Michelottoa, F. Montecassianoa, M. Passaseoa, J. Pazzinia,b,2, M. Pegoraroa, N. Pozzobona,b, P. Ronchesea,b, F. Simonettoa,b, E. Torassaa, M. Tosia,b, S. Vaninia,b, S. Venturaa, M. Zanetti, P. Zottoa,b, A. Zucchettaa,b,2 g g A.T. Meneguzzoa,b, M. Michelottoa, F. Montecassianoa, M. Passaseoa, J. Pazzinia,b,2, M. Pegoraroa, N. Pozzobona,b, P. Ronchesea,b, F. Simonettoa,b, E. Torassaa, M. Tosia,b, S. Vaninia,b, S. Venturaa, M. Zanetti, P. Zottoa,b, A. Zucchettaa,b,2 INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy A. Braghieria, A. Magnania, P. Montagnaa,b, S.P. Rattia,b, V. Rea, C. Riccardia,b, P. Salvinia, I. Vaia, P. Vituloa,b INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L. Alunni Solestizia,b, M. Biasinia,b, G.M. Bileia, D. Ciangottinia,b,2, L. Fan`oa,b, P. Lar NFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy Alunni Solestizia,b M Biasinia,b G M Bileia D Ciangottinia,b,2 INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L. Alunni Solestizia,b, M. Biasinia,b, G.M. Bileia, D. Ciangottinia,b,2, L. Fan`oa,b, P. Lari G Mantovania,b M Menichellia A Sahaa A Santocchiaa,b INFN Sezione di Perugia a, Universita di Perugia b, Perugia, Italy L. Alunni Solestizia,b, M. Biasinia,b, G.M. Bileia, D. Ciangottinia,b,2, L. Fan`oa,b, P. Laricciaa,b, G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b L. Alunni Solestizi , M. Biasini , G.M. Bilei , D. Ciangottini , L. Fano , P. Lariccia , G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b INFN Sezione di Pisa a, Universit`a di Pisa b, Scuola Normale Superiore di Pisa c, Pisa, Italy K. Androsova,33, P. Azzurria,2, G. Bagliesia, J. Bernardinia, T. Boccalia, R. Castaldia, M.A. Cioccia,33, R. Dell’Orsoa, S. Donatoa,c,2, G. Fedi, L. Fo`aa,c†, A. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Giassia, M.T. Grippoa,33, F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy- Navarroa,34, A.T. Serbana, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia INFN Sezione di Roma a, Universit`a di Roma b, Roma, Italy L. Baronea,b, F. Cavallaria, G. D’imperioa,b,2, D. Del Rea,b,2, M. Diemoza, S. Gellia,b, C. Jordaa, E. Longoa,b, F. Margarolia,b, P. Meridiania, G. Organtinia,b, R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b, P. Traczyka,b,2 L. Baronea,b, F. Cavallaria, G. D’imperioa,b,2, D. Del Rea,b,2, M. Diemoza, S. Gellia,b, C. Jordaa, E. Longoa,b, F. Margarolia,b, P. Meridiania, G. Organtinia,b, R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b, P. Traczyka,b,2 E. Longoa,b, F. Margarolia,b, P. Meridiania, G. Organtinia,b, R. Paramattia, F. Pr S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b, P. Traczyka,b,2 INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a del Piemonte Orientale c, Novara, Italy N. Amapanea,b, R. Arcidiaconoa,c,2, S. Argiroa,b, M. Arneodoa,c, R. Bellana,b, C. Biinoa, N. Cartigliaa, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, L. Fincoa,b,2, B. Kiania,b, p , , g , , , , N. Cartigliaa, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, L. Fincoa,b,2, B. Kiania,b, C. Mariottia, S. Masellia, E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M.M. Obertinoa,b, L. Pachera,b, N. Pastronea, M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, A. Solanoa,b, A. Staianoa g L. Pachera,b, N. Pastronea, M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, b b M. Ruspaa,c, R. Sacchia,b, A. Solanoa,b, A. Staianoa INFN Sezione di Trieste a, Universit`a di Trieste b, Trieste, Italy S. Belfortea, V. Candelisea,b,2, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, B. Gobboa, C. La Licataa,b, M. Maronea,b, A. Schizzia,b, A. Zanettia Licataa,b, M. Maronea,b, A. Schizzia,b, A. Zanettia Licataa,b, M. Maronea,b, A. Schizzia,b, A. Zanettia Kangwon National University, Chunchon, Korea A. Kropivnitskaya, S.K. Nam Korea University, Seoul, Korea Seoul National University, Seoul, Korea H.D. Yoo Kangwon National University, Chunchon, Korea A. Kropivnitskaya, S.K. Nam Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, D.J. Kong, S. Lee, Y.D. Oh, A. Sakharov, D.C. Son Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, D.J. Kong, S. Lee, Y.D. Oh, A. Sakharov, D.C. Son Chonbuk National University, Jeonju, Korea Chonbuk National University, Jeonju, Korea J.A. Brochero Cifuentes, H. Kim, T.J. Kim J.A. Brochero Cifuentes, H. Kim, T.J. Kim 37 Seoul National University, Seoul, Korea H.D. Yoo University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu Sungkyunkwan University, Suwon, Korea Y. Choi, J. Goh, D. Kim, E. Kwon, J. Lee, I. Yu Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali35, F. Mohamad Idris36, W.A.T. Wan Abdullah, M.N. Yusli Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico E. Casimiro Linares, H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz37, A. Hernandez-Almada, R. Lopez-Fernandez, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, F. Vazquez Valencia Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, F. Vazquez Valencia Benemerita Universidad Autonoma de Puebla, Puebla, Mexico I. Pedraza, H.A. Salazar Ibarguen Benemerita Universidad Autonoma de Puebla, Puebla, Mexico I. Pedraza, H.A. Salazar Ibarguen Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, T. Khurshid, M. Shoaib National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland G. Brona, K. Bunkowski, A. Byszuk38, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Misiura, M. Olszewski, M. Walczak Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland G. Brona, K. Bunkowski, A. Byszuk38, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Misiura, M. Olszewski, M. Walczak Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, 38 A The CMS Collaboration M. Gallinaro, N. Leonardo, L. Lloret Iglesias, F. Nguyen, J. Rodrigues Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia Joint Institute for Nuclear Research, Dubna, Russia S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. K V Konoplyanikov A Lanev A Malakhov V Matveev39,40 P Moisenz V Palichik V Pe S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavin, V. Konoplyanikov, A. Lanev, A. Malakhov, V. Matveev39,40, P. Moisenz, V. Palichik, V. Perelygin, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, A. Zarubin S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. K V. Konoplyanikov, A. Lanev, A. Malakhov, V. Matveev39,40, P. Moisenz, V. Palichik, V. Pe S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia V. Golovtsov, Y. Ivanov, V. Kim41, E. Kuznetsova, P. Levchenko, V. Murzin, V. Oreshkin, I. Smirnov, V. Sulimov, L. Uvarov, S. Vavilov, A. Vorobyev Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, E. Vlasov, A. Zhokin National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia A. Bylinkin P.N. Lebedev Physical Institute, Moscow, Russia 40 40 y , , V. Andreev, M. Azarkin40, I. Dremin40, M. Kirakosyan, A. Leonidov40, G. Mesyats, S.V. Rusakov Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia A. Baskakov, A. Belyaev, E. Boos, A. Ershov, A. Gribushin, A. Kaminskiy42, O. Ko A. Baskakov, A. Belyaev, E. Boos, A. Ershov, A. Gribushin, A. Kaminskiy42, O. Kodolova, V. Korotkikh, I. Lokhtin, I. Myagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev, I. Vardanyan State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, V. Kachanov, A. Kalinin, D. Konstantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, L. Tourtchanovitch, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P Ad i 43 P Ci k i J Mil i V R k i P. Adzic43, P. Cirkovic, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain , p J. Alcaraz Maestre, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, D. Dom´ınguez V´azquez, A. Escalante Del Valle, C. Universidad de Oviedo, Oviedo, Spain p J. Cuevas, J. Fernandez Menendez, S. Folgueras, I. Gonzalez Caballero, E. Palencia Cortezon, J.M. Vizan Garcia J. Cuevas, J. Fernandez Menendez, S. Folgueras, I. Gonzalez Caballero, E. Palencia Cortezon, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain I.J. Cabrillo, A. Calderon, J.R. Casti˜neiras De Saa, P. De Castro Manzano, M. Fernandez, J. Garcia-Ferrero, G. Gomez, A. Lopez Virto, J. Marco, R. Marco, C. Martinez Rivero, F. Matorras, J. Piedra Gomez, T. Rodrigo, A.Y. Rodr´ıguez-Marrero, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, E. Auffray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney, A. Benaglia, J. Bendavid, L. Benhabib, J.F. Benitez, G.M. Berruti, P. Bloch, A. Bocci, A. Bonato, C. Botta, H. Breuker, T. Camporesi, R. Castello, G. Cerminara, M. D’Alfonso, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, F. De Guio, A. De Roeck, S. De Visscher, E. Di Marco44, M. Dobson, M. Dordevic, B. Dorney, T. du Pree, D. Duggan, M. D¨unser, N. Dupont, A. Elliott-Peisert, G. Franzoni, J. Fulcher, W. Funk, D. Gigi, K. Gill, D. Giordano, M. Girone, F. Glege, R. Guida, S. Gundacker, M. Guthoff, J. Hammer, P. Harris, J. Hegeman, V. Innocente, P. Janot, H. Kirschenmann, M.J. Kortelainen, K. Kousouris, K. Krajczar, P. Lecoq, C. Lourenc¸o, M.T. Lucchini, N. Magini, L. Malgeri, M. Mannelli, A. Martelli, L. Masetti, F. Meijers, S. Mersi, E. Meschi, F. Moortgat, S. Morovic, M. Mulders, M.V. Nemallapudi, H. Neugebauer, S. Orfanelli45, L. Orsini, L. Pape, E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiffer, D. Piparo, A. Racz, T. Reis, G. Rolandi46, M. Rovere, M. Ruan, H. Sakulin, C. Sch¨afer, C. Schwick, M. Seidel, A. Sharma, P. Silva, M. Simon, P. Sphicas47, J. Steggemann, B. Stieger, M. Stoye, Y. Takahashi, D. Treille, A. Triossi, A. Tsirou, G.I. Veres23, N. Wardle, H.K. W¨ohri, A. Zagozdzinska38, W.D. Zeuner Paul Scherrer Institut, Villigen, Switzerland W. Bertl, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, D. Renker, T. Rohe Institute for Particle Physics, ETH Zurich, Zurich, Switzerland F. Bachmair, L. B¨ani, L. Bianchini, B. Casal, G. Dissertori, M. Dittmar, M. Doneg`a, P. Eller, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, W. Lustermann, B. Mangano, M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio, D. Meister, F. Micheli, P. Joint Institute for Nuclear Research, Dubna, Russia Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, J. Santaolalla, M.S. Soares Universidad Aut´onoma de Madrid, Madrid, Spain C. Albajar, J.F. de Troc´oniz, M. Missiroli, D. Moran 39 Universidad de Oviedo, Oviedo, Spain Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, L. Perrozzi, M. Quittnat, M. Rossini, A. Starodumov48, M. Takahashi, V.R. Tavolaro, K. Theoﬁlatos, R. Wallny Universit¨at Z¨urich, Zurich, Switzerland T.K. Aarrestad, C. Amsler49, L. Caminada, M.F. Canelli, V. Chiochia, A. De Cosa, C. Galloni, A. Hinzmann, T. Hreus, B. Kilminster, C. Lange, J. Ngadiuba, D. Pinna, P. Robmann, F.J. Ronga, D. Salerno, Y. Yang National Central University, Chung-Li, Taiwan M. Cardaci, K.H. Chen, T.H. Doan, Sh. Jain, R. Khurana, M. Konyushikhin, C.M. Kuo, W. Lin, Y.J. Lu, S.S. Yu National Taiwan University (NTU), Taipei, Taiwan Arun Kumar, R. Bartek, P. Chang, Y.H. Chang, Y.W. Chang, Y. Chao, K.F. Chen, P.H. Chen, C. Dietz, F. Fiori, U. Grundler, W.-S. Hou, Y. Hsiung, Y.F. Liu, R.-S. Lu, M. Mi˜nano Moya, E. Petrakou, J.f. Tsai, Y.M. Tzeng 40 A The CMS Collaboration Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand B. Asavapibhop, K. Kovitanggoon, G. Singh, N. Srimanobhas, N. Suwonjandee Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand B. Asavapibhop, K. Kovitanggoon, G. Singh, N. Srimanobhas, N. Suwonjandee Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand B. Asavapibhop, K. Kovitanggoon, G. Singh, N. Srimanobhas, N. Suwonjandee Cukurova University, Adana, Turkey A. Adiguzel, S. Cerci50, Z.S. Demiroglu, C. Dozen, I. Dumanoglu, S. Girgis, G. Gokbulut, Y. Guler, E. Gurpinar, I. Hos, E.E. Kangal51, A. Kayis Topaksu, G. Onengut52, K. Ozdemir53, S. Ozturk54, B. Tali50, H. Topakli54, M. Vergili, C. Zorbilmez Cukurova University, Adana, Turkey A. Adiguzel, S. Cerci50, Z.S. Demiroglu, C. Dozen, I. Dumanoglu, S. Girgis, G. Gokbulut, Y. Guler, E. Gurpinar, I. Hos, E.E. Kangal51, A. Kayis Topaksu, G. Onengut52, K. Ozdemir53, S. Ozturk54, B. Tali50, H. Topakli54, M. Vergili, C. Zorbilmez Middle East Technical University, Physics Department, Ankara, Turkey I.V. Akin, B. Bilin, S. Bilmis, B. Isildak55, G. Karapinar56, M. Yalvac, M. Zeyrek Bogazici University, Istanbul, Turkey E. G¨ulmez, M. Kaya57, O. Kaya58, E.A. Yetkin59, T. Yetkin60 Bogazici University, Istanbul, Turkey E. G¨ulmez, M. Kaya57, O. Kaya58, E.A. Yetkin59, T. Yetkin60 Istanbul Technical University, Istanbul, Turkey A. Cakir, K. Cankocak, S. Sen61, F.I. Vardarlı Istanbul Technical University, Istanbul, Turkey A. Cakir, K. Cankocak, S. Sen61, F.I. Vardarlı Institute for Scintillation Materials of National Academy of Science of Ukraine, Kharkov, Ukraine B G Institute for Scintillation Materials of National Academy of Science of Ukraine, Kharkov, Ukraine B Grynyov Ukraine National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk, P. Sorokin University of Bristol, Bristol, United Kingdom y , , g R. Aggleton, F. Ball, L. Beck, J.J. Brooke, E. Clement, D. Cussans, H. Flacher, J. Goldstein, 62 y g R. Aggleton, F. Ball, L. Beck, J.J. Brooke, E. Clement, D. Cussans, H. Flacher, J. Goldstein, M. Grimes, G.P. Heath, H.F. Heath, J. Jacob, L. Kreczko, C. Lucas, Z. Meng, D.M. Newbold62, R. Aggleton, F. Ball, L. Beck, J.J. Brooke, E. Clement, D. Cussans, H. Flacher, J. Goldstein, M. Grimes, G.P. Heath, H.F. Heath, J. Jacob, L. Kreczko, C. Lucas, Z. Meng, D.M. Newbold62, S. Paramesvaran, A. Poll, T. Sakuma, S. Seif El Nasr-storey, S. Senkin, D. Smith, V.J. Smith , , , J J , , , g, , S. Paramesvaran, A. Poll, T. Sakuma, S. Seif El Nasr-storey, S. Senkin, D. Smith, V.J. Smith Rutherford Appleton Laboratory, Didcot, United Kingdom A. Belyaev63, C. Brew, R.M. Brown, L. Calligaris, D. Cieri, D.J.A. Cockerill, J.A. Coughlan, K. Harder, S. Harper, E. Olaiya, D. Petyt, C.H. Shepherd-Themistocleous, A. Thea, I.R. Tomalin, T. Williams, S.D. Worm Imperial College, London, United Kingdom M. Baber, R. Bainbridge, O. Buchmuller, A. Bundock, D. Burton, S. Casasso, M. Citron, D. Colling, L. Corpe, N. Cripps, P. Dauncey, G. Davies, A. De Wit, M. Della Negra, P. Dunne, A. Elwood, W. Ferguson, D. Futyan, G. Hall, G. Iles, M. Kenzie, R. Lane, R. Lucas62, L. Lyons, A.-M. Magnan, S. Malik, J. Nash, A. Nikitenko48, J. Pela, M. Pesaresi, K. Petridis, D.M. Raymond, A. Richards, A. Rose, C. Seez, A. Tapper, K. Uchida, M. Vazquez Acosta64, T. Virdee, S.C. Zenz Brunel University, Uxbridge, United Kingdom J.E. Cole, P.R. Hobson, A. Khan, P. Kyberd, D. Leggat, D. Leslie, I.D. Reid, P. Symonds, L. Teodorescu, M. Turner Baylor University, Waco, USA A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika Baylor University, Waco, USA A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika University of California, Riverside, Riverside, USA University of California, Riverside, Riverside, USA K. Burt, R. Clare, J. Ellison, J.W. Gary, G. Hanson, J. Heilman, M. Ivova PANEVA, P. Jandir, E. Kennedy, F. Lacroix, O.R. Long, A. Luthra, M. Malberti, M. Olmedo Negrete, A. Shrinivas, H. Wei, S. Wimpenny, B. R. Yates University of California, San Diego, La Jolla, USA J.G. Branson, G.B. Cerati, S. Cittolin, R.T. D’Agnolo, M. Derdzinski, A. Holzner, R. Kelley, D. Klein, J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel, A. Vartak, S. Wasserbaech65, C. Welke, F. W¨urthwein, A. Yagil, G. Zevi Della Porta University of California, Santa Barbara, Santa Barbara, USA University of California, Santa Barbara, Santa Barbara, USA J. Bradmiller-Feld, C. Campagnari, A. Dishaw, V. Dutta, K. Flowers, M. Franco Sevilla, P. Geffert, C. George, F. Golf, L. Gouskos, J. Gran, J. Incandela, N. Mccoll, S.D. Mullin, J. Richman, D. Stuart, I. Suarez, C. West, J. Yoo California Institute of Technology, Pasadena, USA D. Anderson, A. Apresyan, A. Bornheim, J. Bunn, Y. Chen, J. Duarte, A. Mott, H.B. Newman, C. Pena, M. Pierini, M. Spiropulu, J.R. Vlimant, S. Xie, R.Y. Zhu Carnegie Mellon University, Pittsburgh, USA M.B. Andrews, V. Azzolini, A. Calamba, B. Carlson, T. Ferguson, M. Paulini, J. Russ, M. Sun, H. Vogel, I. Vorobiev University of Colorado Boulder, Boulder, USA J.P. Cumalat, W.T. Ford, A. Gaz, F. Jensen, A. Johnson, M. Krohn, T. Mulholland, U. Nauenberg, K. Stenson, S.R. Wagner University of Colorado Boulder, Boulder, USA J.P. Cumalat, W.T. Ford, A. Gaz, F. Jensen, A. Johnson, M. Krohn, T. Mulholland, U. Nauenberg, K. Stenson, S.R. Wagner Cornell University, Ithaca, USA J. Alexander, A. Chatterjee, J. Chaves, J. Chu, S. Dittmer, N. Eggert, N. Mirman, G. Nicolas Kaufman, J.R. Patterson, A. Rinkevicius, A. Ryd, L. Skinnari, L. Sofﬁ, W. Sun, S.M. Tan, W.D. Teo, J. Thom, J. Thompson, J. Tucker, Y. Weng, P. Wittich Fermi National Accelerator Laboratory, Batavia, USA S. Abdullin, M. Albrow, G. Apollinari, S. Banerjee, L.A.T. Bauerdick, A. Beretvas, J. Berryhill, P.C. Bhat, G. Bolla, K. Burkett, J.N. Butler, H.W.K. Cheung, F. Chlebana, S. Cihangir, V.D. Elvira, I. Fisk, J. Freeman, E. Gottschalk, L. Gray, D. Green, S. Gr¨unendahl, O. Gutsche, J. Hanlon, D. Hare, R.M. Harris, S. Hasegawa, J. Hirschauer, Z. Hu, B. Jayatilaka, S. Jindariani, M. Johnson, U. Joshi, A.W. Jung, B. Klima, B. Kreis, S. Kwan†, S. Lammel, J. Linacre, D. Lincoln, R. Brown University, Providence, USA y J. Alimena, E. Berry, S. Bhattacharya, D. Cutts, N. Dhingra, A. Ferapontov, A. Garabedian, J. Hakala, U. Heintz, E. Laird, G. Landsberg, Z. Mao, M. Narain, S. Piperov, S. Sagir, R. Syarif J. Alimena, E. Berry, S. Bhattacharya, D. Cutts, N. Dhingra, A. Fer University of California, Davis, Davis, USA R. Breedon, G. Breto, M. Calderon De La Barca Sanchez, S. Chauhan, M. Chertok, J. Conway, R. Conway, P.T. Cox, R. Erbacher, M. Gardner, W. Ko, R. Lander, M. Mulhearn, D. Pellett, J. Pilot, F. Ricci-Tam, S. Shalhout, J. Smith, M. Squires, D. Stolp, M. Tripathi, S. Wilbur, R. Yohay University of California, Los Angeles, USA University of California, Los Angeles, USA R. Cousins, P. Everaerts, C. Farrell, J. Hauser, M. Ignatenko, D. Saltzberg, E. Takasugi, V. Valuev, M. Weber University of California, Riverside, Riverside, USA The University of Alabama, Tuscaloosa, USA O. Charaf, S.I. Cooper, C. Henderson, P. Rumerio The University of Alabama, Tuscaloosa, USA O. Charaf, S.I. Cooper, C. Henderson, P. Rumerio Boston University, Boston, USA D. Arcaro, A. Avetisyan, T. Bose, C. Fantasia, D. Gastler, P. Lawson, D. Rankin, C. Richardson, J. Rohlf, J. St. John, L. Sulak, D. Zou J. Rohlf, J. St. John, L. Sulak, D. Zou 41 Brown University, Providence, USA J. Alimena, E. Berry, S. Bhattacharya, D. Cutts, N. Dhingra, A. Ferapontov, A. Garabedian, J. Hakala, U. Heintz, E. Laird, G. Landsberg, Z. Mao, M. Narain, S. Piperov, S. Sagir, R. Syarif Brown University, Providence, USA University of California, Riverside, Riverside, USA Lipton, T. Liu, R. Lopes De S´a, J. Lykken, K. Maeshima, J.M. Marrafﬁno, V.I. Martinez Outschoorn, S. Maruyama, D. Mason, P. McBride, P. Merkel, K. Mishra, S. Mrenna, S. Nahn, C. Newman-Holmes, V. O’Dell, K. Pedro, O. Prokofyev, G. Rakness, E. Sexton-Kennedy, A The CMS Collaboration 42 A. Soha, W.J. Spalding, L. Spiegel, N. Strobbe, L. Taylor, S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal, H.A. Weber, A. Whitbeck, F. Yang Lawrence Livermore National Laboratory, Livermore, USA D. Lange, F. Rebassoo, D. Wright D. Lange, F. Rebassoo, D. Wright University of Maryland, College Park, USA C. Anelli, A. Baden, O. Baron, A. Belloni, B. Calvert, S.C. Eno, C. Ferraioli, J.A. Gomez, N.J. Hadley, S. Jabeen, R.G. Kellogg, T. Kolberg, J. Kunkle, Y. Lu, A.C. Mignerey, Y.H. Shin, A. Skuja, M.B. Tonjes, S.C. Tonwar University of Florida, Gainesville, USA D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Carnes, M. Carver, D. Curry, S. Das, R.D. Field, I.K. Furic, S.V. Gleyzer, J. Hugon, J. Konigsberg, A. Korytov, J.F. Low, P. Ma, K. Matchev, H. Mei, P. Milenovic66, G. Mitselmakher, D. Rank, R. Rossin, L. Shchutska, M. Snowball, D. Sperka, N. Terentyev, L. Thomas, J. Wang, S. Wang, J. Yelton R.D. Field, I.K. Furic, S.V. Gleyzer, J. Hugon, J. Konigsberg, A. Korytov, J.F. Low, P. M K. Matchev, H. Mei, P. Milenovic66, G. Mitselmakher, D. Rank, R. Rossin, L. Shchutsk M. Snowball, D. Sperka, N. Terentyev, L. Thomas, J. Wang, S. Wang, J. Yelton Florida International University, Miami, USA Florida International University, Miami, USA S. Hewamanage, S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez y S. Hewamanage, S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida State University, Tallahassee, USA y, , A. Ackert, J.R. Adams, T. Adams, A. Askew, S. Bein, J. Bochenek, B. Diamond, J. Haas, S. Hagopian, V. Hagopian, K.F. Johnson, A. Khatiwada, H. Prosper, M. Weinberg S. Hagopian, V. Hagopian, K.F. Johnson, A. Khatiwada, H. Prospe Florida Institute of Technology, Melbourne, USA Florida Institute of Technology, Melbourne, USA M.M. Baarmand, V. Bhopatkar, S. Colafranceschi67, M. Hohlmann, H. Kalakhety, D. Noonan, T. Roy, F. Yumiceva University of Illinois at Chicago (UIC), Chicago, USA M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, I. Bucinskaite, R. Cavanaugh, O. Evdokimov, L. Gauthier, C.E. Gerber, D.J. Hofman, P. Kurt, C. O’Brien, I.D. Sandoval Gonzalez, C. Silkworth, H. Trauger, P. Turner, N. Varelas, Z. Wu, M. Zakaria University of Illinois at Chicago (UIC), Chicago, USA M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, I. Bucinskaite, R. Cavanaugh, O. Evdokimov, L. Gauthier, C.E. Gerber, D.J. Hofman, P. Kurt, C. O’Brien, I.D. Sandoval Gonzalez, p y g L. Gauthier, C.E. Gerber, D.J. Hofman, P. Kurt, C. O’Brien, I.D. Sandoval Gonzalez, C. Silkworth, H. Trauger, P. Turner, N. Varelas, Z. Wu, M. Zakaria The University of Iowa, Iowa City, USA B. Bilki68, W. Clarida, K. Dilsiz, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khristenko, J.-P. Merlo, H. Mermerkaya69, A. Mestvirishvili, A. Moeller, J. Nachtman, H. Ogul, Y. Onel, F. Ozok59, A. Penzo, C. Snyder, E. Tiras, J. Wetzel, K. Yi Johns Hopkins University, Baltimore, USA I. Anderson, B.A. Barnett, B. Blumenfeld, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan, P. Maksimovic, C. Martin, M. Osherson, J. Roskes, A. Sady, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You The University of Kansas, Lawrence, USA P. Baringer, A. Bean, G. Benelli, C. Bruner, R.P. Kenny III, D. Majumder, M. Malek, M. Murray, S. Sanders, R. Stringer, Q. Wang Kansas State University, Manhattan, USA y A. Ivanov, K. Kaadze, S. Khalil, M. Makouski, Y. Maravin, A. Mohammadi, L.K. Saini, N. Skhirtladze, S. Toda Lawrence Livermore National Laboratory, Livermore, USA D. Lange, F. Rebassoo, D. Wright Lawrence Livermore National Laboratory, Livermore, USA Purdue University, West Lafayette, USA V.E. Barnes, D. Benedetti, D. Bortoletto, L. Gutay, M.K. Jha, M. Jones, K. Jung, D.H. Miller, N. Neumeister, B.C. Radburn-Smith, X. Shi, I. Shipsey, D. Silvers, J. Sun, A. Svyatkovskiy, F. Wang, W. Xie, L. Xu F. Wang, W. Xie, L. Xu Purdue University Calumet, Hammond, USA N. Parashar, J. Stupak Purdue University Calumet, Hammond, USA N. Parashar, J. Stupak University of Nebraska-Lincoln, Lincoln, USA University of Nebraska-Lincoln, Lincoln, USA y E. Avdeeva, K. Bloom, S. Bose, D.R. Claes, A. Dominguez, C. Fangmeier, R. Gonzalez Suarez, R. Kamalieddin, J. Keller, D. Knowlton, I. Kravchenko, F. Meier, J. Monroy, F. Ratnikov, J.E. Siado, G.R. Snow g g R. Kamalieddin, J. Keller, D. Knowlton, I. Kravchenko, F. Meier, J. Monroy, F. Ratnikov, J.E. Siado, G.R. Snow J.E. Siado, G.R. Snow State University of New York at Buffalo, Buffalo, USA Northwestern University, Evanston, USA y K.A. Hahn, A. Kubik, N. Mucia, N. Odell, B. Pollack, A. Pozdnyakov, M. Schmitt, S. Stoynev, K. Sung, M. Trovato, M. Velasco State University of New York at Buffalo, Buffalo, USA State University of New York at Buffalo, Buffalo, USA M. Alyari, J. Dolen, J. George, A. Godshalk, C. Harrington, I. Iashvili, J. Kaisen, A. Kharchilava, A. Kumar, S. Rappoccio, B. Roozbahani y , , M. Alyari, J. Dolen, J. George, A. Godshalk, C. Harrington, I. Iashvili, J. Kaisen, A. Kharchilava, A. Kumar, S. Rappoccio, B. Roozbahani A. Kumar, S. Rappoccio, B. Roozbahani Northeastern University, Boston, USA G. Alverson, E. Barberis, D. Baumgartel, M. Chasco, A. Hortiangtham, A. Massironi, D.M. Morse, D. Nash, T. Orimoto, R. Teixeira De Lima, D. Trocino, R.-J. Wang, D. Wood, J. Zhang Northwestern University, Evanston, USA Massachusetts Institute of Technology, Cambridge, USA Massachusetts Institute of Technology, Cambridge, USA A. Apyan, R. Barbieri, A. Baty, K. Bierwagen, S. Brandt, W. Busza, I.A. Cali, Z. Demiragli, L. Di Matteo, G. Gomez Ceballos, M. Goncharov, D. Gulhan, Y. Iiyama, G.M. Innocenti, M. Klute, D. Kovalskyi, Y.S. Lai, Y.-J. Lee, A. Levin, P.D. Luckey, A.C. Marini, C. Mcginn, 43 C. Mironov, S. Narayanan, X. Niu, C. Paus, D. Ralph, C. Roland, G. Roland, J. Salfeld- Nebgen, G.S.F. Stephans, K. Sumorok, M. Varma, D. Velicanu, J. Veverka, J. Wang, T.W. Wang, B. Wyslouch, M. Yang, V. Zhukova University of Minnesota, Minneapolis, USA B. Dahmes, A. Evans, A. Finkel, A. Gude, P. Hansen, S. Kalafut, S.C. Kao, K. Klapoetke, Y. Kubota, Z. Lesko, J. Mans, S. Nourbakhsh, N. Ruckstuhl, R. Rusack, N. Tambe, J. Turkewitz University of Mississippi, Oxford, USA J.G. Acosta, S. Oliveros ir, B. Akgun, Z. Chen, K.M. Ecklund, F.J.M. Geurts, M. Guilbaud, W. Li, B. Michlin, thup, B.P. Padley, R. Redjimi, J. Roberts, J. Rorie, Z. Tu, J. Zabel University of Notre Dame, Notre Dame, USA University of Notre Dame, Notre Dame, USA University of Notre Dame, Notre Dame, USA A. Brinkerhoff, N. Dev, M. Hildreth, C. Jessop, D.J. Karmgard, N. Kellams, K. Lannon, N. Marinelli, F. Meng, C. Mueller, Y. Musienko39, M. Planer, A. Reinsvold, R. Ruchti, G. Smith, S. Taroni, N. Valls, M. Wayne, M. Wolf, A. Woodard The Ohio State University, Columbus, USA L. Antonelli, J. Brinson, B. Bylsma, L.S. Durkin, S. Flowers, A. Hart, C. Hill, R. Hughes, W. Ji, K. Kotov, T.Y. Ling, B. Liu, W. Luo, D. Puigh, M. Rodenburg, B.L. Winer, H.W. Wulsin The Ohio State University, Columbus, USA Princeton University, Princeton, USA Princeton University, Princeton, USA O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, S.A. Koay, P. Lujan, D. Marlow, T. Medvedeva, M. Mooney, J. Olsen, C. Palmer, P. Pirou´e, H. Saka, D. Stickland, C. Tully, A. Zuranski O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, S.A. Koay, P. Lujan, D. Marlow, T. Medvedeva, M Mooney J Olsen C Palmer P Pirou´e H Saka D Stickland C Tully A Zuranski O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, S.A. Koay, P. Lujan, D. Marlow, T. Med O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, S.A. Koay, P. Lujan, D. Marlow, T. Medvedeva, M. Mooney, J. Olsen, C. Palmer, P. Pirou´e, H. Saka, D. Stickland, C. Tully, A. Zuranski M. Mooney, J. Olsen, C. Palmer, P. Pirou´e, H. Saka, D. Stickland, C. Tully, A. Zuranski M. Mooney, J. Olsen, C. Palmer, P. Pirou´e, H. Saka, D. Stickland, C University of Puerto Rico, Mayaguez, USA S. Malik University of Wisconsin, Madison, USA y , , D.A. Belknap, D. Carlsmith, M. Cepeda, S. Dasu, L. Dodd, S. Duric, B. Gomber, M. Grothe, R Hall Wilton M Herndon A Herv´e P Klabbers A Lanaro A Levine K Long R Loveless D.A. Belknap, D. Carlsmith, M. Cepeda, S. Dasu, L. Dodd, S. Duric, B. Gomber, M. Grothe, R. Hall-Wilton, M. Herndon, A. Herv´e, P. Klabbers, A. Lanaro, A. Levine, K. Long, R. Loveless, g A. Mohapatra, I. Ojalvo, T. Perry, G.A. Pierro, G. Polese, T. Ruggles, T. Sarangi, A. Savin, A. Sharma, N. Smith, W.H. Smith, D. Taylor, N. Woods A. Mohapatra, I. Ojalvo, T. Perry, G.A. Pierro, G. Polese, T. Ruggles, T. Sarangi, A. Savin, University of Virginia, Charlottesville, USA y g M.W. Arenton, B. Cox, B. Francis, J. Goodell, R. Hirosky, A. Ledovskoy, H. Li, C. Lin, C. Neu, T. Sinthuprasith, X. Sun, Y. Wang, E. Wolfe, J. Wood, F. Xia M.W. Arenton, B. Cox, B. Francis, J. Goodell, R. Hirosky, A. Ledovskoy, H. Li, C. Lin, h h lf d Wayne State University, Detroit, USA C. Clarke, R. Harr, P.E. Karchin, C. Kottachchi Kankanamge Don, P. Lamichhane, J. Sturdy University of Wisconsin, Madison, USA Rice University, Houston, USA A. Adair, B. Akgun, Z. Chen, K.M. Ecklund, F.J.M. Geurts, M. Guilbaud, W. Li, B. Michlin, M. Northup, B.P. Padley, R. Redjimi, J. Roberts, J. Rorie, Z. Tu, J. Zabel g M. Northup, B.P. Padley, R. Redjimi, J. Roberts, J. Rorie, Z. Tu, J. Zabel A The CMS Collaboration 44 University of Rochester, Rochester, USA University of Rochester, Rochester, USA B. Betchart, A. Bodek, P. de Barbaro, R. Demina, Y. Eshaq, T. Ferbel, M. Galanti, A. Garcia- Bellido, J. Han, A. Harel, O. Hindrichs, A. Khukhunaishvili, G. Petrillo, P. Tan, M. Verzetti B. Betchart, A. Bodek, P. de Barbaro, R. Demina, Y. Eshaq, T. Ferbel, M. Galanti, A. Garcia- Bellido, J. Han, A. Harel, O. Hindrichs, A. Khukhunaishvili, G. Petrillo, P. Tan, M. Verzetti Rutgers, The State University of New Jersey, Piscataway, USA Rutgers, The State University of New Jersey, Piscataway, USA S. Arora, A. Barker, J.P. Chou, C. Contreras-Campana, E. Contreras-Campana, D. Ferencek, Y. Gershtein, R. Gray, E. Halkiadakis, D. Hidas, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, A. Lath, K. Nash, S. Panwalkar, M. Park, S. Salur, S. Schnetzer, D. Shefﬁeld, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker University of Tennessee, Knoxville, USA M. Foerster, G. Riley, K. Rose, S. Spanier, A. York Texas A&M University, College Station, USA O. Bouhali70, A. Castaneda Hernandez70, A. Celik, M. Dalchenko, M. De Mattia, A. Delgado, S. Dildick, R. Eusebi, J. Gilmore, T. Huang, T. Kamon71, V. Krutelyov, R. Mueller, I. Osipenkov, Y. Pakhotin, R. Patel, A. Perloff, A. Rose, A. Safonov, A. Tatarinov, K.A. Ulmer2 Texas Tech University, Lubbock, USA Texas Tech University, Lubbock, USA N. Akchurin, C. Cowden, J. Damgov, C. Dragoiu, P.R. Dudero, J. Faulkner, S. Kunori, K. Lamichhane, S.W. Lee, T. Libeiro, S. Undleeb, I. Volobouev Vanderbilt University, Nashville, USA E. Appelt, A.G. Delannoy, S. Greene, A. Gurrola, R. Janjam, W. Johns, C. Maguire, Y. Mao, A. Melo, H. Ni, P. Sheldon, B. Snook, S. Tuo, J. Velkovska, Q. Xu Vanderbilt University, Nashville, USA University of Virginia, Charlottesville, USA M.W. Arenton, B. Cox, B. Francis, J. Goodell, R. Hirosky, A. Ledovskoy, H. Li, C. Lin, C. Neu, T. Sinthuprasith, X. Sun, Y. Wang, E. Wolfe, J. Wood, F. Xia 52: Also at Cag University, Mersin, Turkey 53: Also at Piri Reis University, Istanbul, Turkey 53: Also at Piri Reis University, Istanbul, Turkey †: Deceased Petersburg State Polytechnical University, St. Petersburg, Russia 42: Also at INFN Sezione di Padova; Universit`a di Padova; Universit`a di Trento (Trento), Padova, Italy 42: Also at INFN Sezione di Padova; Universit`a di Padova; Universit`a di Trento (Trento), Padova, Italy 43: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 43: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 44: Also at INFN Sezione di Roma; Universit`a di Roma, Roma, Italy 44: Also at INFN Sezione di Roma; Universit`a di Roma, Roma, Italy 45: Also at National Technical University of Athens, Athens, Greece 45: Also at National Technical University of Athens, Athens, Greece 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 47: Also at University of Athens, Athens, Greece 48: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 48: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 49: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 49: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 50: Also at Adiyaman University, Adiyaman, Turkey 50: Also at Adiyaman University, Adiyaman, Turkey 51: Also at Mersin University, Mersin, Turkey 51: Also at Mersin University, Mersin, Turkey 52: Also at Cag University, Mersin, Turkey †: Deceased 1: Also at Vienna University of Technology, Vienna, Austria 2: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 2: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 2: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 3: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China 3: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China o at Institut Pluridisciplinaire Hubert Curien, Universit´e de Strasbourg, Universit´e de 4: Also at Institut Pluridisciplinaire Hubert Curien, Universite de Strasbourg, Universite de Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 5: Also at National Institute of Chemical Physics and Biophysics, Tallinn, Estonia 6: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 6: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 7: Also at Universidade Estadual de Campinas, Campinas, Brazil p p 8: Also at Centre National de la Recherche Scientiﬁque (CNRS) - IN2P3, Paris, France q ( ) 9: Also at Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau, France q 9: Also at Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau, France 10: Also at Joint Institute for Nuclear Research, Dubna, Russia 10: Also at Joint Institute for Nuclear Research, Dubna, Russia 11: Also at Helwan University, Cairo, Egypt 11: Also at Helwan University, Cairo, Egypt 45 12: Now at Zewail City of Science and Technology, Zewail, Egypt 12: Now at Zewail City of Science and Technology, Zewail, Egypt y gy 13: Also at Beni-Suef University, Bani Sweif, Egypt 13: Also at Beni-Suef University, Bani Sweif, Egypt 14: Now at British University in Egypt, Cairo, Egypt 14: Now at British University in Egypt, Cairo, Egypt 15: Now at Ain Shams University, Cairo, Egypt 15: Now at Ain Shams University, Cairo, Egypt y gyp 16: Also at Universit´e de Haute Alsace, Mulhouse, France y gyp 16: Also at Universit´e de Haute Alsace, Mulhouse, France 17: Also at Tbilisi State University, Tbilisi, Georgia 17: Also at Tbilisi State University, Tbilisi, Georgia y y y 19: Also at Indian Institute of Science Education and Research, Bhopal, India y y 19: Also at Indian Institute of Science Education and Re y y 19: Also at Indian Institute of Science Education and Rese 20: Also at University of Hamburg, Hamburg, Germany y g g y 21: Also at Brandenburg University of Technology, Cottbus, Germany y g g y 21: Also at Brandenburg University of Technology, Cottbus, Germany 22: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 22: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 23: Also at E¨otv¨os Lor´and University, Budapest, Hungary 23: Also at E¨otv¨os Lor´and University, Budapest, Hungary 24: Also at University of Debrecen, Debrecen, Hungary 24: Also at University of Debrecen, Debrecen, Hungary 25: Also at Wigner Research Centre for Physics, Budapest, Hungary 25: Also at Wigner Research Centre for Physics, Budapest, Hungary 26: Also at University of Visva-Bharati, Santiniketan, India 26: Also at University of Visva-Bharati, Santiniketan, India 27: Now at King Abdulaziz University, Jeddah, Saudi Arabia 27: Now at King Abdulaziz University, Jeddah, Saudi Arabia 28: Also at University of Ruhuna, Matara, Sri Lanka 29: Also at Isfahan University of Technology, Isfahan, Iran 29: Also at Isfahan University of Technology, Isfahan, Iran 30: Also at University of Tehran, Department of Engineering Science, Tehran, Iran 30: Also at University of Tehran, Department of Engineerin 31: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran 2: Also at Laboratori Nazionali di Legnaro dell’INFN, Legnaro, Italy 32: Also at Laboratori Nazionali di Legnaro dell’INFN, Legnaro, Italy 33: Also at Universit`a degli Studi di Siena, Siena, Italy 34: Also at Purdue University, West Lafayette, USA y y 35: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 36: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 37: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 38: Also at Warsaw University of Technology, Institute of Electronic Systems, Wa 39: Also at Institute for Nuclear Research, Moscow, Russia 39: Also at Institute for Nuclear Research, Moscow, Russia 40: Now at National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia 40: Now at National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia 41: Also at St. 53: Also at Piri Reis University, Istanbul, Turkey 54: Also at Gaziosmanpasa University, Tokat, Turkey 54: Also at Gaziosmanpasa University, Tokat, Turkey 55: Also at Ozyegin University, Istanbul, Turkey 55: Also at Ozyegin University, Istanbul, Turkey 56: Also at Izmir Institute of Technology, Izmir, Turkey 56: Also at Izmir Institute of Technology, Izmir, Turkey 57: Also at Marmara University, Istanbul, Turkey 57: Also at Marmara University, Istanbul, Turkey A The CMS Collaboration 46 58: Also at Kafkas University, Kars, Turkey 58: Also at Kafkas University, Kars, Turkey y y 59: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 59: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 60: Also at Yildiz Technical University, Istanbul, Turkey 60: Also at Yildiz Technical University, Istanbul, Turkey 61: Also at Hacettepe University, Ankara, Turkey 61: Also at Hacettepe University, Ankara, Turkey p y y 62: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 62: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom pp y g 63: Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom 63: Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom g 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain : Also at Utah Valley University, Orem, USA y y 66: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 66: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia g 67: Also at Facolt`a Ingegneria, Universit`a di Roma, Roma, Italy g 67: Also at Facolt`a Ingegneria, Universit`a di Roma, Roma, Italy 68: Also at Argonne National Laboratory, Argonne, USA 68: Also at Argonne National Laboratory, Argonne, USA 69: Also at Erzincan University, Erzincan, Turkey 69: Also at Erzincan University, Erzincan, Turkey 70: Also at Texas A&M University at Qatar, Doha, Qatar 70: Also at Texas A&M University at Qatar, Doha, Qatar 71: Also at Kyungpook National University, Daegu, Korea 71: Also at Kyungpook National University, Daegu, Korea
https://openalex.org/W2883377420	https://europepmc.org/articles/pmc6133269?pdf=render	English	null	Survival and development of potato psyllid (Hemiptera: Triozidae) on Convolvulaceae: effects of a plant-fungus symbiosis (<i>Periglandula</i>)	bioRxiv (Cold Spring Harbor Laboratory)	2,018	public-domain	11,473	RESEARCH ARTICLE Navneet Kaur1,2, William Rodney Cooper2, Jennifer M. Duringer3, Ismael E. Badillo- Vargas4, Gabriela Esparza-Dı´az4¤, Arash Rashed1, David R. Horton2 Navneet Kaur1,2, William Rodney Cooper2, Jennifer M. Duringer3, Ismael E. Badillo- Vargas4, Gabriela Esparza-Dı´az4¤, Arash Rashed1, David R. Horton2 1 Department of Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, Idaho, United States of America, 2 USDA-ARS, Temperate Tree Fruit and Vegetable Research Unit, Wapato, Washington, United States of America, 3 Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, United States of America, 4 Department of Entomology, Texas A&M AgriLife Research and Extension Center, Weslaco, Texas, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: AmerStem Inc., Camarillo, California, United States of America * Navneet.kaur2@ars.usda.gov OPEN ACCESS Plant species in the family Solanaceae are the usual hosts of potato psyllid, Bactericera cockerelli (Sˇ ulc) (Hemiptera: Psylloidea: Triozidae). However, the psyllid has also been shown to develop on some species of Convolvulaceae (bindweeds and morning glories). Developmental success on Convolvulaceae is surprising given the rarity of psyllid species worldwide associated with this plant family. We assayed 14 species of Convolvulaceae across four genera (Convolvulus, Calystegia, Ipomoea, Turbina) to identify species that allow development of potato psyllid. Two populations of psyllids were assayed (Texas, Washington). The Texas population overlaps extensively with native Convolvulaceae, whereas Washington State is noticeably lacking in Convolvulaceae. Results of assays were overlain on a phylogenetic analysis of plant species to examine whether Convolvulaceae distantly related to the typical host (potato) were less likely to allow development than spe- cies of Convolvulaceae more closely related. Survival was independent of psyllid population and location of the plant species on our phylogenetic tree. We then examined whether pres- ence of a fungal symbiont of Convolvulaceae (Periglandula spp.) affected psyllid survival. These fungi associate with Convolvulaceae and produce a class of mycotoxins (ergot alka- loids) that may confer protection against plant-feeding arthropods. Periglandula was found in 11 of our 14 species, including in two genera (Convolvulus, Calystegia) not previously known to host the symbiont. Of these 11 species, leaf tissues from five contained large quantities of two classes of ergot alkaloids (clavines, amides of lysergic acid) when evalu- ated by LC-MS/MS. All five species also harbored Periglandula. No ergot alkaloids were detected in species free of the fungal symbiont. Potato psyllid rapidly died on the five spe- cies that harbored Periglandula and contained ergot alkaloids, but survived to adulthood on seven of the nine species in which ergot alkaloids were not detected. These results support Citation: Kaur N, Cooper WR, Duringer JM, Badillo-Vargas IE, Esparza-Dı´az G, Rashed A, et al. (2018) Survival and development of potato psyllid (Hemiptera: Triozidae) on Convolvulaceae: Effects of a plant-fungus symbiosis (Periglandula). PLoS ONE 13(9): e0201506. Introduction The potato psyllid, Bactericera cockerelli (Sˇulc) (Hemiptera: Psylloidea: Triozidae) is a pest of solanaceous crops such as potatoes, tomatoes, and peppers. The psyllid occurs throughout the western and central United States, Canada, Mexico, and Central America [1], and as an intro- duction in New Zealand and Australia [2, 3]. High densities of the psyllid may lead to plant disorders known as “psyllid yellows” [4, 5] caused by a toxin that is injected into plants during the psyllid’s feeding activities [6]. However, recent crop losses have been caused primarily by a bacterial pathogen, ‘Candidatus Liberibacter solanacearum’ (Lso), that is transmitted by the psyllid [1]. Difficulties in managing potato psyllid and its associated Liberibacter are in part due to poor understanding of the role that non-crop species have in the biology of the vector. Most species of psyllids are monophagous or oligophagous, limited to development on plants within a single genus or family [7, 8]. Potato psyllid is unusual in being able to develop on plants across more than a single family [9, 10, 11, 12]. Non-crop plant species act as reservoirs of the insect during the growing season and may help the psyllid bridge intervals in which crop hosts are unavailable [10, 13, 14, 15, 16, 17]. It is therefore important to know what non- crop species of plants found in potato or tomato growing regions also support the reproduc- tion and development of potato psyllid. Although plant species in the family Solanaceae (Solanales) are the typical developmental hosts for potato psyllid, at least some species in the Convolvulaceae (Solanales) also support development [9, 10, 11, 12, 14, 18]. Observations leading to this conclusion include rearing tri- als [9, 10, 11, 12] and field records [14, 18]. Developmental success on Convolvulaceae is unex- pected given that Convolvulaceae is substantially underrepresented among plant families as hosts of Psylloidea. Despite its extensive diversity and widespread distribution [19] Convolvu- laceae is listed as a developmental host for only five species of psyllids worldwide, including potato psyllid [20]. Rearing trials with potato psyllid have been limited to two species, Convol- vulus arvensis L. (field bindweed) and Ipomoea batatas (L.) Lam. (sweet potato). While potato psyllid is able to complete development on these species, development rates are slow and may be accompanied by nymphal mortality [10, 12]. A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid the hypothesis that a plant-fungus symbiotic relationship affects the suitability of certain Convolvulaceae to potato psyllid. the hypothesis that a plant-fungus symbiotic relationship affects the suitability of certain Convolvulaceae to potato psyllid. design, data collection and analysis, decision to publish, or preparation of the manuscript. design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. OPEN ACCESS https://doi.org/10.1371/ journal.pone.0201506 Editor: Sean Michael Prager, University of Saskatchewan College of Agriculture and Bioresources, CANADA Received: July 12, 2018 Accepted: August 29, 2018 Published: September 11, 2018 Editor: Sean Michael Prager, University of Saskatchewan College of Agriculture and Bioresources, CANADA Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the ARS Federal-State Partnership Potato Research Grants program, the Northwest Potato Research Consortium, and USDA-NIFA-SCRI (#2015-51181- 24292) to DH. The funders had no role in study 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Introduction In this study, we examined the development of potato psyllid on species and genera of Con- volvulaceae that have not previously been assayed. Our assays targeted species that are native to North America and are thus likely to have an evolutionary history with at least some popula- tions of potato psyllid. Our first objective was to assay a taxonomically broader group of Con- volvulaceae than previously done, to determine whether plant suitability extends beyond Co. arvensis and I. batatas. Part of this objective included a comparison of two haplotypes of the psyllid on each plant species. Potato psyllid occurs as a minimum of four unique genetic types or “haplotypes” [21, 22] that we now know differ biologically [23, 24, 25, 26, 27]. We compared developmental success on Convolvulaceae between two of these haplotypes, the Central haplo- type and the Northwestern haplotype. Convolvulaceae is highly diverse in the southern US and Mexico [28] where its presence overlaps extensively with the distribution of the Central haplotype [29]. In contrast, native Convolvulaceae are almost completely absent from the Pacific Northwest region of the US [28] where the Northwestern haplotype of potato psyllid is endemic [21, 22, 30]. Thus, the Northwestern haplotype is likely to have a much-reduced field history with native Convolvulaceae in comparison to the Central haplotype. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 2 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Our second objective was to look for traits that predict whether a given plant species allows psyllid development. We addressed two separate questions in this objective. First, we examined whether suitability is predicted by the location of plant species within a phylogenetic tree. Because of the strong tendency towards host specificity among species of Psylloidea, host switching or dietary expansion by psyllids tends to be phylogenetically conserved [31] such that evolutionary shifts in diets by psyllids are often between closely related plant species [8, 31, 32]. This specialism prompted us to examine whether plant suitability tracked plant phy- logeny. We constructed a phylogenetic tree from DNA-sequence data to examine whether plant species allowing successful development of potato psyllid clustered together in the tree, as would be expected if plant chemistry or other traits affecting psyllid host use also grouped phylogenetically [33]. We then examined whether psyllid development was affected by the presence of a plant- fungus mutualism found in Convolvulaceae. Introduction The Convolvulaceae is unusual among dicotyle- donous plant families in its association with a class of chemicals known as ergot alkaloids [34]. Many species of Convolvulaceae have formed a symbiotic association with clavicipitaceous fungi in the genus Periglandula [35, 36, 37, 38]. This fungus is vertically transmitted, and is present systemically in members of the family Convolvulaceae [39] often forming epiphytic colonies surrounding peltate glandular trichomes on the adaxial leaf surfaces [35, 40] where the colonies produce ergot alkaloids [41]. This symbiosis appears to be most common in Ipo- moea and related genera, with possibly 450 or more plant species worldwide having the associ- ation [34, 42]. Similar alkaloids produced by clavicipitaceous fungi in grasses have been shown to have deleterious effects against herbivorous insects [43, 44, 45]. The defensive properties of ergot alkaloids associated with the Convolvulaceae-Periglandula symbiosis have received almost no attention, although extracts from Ipomoea parasitica (H.B.K.) G. Don, have been found to reduce feeding and digestive efficiency of caterpillars [46]. Our overall goal therefore was to examine whether survival and development of the potato psyllid was correlated with the presence or absence of Periglandula, and to determine whether psyllid development was affected by the types and quantities of fungal alkaloids produced by this symbiosis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 silvatica overlaps geographically with potato psyllid given historical uncertainties in distribution of the plant in the western U.S. ific Northwest plant is grown only as a summer ornamental. This may result in some level of sympatry with the Northwester https://doi.org/10.1371/journal.pone.0201506.t001 lights. Assays were done at the USDA–ARS in Wapato, WA. Plants at 1 to 4 fully expanded leaf stage were used in the assays. Potato psyllids to be used in assays were obtained from colonies maintained at the USDA-ARS facility in Wapato, WA. The parental insects for colonies were collected from potato fields near Weslaco, TX in March 2017 (Central haplotype, APHIS permit P526P-17- 00366) and from solanaceous weeds growing near Prosser, WA in the summer and autumn of 2016 (Northwestern haplotype). The colonies were maintained on potato (‘Russet Burbank’) at 22˚C and a 16:8 h light: dark cycle. Colonies were assayed preceding the study using high reso- lution melting analysis to confirm haplotype status [21]. Colonies were checked periodically for Lso infection using PCR detection methods [50]. Colonies were Lso-free for the duration of the study. Source of plants and insects Insect bioassays included a screening of 11 species of native Convolvulaceae distributed across three plant genera (Convolvulus, Ipomoea, and Turbina), and three introduced species in Con- volvulus and Calystegia including the widespread pest field bindweed, Co. arvensis (Table 1). All species except Calystegia silvatica overlap geographically with psyllids of the Central haplo- type (Table 1). The Northwestern haplotype overlaps geographically with Co. arvensis, possibly with Ca. silvatica, and is likely to have some overlap with the four species of Ipomoea that are grown extensively as summer ornamentals (Table 1). It is unlikely that these ornamentals are able to survive the winter conditions of the Pacific Northwest. Test plants were examined in side-by-side comparisons with potato, Solanum tuberosum L. (‘Russet Burbank’) (Solanaceae), a typical and highly suitable host for potato psyllid. Plants were grown either from seeds or from stem cuttings (sources listed in Table 1). Seeds were scarified using sandpaper and soaked in gibberellic acid (1000 ppm in water) for 24 h prior to planting. Plants were grown in 10-cm pots (volume ~ 473.3 cm3) containing four parts com- mercial potting soil (Miracle-Gro Moisture Control Potting Mix, Scotts Company, Marysville, OH), one part perlite (Miracle-Gro Perlite, Scotts Company, Marysville, OH), and one part clean sand, and maintained in a greenhouse under ambient light supplemented with grow PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 3 / 19 C: Central haplotype occurs within geographic range of plant; N: Northwestern haplotype occurs within geographic range of plant. Haplotype distribution data [21, 22, 29, 30]. Plant distribution data [28]. Calystegia is a taxonomically difficult genus with species often exhibiting substantial geographic variation in morphological traits [47]. We believe that the Calystegia assayed in this study is Calystegia silvatica (Kit.) Griseb. subsp. disjuncta Brummitt [48, 49]. Calystegia silvatica subsp. disjuncta is likely of Mediterranean origin, although there have been suggestions (probably incorrect) that it is native to North America [49]. It is unclear whether Ca. silvatica overlaps geographically with potato psyllid given historical uncertainties in distribution of the plant in the western U.S. ǂ h f h l l l h l l l f h h h h l A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Table 1. List of plant species used in assays (origin, geographic overlap with psyllid haplotype, and source). Species North America Origin Overlap of insect haplotype and plant Source Convolvulus equitans Benth. Native C Western Region Plant Introduction Station, Pullman WA Convolvulus tricolor L. ǂ Introduced C+N J.L. Hudson, Seedsman, La Honda, CA Convolvulus arvensis L. Introduced C+N Prosser, WA Calystegia silvatica (Kit.) Griseb. Introduced N? Tillamook Co., OR Ipomoea alba L. ǂ Native C+N The Sample Seed Shop, Buffalo, NY Ipomoea cordatotriloba Dennstedt Native C Georgia Vines, Claxton, GA Ipomoea hederacea L. Native C J.L. Hudson, Seedsman, La Honda, CA Ipomoea ternifolia Torrey Native C Southwest Seeds, Dolores, CO Ipomoea nil (L.) Rothǂ Native C+N The Sample Seed Shop, Buffalo, NY Ipomoea imperati (Vahl) Grisebach Native C South Padre Island, TX Ipomoea leptophylla Torrey Native C Georgia Vines, Claxton, GA Ipomoea pandurata (L.) G.F. Meyer Native C Georgia Vines, Claxton, GA Ipomoea tricolor Cavanillesǂ Native C+N J.L. Hudson, Seedsman, La Honda, CA Turbina corymbosa (L.) Rafinesque Native C J.L. Hudson, Seedsman, La Honda, CA Solanum tuberosum L. Native C+N Skone & Conners, Warden, WA Table 1. List of plant species used in assays (origin, geographic overlap with psyllid haplotype, and sourc C: Central haplotype occurs within geographic range of plant; N: Northwestern haplotype occurs within geographic range of plant. Haplotype distribution data [21, 22, 29, 30]. Plant distribution data [28]. Calystegia is a taxonomically difficult genus with species often exhibiting substantial geographic variation in morphological traits [47]. We believe that the Calystegia assayed in this study is Calystegia silvatica (Kit.) Griseb. subsp. disjuncta Brummitt [48, 49]. Calystegia silvatica subsp. disjuncta is likely of Mediterranean origin, although there have been suggestions (probably incorrect) that it is native to North America [49]. It is unclear whether Ca. silvatica overlaps geographically with potato psyllid given historical uncertainties in distribution of the plant in the western U.S. Calystegia is a taxonomically difficult genus with species often exhibiting substantial geographic variation in morphological traits [47]. We believe that the Calystegia assayed in this study is Calystegia silvatica (Kit.) Griseb. subsp. disjuncta Brummitt [48, 49]. Calystegia silvatica subsp. disjuncta is likely of Mediterranean origin, although there have been suggestions (probably incorrect) that it is native to North America [49]. It is unclear whether Ca. A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid adults were removed. Containers were monitored every 2–3 days for hatching of eggs and sub- sequent development of nymphs. For plant species on which psyllids developed successfully, we recorded the number of days required to develop from egg deposition to production of the first adult (i.e., the minimum time required to complete development). We did not attempt to compare survival rates among plant species due to difficulties in obtaining accurate estimates of egg numbers on plants with- out also damaging the plant. Once new adults were seen in a container, that plant and con- tainer was dismantled. A leaf was collected from the plant for DNA extraction and biochemical analysis (described below). On species which failed to support development, mortality almost invariably occurred as first instar nymphs often within 48 h of hatch. When monitoring showed that all nymphs on a given plant were dead, the assay for that plant and container was dismantled, and leaf samples were collected for DNA extraction and ergot alkaloid quantification. We had five replicates per plant species per psyllid haplotype combination. The large number of treatments, com- bined with uneven germination of seed, did not allow us to conduct the five replicates simulta- neously. Thus, each replicate was initiated on a separate date, with date of assay included in the statistical analyses as a blocking factor (see Statistical analyses). Phylogenetic mapping of Convolvulaceae DNA was extracted using a cetyltrimethylammonium bromide (CTAB) precipitation method [51]. Two different universal plant barcoding primer sets were used. The first primer set tar- geted approximately 500 bp of the internal transcribed spacer region (ITS): ITS2F (ATGCGA TACTTGGTGTGAAT)and ITS3R (GACGCTTCTCCAGACTACAAT) [52]. The second primer set targeted approximately 684 bp region of the chloroplast maturase K gene (matK): matK 472-F (CCCRTYCATCTGGAAATCTTGGTT) and matK 1248-R (GCTRTRATAATGAGAAAGA TTTCTGC) [53]. PCR conditions used for both primer sets were similar, consisting of an initial denaturation step of 94˚C for 5 min followed by 35 cycles of 94˚C for 30 s, 56˚C for 30 s, and 72˚C for 42 s, followed by a final extension at 72˚C for 10 min. Each 20μl reaction contained Amplitaq Gold 360 PCR Master Mix (Invitrogen, Carsbad, CA), 500nM of each primer, and DNA template (10–20 ng). Upon amplification, bands were excised from agarose gels, purified using GenElute minus ethidium bromide spin columns (Sigma, St. Louis, MO), and were cloned using a TOPO TA cloning kit with TOP10 E. coli chemical competent cells (Invitrogen, Carlsbad, CA). The QIAprep spin mini prep kit (Qiagen, Valencia, CA) was used to prepare plasmid DNA for sequencing by MC Laboratories (MC Lab, San Francisco, CA). Sequences were deposited into GenBank (Table 2). DNA sequences were aligned and consensus sequences were made using Geneious R10 software (North America Biomatters Inc, Newark, NJ). The phylogenetic tree was constructed using a Tamura-Nei model and neighbor-Joining method with the Tree Builder function of Geneious R10 [54]. Phylogenetic distances for tree construction were estimated based upon concatenated sequences of ITS and matK regions. Potato was treated as an outgroup. Suitability of Convolvulaceae to potato psyllid Our primary objective was to determine whether the potato psyllid is able to complete devel- opment on targeted plant species. Given the large size of the experimental design (two psyllid haplotypes x 15 plant species), we limited our measures of psyllid performance to two traits: egg-to-adult survival (as a yes/no variable), and egg-to-adult development time (in days). Ten adults (unsexed) from both haplotypes were collected from their respective colony cages. The ten psyllids of a given haplotype were confined for egg-laying on a test plant kept individually in a 7.5 L plastic container (Cambro1, Huntington Beach, CA) modified to allow ventilation at 22˚C and a 16:8 h light: dark cycle. Once 20 or more eggs were present on a test plant, the PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 4 / 19 Quantification of ergot alkaloids Acetonitrile (ACN) and methanol (LC-MS grade) as well as acetic acid (OmniTrace Ultra), were purchased from EMD Millipore (Darmstadt, Germany). Ammonium acetate (>99.0%, HPLC grade) was obtained from Sigma Aldrich (St. Louis, MO USA). Ergot alkaloid standards were purchased from Romer Labs (Tulln, Austria) (biopure mix 6-ergocornine, ergocristine, α-ergocryptine, ergometrine, ergosine and ergotamine) and Sigma-Aldrich (St. Louis, MO USA) (ergonovine, agroclavine, lysergic acid and lysergol). Ultrapure 18 mO cm-1 water was obtained from an Elga (Marlow, Buckinghamshire, UK.) PURELAB Ultra Genetic system. Acetonitrile (ACN) and methanol (LC-MS grade) as well as acetic acid (OmniTrace Ultra), were purchased from EMD Millipore (Darmstadt, Germany). Ammonium acetate (>99.0%, HPLC grade) was obtained from Sigma Aldrich (St. Louis, MO USA). Ergot alkaloid standards were purchased from Romer Labs (Tulln, Austria) (biopure mix 6-ergocornine, ergocristine, α-ergocryptine, ergometrine, ergosine and ergotamine) and Sigma-Aldrich (St. Louis, MO USA) (ergonovine, agroclavine, lysergic acid and lysergol). Ultrapure 18 mO cm-1 water was obtained from an Elga (Marlow, Buckinghamshire, UK.) PURELAB Ultra Genetic system. Fully expanded leaves were collected from assayed plants at the end of suitability tests and subjected to air drying at the room temperature ~22–25˚C for 3-5d. Dried tissue was ground using either a mortar and pestle or a cyclone sample mill with a 0.5 mm screen (UDY Corpora- tion, Fort Collins CO). Extraction solution (79:20:1 ACN:water:acetic acid) was added to ground sample at a ratio of 4 mL/g and turned for 90 min in the dark [56]. The sample was then centrifuged for 2 min at 1462 x g. Dilution solution (250 μL 20:79:1 ACN:water:acetic acid) was added to 250 μL supernatant, vortexed for 10 sec, then placed in an amber HPLC vial for ergot alkaloid analysis by LC-MS/MS. Fully expanded leaves were collected from assayed plants at the end of suitability tests and subjected to air drying at the room temperature ~22–25˚C for 3-5d. Dried tissue was ground using either a mortar and pestle or a cyclone sample mill with a 0.5 mm screen (UDY Corpora- tion, Fort Collins CO). Extraction solution (79:20:1 ACN:water:acetic acid) was added to ground sample at a ratio of 4 mL/g and turned for 90 min in the dark [56]. The sample was then centrifuged for 2 min at 1462 x g. A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Table 2. GenBank accession numbers. Species ITS matK dmaW Convolvulus equitans MG889580 MH198126 MH195190 Convolvulus tricolor MG889582 MH198117 MH195191 Convolvulus arvensis MG889579 MH198115 nd Calystegia silvatica MG889581 MH198116 MH195189 Ipomoea alba MG910322 MH198118 nd Ipomoea cordatotriloba MG910323 MH198119 MH195192 Ipomoea hederacea MG910324 MH198127 MH195193 Ipomoea ternifolia MG910327 MH198121 MH195196 Ipomoea nil MG910328 MH198122 nd Ipomoea imperati MG910325 MH198120 MH195194 Ipomoea leptophylla MG910326 MH198128 MH195199 Ipomoea pandurata MG910329 MH198123 MH195195 Ipomoea tricolor MG910330 MH198124 MH195197 Turbina corymbosa MG910332 MH198125 MH195198 Solanum tuberosum MG910331 MH198129 nd Not detected (nd) https://doi org/10 1371/journal pone 0201506 t002 Table 2. GenBank accession numbers. https://doi.org/10.1371/journal.pone.0201506.t002 PCR conditions consisted of an initial denaturation step of 95˚C for 5 min followed by 40 cycles of 95˚C for 1 min, 52˚C for 1 min, and 72˚C for 45 s, followed by a final extension at 72˚C for 5 min. Each 20μl reaction contained Amplitaq Gold 360 PCR Master Mix (Invitrogen, Carsbad, CA), 500nM of each primer, and DNA template (10–20 ng). Upon amplification, bands were excised from agarose gels, purified using GenElute minus ethidium bromide spin columns, and were cloned (methods described in previously). Sequencing again was done by MC Laboratories. Sequences were deposited into GenBank (Table 2). Detection of the Convolvulaceae-Periglandula association Because Periglandula is not always readily visible on plants even when the fungus is present, extraction and analysis of DNA-sequences is often used to confirm infestation. Presence or absence of the dmaW gene, encoding 4- (γ,γ –dimethylallyl) tryptophan synthase and required for the determinant step of ergot alkaloid synthesis, was evaluated using PCR [35, 55]. Plant DNA extracted using CTAB method was used to amplify approximately 1050 bp region using dmaWF5 (GACCGTAAACGAGTCAGGAA) and dmaWR2 (AAATACACCTGGGGCTCG)primers. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 5 / 19 https://doi.org/10.1371/journal.pone.0201506.t002 Quantification of ergot alkaloids Dilution solution (250 μL 20:79:1 ACN:water:acetic acid) was added to 250 μL supernatant, vortexed for 10 sec, then placed in an amber HPLC vial for ergot alkaloid analysis by LC-MS/MS. An ABI/SCIEX 3200 QTRAP LC-MS/MS system (Applied Biosystems, Foster City, CA USA) was used to monitor for ergoline and ergopeptide compounds via positive electrospray ionization, with separation performed using a Perkin Elmer (Waltham, MA USA) Series 200 PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 6 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid autosampler and HPLC connected to a Gemini C18 column (150 x 4.6 mm, 5 μ, Phenomenex (Torrance, CA USA)) with a 4 x 3 mm security guard cartridge of similar packing [56]. Mobile phases consisted of 5 mM ammonium acetate and methanol:water:acetic acid in a ratio of 10:89:1(v/v/v) (A) or 97:2:1 (B) and were run in a gradient program at 1 mL/min. Multiple reaction monitoring (MRM) of two transitions (quantitative and qualitative) per compound was used to detect the ergot alkaloids ergonovine, ergotamine, ergocornine, α-ergocryptine, ergocristine, ergovaline, ergine, ergosine and their epimers, as well as agroclavine, chanocla- vine, lysergol, lysergic acid, oxidized luol, dihydrolysergol, chanoclavine, dihydroergosine, dihydroergotamine, festuclavine, fumigaclavine and elymoclavine. The presence of a mycotoxin was confirmed when the signal was equal to or greater than a signal-to-noise (S/N) ratio of 3:1 (limit of detection (LOD)), and both quantitative and qualita- tive transitions were present. Samples were quantitated blind as to sample identity against a standard curve using Analyst 1.6.2 and MultiQuant 3.0.1 (Applied Biosystems). The limit of quantitation (LOQ) was defined as the concentration at which the analyte had a precision and accuracy that did not exceed greater than 20% of the coefficient of variation [57]. LOD and LOQ for detected mycotoxins were as follows: ergotamine, ergocornine, ergosine ergocristine and agroclavine (1, 1 ng/mL); ergonovine (1, 2 ng/mL); lysergol (2, 2 ng/mL); lysergic acid (20 and 50 ng/mL). No commercial standards were available for chanoclavine, festuclavine, elymo- clavine, elymoclavine fructoside, ergine and dihydrolysergol. These compounds were com- pared on a scale of present (“+” indicating low, “++” indicating high) or not present (“-“) amongst the plant species extracted based on relative peak area. Statistical analyses Effects of plant species and psyllid haplotype on mean psyllid development time were exam- ined using a generalized linear mixed model (Proc GLIMMIX) [58]. Plant species, psyllid hap- lotype, and the species x haplotype interaction were included as fixed effects. The analysis was limited to plant species on which psyllids completed development to the adult stage. We speci- fied an underlying gamma distribution using a DIST = gamma statement. This distribution is useful for modeling time-to-occurrence data [59]. The ILINK function was used to back-trans- form means into the original units (number of days to first adult). The CONTRAST statement was used to examine a priori defined comparisons among plant species following a significant plant species effect in the overall model (see results). The survival data (yes/no) were not ana- lyzed statistically, as the two haplotypes showed identical results as to what plant species sup- ported development (see results). Genetic distances between species in a tree were calculated automatically by the Geneious R10 software using the Tamura-Nei model. This approach expresses distance as nucleotide substitutions per site. We used these distances to determine whether plant suitability for psyl- lids decreased as genetic distance from potato increased. We conducted a two-sample t-test [58] to determine whether mean genetic distance from potato differed between plant species allowing development and plant species not allowing development. If suitability was affected by genetic distance from the typical host (potato), we expected mean distance to be smaller for plants on which psyllids survived than plants on which psyllids failed to survive. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Psyllid developmental success and plant phylogeny Eggs were present within 24–48 h of adding egg-laying psyllids on all plant species except for Ipomoea pandurata and Turbina corymbosa which generally required 72 h before eggs were present. Egglayers were noticeably reluctant to settle on these two plant species, and instead PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 7 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Fig 1. Phylogeny of assayed Convolvulaceae based on ITS and matK sequences. Node confidence was calculated using Neighbor Joining tree (Bootstrap replicates = 100). Species in red font followed by an insect kill icon failed to allow survival to adult stage; species in green font followed by a psyllid adult icon allowed egg-to-adult development. Fig 1. Phylogeny of assayed Convolvulaceae based on ITS and matK sequences. Node confidence was calculated using Neighbor Joining tree (Bootstrap replicates = 100). Species in red font followed by an insect kill icon failed to allow survival to adult stage; species in green font followed by a psyllid adult icon allowed egg-to-adult development. https://doi.org/10.1371/journal.pone.0201506.g001 https://doi.org/10.1371/journal.pone.0201506.g001 were seen to spend considerable time wandering on the sides of the cages. Psyllids of both hap- lotypes failed to complete development in all five replicates on Convolvulus equitans, Calystegia silvatica, Ipomoea imperati, Ipomoea leptophylla, Ipomoea pandurata, Ipomoea tricolor, and Turbina corymbosa. Nymphs invariably died within a week of hatch on these species. It was not clear from our observations whether nymphs fed to any extent prior to death. On some species, notably I. imperati, I. pandurata, and T. corymbosa, mortality occurred within 24–48 h of egg hatch. Psyllids of both haplotypes completed development on potato, Convolvulus arvensis, Convolvulus tricolor, Ipomoea alba, Ipomoea cordatotriloba, Ipomoea hederacea, Ipo- moea ternifolia, and Ipomoea nil. A tree generated from ITS and matK sequences resolved the 14 species of Convolvulaceae into two major groups (Fig 1: Convolvuleae, Ipomoeeae), consistent with subfamilial group- ings shown elsewhere in substantially more detailed taxonomic work [60]. The assay data were overlain on the tree to search for evidence that plant phylogeny predicted survival. Within PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 8 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Tribe Convolvuleae, psyllids of both haplotypes developed successfully on Co. arvensis and Co. tricolor, but failed to survive on Ca. silvatica and Co. equitans, despite their phylogenetic close- ness to Co. arvensis (Fig 1). Psyllid developmental success and plant phylogeny Within Tribe Ipomoeeae, psyllids of both haplotypes developed successfully on five species of Ipomoea, but failed to develop on four other Ipomoea or on T. corymbosa. Phylogenetic distance from the control host plant (potato) was calculated for each species of Convolvulaceae in the phylogenetic tree (distances calculated in Geneious1, S1 Table). A two-sample t-test demonstrated that mean phylogenetic distance from potato was statistically identical between plant species that allowed psyllid survival versus species on which psyllids failed to survive (P = 0.4563; Fig 2), confirming observations in the phylogenetic tree that phylogenetic nearness of a species to potato did not predict whether the psyllid would complete development on the plant (Fig 1). We did not record actual rates of survival, so the assays cannot tell us whether percent sur- vival on plant species that allowed egg-to-adult development was similar to survival on potato. To determine if developmental rates on Convolvulaceae were similar to rates on potato, we compared mean number of days from oviposition to production of the first adult (N = 5 repli- cates per plant species and psyllid haplotype) between psyllids on potato and on those Convol- vulaceae allowing survival to the adult stage. Development times varied between ~20–35 days depending upon psyllid haplotype and plant species (Fig 3). Mean number of days between egg deposition and emergence of the first adult differed statistically between psyllid haplotypes (F = 15.5; df = 1, 57.0; P <0.001) and among plant species (F = 2.8; df = 7, 57.1; P = 0.013); the haplotype x plant species interaction was not significant (F = 1.4; df = 7, 57.1; P = 0.22) indicat- ing that the effects of plant species on psyllid development time was similar between the two haplotypes. The Central haplotype developed more rapidly (mean = 24.7 ± 1.1 d) than the Northwestern haplotype (mean = 29.4 ± 1.3 d), when averaged across host plant. We extracted contrasts to examine two a priori defined comparisons of interest. A test of mean development time on potato vs. Convolvulaceae was significant (F = 18.01; df = 1, 57.02; P < 0.001), and showed that mean development time on potato was statistically shorter than development time on Convolvulaceae, averaged over haplotype (Fig 3). A second set of contrasts was extracted to examine whether there was evidence for plant effects within the Convolvulaceae, ignoring potato. Psyllid developmental success and plant phylogeny Averaged over the two haplotypes, there was no evidence that development time of psyllids varied among species of Convolvulaceae (Fig 3: F = 0.31; df = 6, 57.1; P = 0.93). PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Psyllid developmental success and a plant-fungus symbiosis Visible evidence for the presence of Periglandula was most pronounced in two species, T. cor- ymbosa and I. leptophylla (Fig 4A and 4B). The fungal colonies were found on the adaxial sur- faces of younger leaves. Because visible evidence for presence of fungal colonies was rare, we used a molecular approach for detection of the fungus. Analysis of DNA-sequences led to detection of the dmaW gene in 11 of 14 plant species (Fig 4C and 4D; Table 2), indicating widespread presence of Periglandula across species despite absence of visible evidence. Only three species (Co. arvensis, I. alba, I. nil) failed to show presence of Periglandula. Presence of Periglandula in Convolvulus and Calystegia (Convolvuleae) was unexpected, as there had been no previous unambiguous evidence suggesting an association between Periglandula and plant species outside of the Ipomoeeae [34, 42]. Ergot alkaloids are categorized into three classes (clavines, simple amides of lysergic acid, and ergopeptines) based on their structural complexity and occurrence in the biochemical pathway [61, 62]. Compounds from two classes (clavines, amides of lysergic acid) were detected in leaf tissues of plant species in which the dmaW gene (indicating presence of Peri- glandula) was also detected (Table 3). Compounds included eight clavines and two lysergic PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 9 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Table 3. Plant species assayed, psyllid survival (Y/N), and detection of ergot alkaloids (mean ± S.E., n = 3) by HPLC-MS. Plant species Survival Clavines Simple Amides of Lysergic Acid Ergopeptines Chanoclavine Lysergic acid (μg/g) Agroclavine (μg/g) Lysergol (μg/g) Festuclavine Elymoclavine Elymoclavine fructoside Dihydrolysergol Ergonovine (μg/g) Ergine Ergotamine Ergocristine Ergocornine Ca. silvatica (+) N - - - - - - - - - - - - - Co. equitans (+) N - - - - - - - - - - - - - I. imperati (+) N ++ 4.4 ± 0.5 0.3 ± 0.04 0.4 ± 0.05 ++ ++ ⁻ ++ 5.5 ± 1.0 ++ ⁻ ⁻ ⁻ I. leptophylla (+) N ++ 0.8 ± 0.02 - - + ++ ⁻ ++ 7.1 ± 0.3 ++ - ⁻ ⁻ I. pandurata (+) N + - - - - ⁻ ⁻ ⁻ ⁻ ⁻ ⁻ ⁻ ⁻ I. tricolor (+) N ++ 0.9 ± 0.02 - - + + ++ ++ 0.3 ± 0.01 ++ ⁻ ⁻ ⁻ T. Psyllid developmental success and a plant-fungus symbiosis corymbosa (+) N ++ 3.0 ± 0.03 - - - + - + 0.7 ± 0.04 ++ ⁻ ⁻ ⁻ Co. arvensis Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ Co. tricolor (+) Y - - - - - - - - ⁻ ⁻ - - - I. alba Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ I. cordatotriloba (+) Y - - - - - - - - - - - - - I. hederacea (+) Y - - - - - - - - - - - - - I. ternifolia (+) Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ I. nil Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ Potato Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ Periglandula detected (+) https://doi.org/10.1371/journal.pone.0201506.t003 Table 3. Plant species assayed, psyllid survival (Y/N), and detection of ergot alkaloids (mean ± S.E., n = 3) by HPLC-MS. Plant species Survival Clavines Simple Amides of Lysergic Acid Ergopeptines Chanoclavine Lysergic acid (μg/g) Agroclavine (μg/g) Lysergol (μg/g) Festuclavine Elymoclavine Elymoclavine fructoside Dihydrolysergol Ergonovine (μg/g) Ergine Ergotamine Ergocristine Ergocornine Ca. silvatica (+) N - - - - - - - - - - - - - Co. equitans (+) N - - - - - - - - - - - - - I. imperati (+) N ++ 4.4 ± 0.5 0.3 ± 0.04 0.4 ± 0.05 ++ ++ ⁻ ++ 5.5 ± 1.0 ++ ⁻ ⁻ ⁻ I. leptophylla (+) N ++ 0.8 ± 0.02 - - + ++ ⁻ ++ 7.1 ± 0.3 ++ - ⁻ ⁻ I. pandurata (+) N + - - - - ⁻ ⁻ ⁻ ⁻ ⁻ ⁻ ⁻ ⁻ I. tricolor (+) N ++ 0.9 ± 0.02 - - + + ++ ++ 0.3 ± 0.01 ++ ⁻ ⁻ ⁻ T. corymbosa (+) N ++ 3.0 ± 0.03 - - - + - + 0.7 ± 0.04 ++ ⁻ ⁻ ⁻ Co. arvensis Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ Co. tricolor (+) Y - - - - - - - - ⁻ ⁻ - - - I. alba Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ I. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Psyllid developmental success and a plant-fungus symbiosis cordatotriloba (+) Y - - - - - - - - - - - - - I. hederacea (+) Y - - - - - - - - - - - - - I. ternifolia (+) Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ I. nil Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ Potato Y - - - - - - - - ⁻ ⁻ ⁻ ⁻ ⁻ Periglandula detected (+) PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 10 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Fig 2. Scatter plot showing relationship between genetic distance of plant from potato (control) and survival of potato psyllid to the adult stage. Horizontal lines indicate mean distances. https://doi.org/10.1371/journal.pone.0201506.g002 Fig 2. Scatter plot showing relationship between genetic distance of plant from potato (control) and survival of potato psyllid to the adult stage. Horizontal lines indicate mean distances. Fig 2. Scatter plot showing relationship between genetic distance of plant from potato (control) and survival of potato psyllid to the adult stage. Horizontal lines indicate mean distances. https://doi.org/10.1371/journal.pone.0201506.g002 https://doi.org/10.1371/journal.pone.0201506.g002 https://doi.org/10.1371/journal.pone.0201506.g002 acid amides (Table 3). No ergopeptines were detected. Additionally, no ergot alkaloids were detected in species not shown to host Periglandula (Co. arvensis, I. nil, I. alba). However, the presence of Periglandula did not always lead to detection of alkaloids in plant tissues. Alkaloid content may vary with plant age or organ, with higher concentrations typically occurring in seeds and seedlings over vegetative parts [46, 63]. This variation, combined with the possibility that ergot alkaloid concentrations can fall below detection limits, may lead to a failure in con- firming presence of ergot alkaloids despite detection of Periglandula by molecular methods [55]. acid amides (Table 3). No ergopeptines were detected. Additionally, no ergot alkaloids were detected in species not shown to host Periglandula (Co. arvensis, I. nil, I. alba). However, the presence of Periglandula did not always lead to detection of alkaloids in plant tissues. Alkaloid content may vary with plant age or organ, with higher concentrations typically occurring in seeds and seedlings over vegetative parts [46, 63]. This variation, combined with the possibility that ergot alkaloid concentrations can fall below detection limits, may lead to a failure in con- firming presence of ergot alkaloids despite detection of Periglandula by molecular methods [55]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Psyllid developmental success and a plant-fungus symbiosis We observed often striking differences in alkaloid profiles between plant species that allowed psyllid development and species on which the psyllid failed to develop (Table 3). Plants in which clavines and amides of lysergic acid were readily detected were invariably fatal to nymphal psyllids (Table 3). Mortality was quite rapid on these species. Nymphs always died as first instars generally within 24–48 h following egg hatch (Kaur and Horton pers. observa- tion). With two exceptions (Ca. silvatica, Co. equitans), plant species in which alkaloids were not detected allowed egg-to-adult development (Table 3). Psyllids failed to develop successfully on these two species despite a failure to detect alkaloids and despite detection of Periglandula in host tissues (Fig 4C, Table 3). Whether ergot alkaloids were actually present, but not detected, is not known. Lack of survival on Co. equitans may have been caused in part by the PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 11 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Fig 3. Number of days required to complete development from egg to adult stage by psyllids of the Northwestern and the Central haplotypes on potato and Convolvulaceae. Error bars represent standard error of mean. Mean development times differed statistically between psyllid haplotypes (F = 15.5; df = 1, 57.0; P <0.001) and among plant species (F = 2.8; df = 7, 57.1; P = 0.013). Fig 3. Number of days required to complete development from egg to adult stage by psyllids of the Northwestern and the Central haplotypes on potato and Convolvulaceae. Error bars represent standard error of mean. Mean development times differed statistically between psyllid haplotypes (F = 15.5; df = 1, 57.0; P <0.001) and among plant species (F = 2.8; df = 7, 57.1; P = 0.013). https://doi.org/10.1371/journal.pone.0201506.g003 plant’s extreme hairiness, as the pubescence was found to interfere with the ability of psyllids to feed and settle (from visual observations). Psyllids did successfully develop on four other species in which the dmaW gene was detected (Co. tricolor, I. cordatotriloba, I. hederacea, I. ter- nifolia). However, no ergot alkaloids were detected in leaf tissues from these four species, despite presence of the fungus (Table 3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Discussion This study adds to the list of Convolvulaceae that support egg-to-adult development of potato psyllid, and shows conclusively that the psyllid is able to develop on Convolvulaceae other than the two species (Co. arvensis and I. batatas) previously listed in literature accounts [9, 11, 12]. These additional taxa included an ornamental species of Convolvulus (Co. tricolor) likely of Mediterranean origin [64] and five species of New World Ipomoea. The Ipomoea comprised a mix of species that are grown as ornamentals (I. alba, I. nil, I. hederacea), and two species (I. cordatotriloba, I. ternifolia) that are present naturally in regions of Central America, Mexico, and the southwestern U.S. [28, 65, 66, 67]. Previous accounts of association between potato psyllid and Convolvulaceae include rearing trials and field observations. Some care must be taken in interpretation of the field records, as field observations can lead to inflated ideas of true host range of psyllids due to the willingness of these insects to colonize and feed upon plant species that nonetheless fail to support nymphal development [7, 11, 68]. In this study, we followed the strict published guidelines of Burckhardt et al. [7] in defining psyllid “host plant” as a species that allows egg-to-adult development. A failure to appreciate this distinction has led to confusion about the host range of potato psyllid [11]. We obtained egg-laying and PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 12 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Fig 4. Colonies of Periglandula spp. on (A) Turbina corymbosa and (B) Ipomoea leptophylla, (C) Agarose gel showing detection of Periglandula dmaW gene ~ 1050bp amplicon, (D) List of species in which the dmaW gene was detected or not detected corresponding to lane numbers designated in the gel picture. https://doi.org/10.1371/journal.pone.0201506.g004 Fig 4. Colonies of Periglandula spp. on (A) Turbina corymbosa and (B) Ipomoea leptophylla, (C) Agarose gel showing detection of Periglandula dmaW gene ~ 1050bp amplicon, (D) List of species in which the dmaW gene was detected or not detected corresponding to lane numbers designated in the gel picture. https://doi.org/10.1371/journal.pone.0201506.g004 Fig 4. Colonies of Periglandula spp. on (A) Turbina corymbosa and (B) Ipomoea leptophylla, (C) Agarose gel showing detection of Periglandula dmaW gene ~ 1050bp amplicon, (D) List of species in which the dmaW gene was detected or not detected corresponding to lane numbers designated in the gel picture. PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Discussion https://doi.org/10.1371/journal.pone.0201506.g004 egg hatch (presence of nymphs) on all 14 species of Convolvulaceae that were assayed in this study, but development to the adult stage was limited to seven of these species. Psyllids of the Central and Northwestern haplotypes were identical with respect to what plant species allowed successful development. The haplotypes did differ in development rates on species allowing development, with psyllids of the Central haplotype developing more rap- idly than psyllids of the Northwestern haplotype. Other studies have shown that haplotypes of potato psyllid differ in biological traits, including settling and oviposition behavior [69], devel- opment rates [25], body size [24, 25], and composition of endosymbiont communities [27]. The Central haplotype developed more rapidly on cultivated and weedy Solanaceae than psyl- lids of the Northwestern haplotype [25], which is consistent with our observations. It is likely that differences in development times were partly or largely due to differences between haplo- types in body size. Psyllids of the Northwestern haplotype are conspicuously larger than psyl- lids of the Central haplotype [24, 25] and it seems likely that the size differences translated into differences in development times between the haplotypes. We examined whether survival of psyllids on a given plant species could be predicted by location of the species in a phylogenetic tree. The Psylloidea have shown the ability to track phylogenetic diversification of plants within lineages, and host switching or dietary expansion in evolutionary or ecological time by psyllids appear to occur most often between phylogeneti- cally related plants species [8, 31, 32]. One outcome of this sort of phylogenetic tracking is the expectation that dietary breadth for a given psyllid species would likely encompass phylogenet- ically related plant species rather than a mixture of less-related species. The phylogenetic tree developed from our sequencing work is consistent with trees constructed by earlier phyloge- netic work for the Convolvulaceae [60]. Our sequences resolved the fourteen assayed species PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 13 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid into two clades which fall respectively into two major tribes [60], the Convolvuleae and Ipo- moeeae. The Ipomoeeae was further resolved into two clades [60]: the Argyreiinae, which includes one of our assayed species (Turbina corymbosa); and the Astripomoeinae clade, which contains the remaining Ipomoeeae (all species of Ipomoea) that were assayed. Discussion Our data failed to show that developmental success of psyllids was affected by location of plants in our phylogenetic tree. Plant species that allowed development were represented in both Tribes of Convolvulaceae that were assayed here, as were species that failed to allow development (Fig 1). Observations in the literature indicate that species of Convolvulaceae may often harbor a class of alkaloids (ergot alkaloids) known in grasses to confer resistance to insect herbivory [43, 45, 61]. These compounds are produced in grasses by fungal species in the family Clavici- pitaceae (genus Epichloё) which have formed a mutualistic relationship with grasses. A similar mutualistic association between Convolvulaceae and clavicipitaceous fungi in a different genus (Periglandula) has been shown to explain the presence of ergot alkaloids in Convolvulaceae [35, 36, 37, 38, 39]. The visual presence of fungal colonies on at least some of our targeted spe- cies (Fig 4A and 4B), combined with extensive literature confirming the presence of ergot alka- loids in Convolvulaceae, prompted us to examine whether psyllid development or lack of development was correlated with the presence or absence of ergot alkaloids. We detected Periglandula in a surprisingly large proportion of assayed plants (11 of 14 spe- cies), including in two genera (Convolvulus, Calystegia) not previously known to host this fun- gal symbiont. Previous surveys have suggested that the occurrence of ergot alkaloids (and thus this mutualistic association) was limited to the tribe Ipomoeeae and two clades (Argyreiinae and Astripomoeinae) within this tribe (data based on analyses of 46 species) [34, 42]. It has now been estimated that approximately 50% of Ipomoeeae species, or upwards of 450 species worldwide, could contain ergot alkaloids. These observations understandably have led researchers to focus on the tribe Ipomoeeae in efforts to document presence of ergot alkaloids [34, 42, 37] and it is possible that this focus has led workers to substantially underestimate the taxonomic diversity of Convolvulaceae actually harboring ergot alkaloids. The few reports in the literature suggesting that ergot alkaloids in the Convolvulaceae occur outside of the Ipo- moeeae, including in Calystegia and Convolvulus, have been categorized as “unverified” [34]. Our results are the first to demonstrate that the presence of Periglandula in Convolvulaceae does indeed extend outside of Ipomoeeae. Ergot alkaloids representing two classes (clavines and amides of lysergic acid) were detected in five of our assayed plant species (Table 3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 Discussion Efforts to detect ergot alkaloids in Convolvulaceae can lead to inconsistent results, even in assays of plant species known from previous studies to harbor the chemicals [34, 55]. These inconsistencies may be the consequence of any of a number of factors, including sensitivity of the analytical approach chosen to look for alkaloids, age of the plant seed or conditions under which the seed was stored, age of the plant, which plant structures are examined, and incorrect taxonomic work leading to mistakes in species identification [34, 71]. Alkaloid levels within a single plant may vary with plant structure. Levels in vegetative tissues, as were targeted here, may be lower than levels in other plant parts, such as seed or newly expanded cotyledons [46, 63]. It may be that analysis and extraction of plant structures other than those that were targeted here (the fully expanded leaf) would have led to detection of ergot alkaloids in those species found to harbor Periglandula but in which we failed to detect the chemicals. Potato psyllid successfully com- pleted development on five species in which Periglandula was present but in which ergot alka- loids were not detected. If ergot alkaloids do have psyllicidal effects, as suggested by our results in Fig 4C and 4D and Table 3, then successful development by psyllids on those five Periglan- dula-positive species from which we failed to detect alkaloids may indicate that alkaloids were indeed not present, or that they were at levels low enough to allow psyllid development and to escape biochemical detection. Symbiotic association between plants and clavicipitaceous fungi is best known for monocot- yledonous plants (Poaceae, Cyperaceae and Junaceae), where (as with Convolvulaceae) the sym- bioses lead to production of ergot alkaloids [41, 72, 73]. These associations may lead to any of several benefits for the plant, notably protection against herbivores, but including also nonde- fense type functions such as enhanced growth rates of the plant or increased ability to withstand drought or other abiotic stresses [74, 75, 76, 77]. Observations that benefits to plants may include multiple types of effects, combined with observations showing that these effects are not always predictable across studies, species, or environments, have led to a large body of literature debating the actual evolutionary processes leading to these associations [77, 78, 79, 80]. Discussion Previous literature accounts summarized in Eich (2008) report these same two classes of alkaloids in four of these five species (failing to list only I. pandurata). These same accounts identified many of the same specific compounds that were identified in this study [34, 37]. All species in this study which showed presence of ergot alka- loids were shown (with PCR) to also host Periglandula. No ergot alkaloids were detected in the three species in which we failed to detect Periglandula (I. nil, I. alba, Co. arvensis). This result is consistent with other studies of these three species [34]. We invariably observed 100% mortality of psyllid nymphs on species in which ergot alka- loids were detected (Table 3). Mortality occurred very rapidly following egg hatch, generally within 24–48 h of hatch. Assuming that nymphal mortality was due to the presence of these alkaloids, the next logical question is what mode of action explains our results? Absence of development could have been caused by direct toxicity of the alkaloids or because the com- pounds deter feeding. At this time, we cannot separate these effects. Insecticidal activity of this class of alkaloids could arise from their capacity to act as agonists or antagonists to neurotrans- mitter receptors and subsequent malfunctioning of the central nervous system [61]. However, it is also possible that the compounds deterred feeding enough that newly hatched nymphs PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 14 / 19 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid rapidly desiccated and died. An evaluation of these competing effects will require additional assays, likely including assays that allow measurement of feeding rates (e.g., production of honeydew). Studies in which synthetic analogues of targeted compounds are assayed would also be useful, as use of synthesized compounds would allow insect responses to be examined relative to specific concentrations of compounds or to mixtures of compounds [45, 70]. We failed to detect ergot alkaloids in six species that nonetheless were shown by PCR to harbor Periglandula (Table 3). It is unclear if the alkaloids were actually present but were not at detectable levels, if ergot alkaloids were present but were different compounds than targeted by our biochemistry work, or if indeed alkaloids were not present at all. Discussion Our results provide correlative evidence that presence of ergot alkaloids in Convolvulaceae prevents development of psyllid nymphs, suggesting that the Periglandula-Convolvulaceae symbiosis does lead to protection of plants against insect herbivores. Our results also showed, however, that presence of the fungus does not necessarily indicate that psyllids would not survive on the plant host, as species in which Periglandula was present but from which alkaloids were not detected did allow egg-to-adult development by psyllids. Future studies will include screening of a larger diversity of Convolvulaceae than assayed here, comprising both Periglandula-positive and Periglandula-negative species, and we believe that this larger study will shed additional light on the role of this fungal symbiosis in affecting fitness of phloem-feeding insects. S1 Table. Genetic distance from potato as calculated by Geneious1. (DOCX) Author Contributions Conceptualization: Navneet Kaur, William Rodney Cooper, David R. Horton. Conceptualization: Navneet Kaur, William Rodney Cooper, David R. Horton. Formal analysis: Navneet Kaur, Jennifer M. Duringer. Formal analysis: Navneet Kaur, Jennifer M. Duringer. Investigation: Navneet Kaur, William Rodney Cooper, David R. Horton. Methodology: Navneet Kaur, William Rodney Cooper, Jennifer M. Duringer, David R. Horton. Supporting information S1 Table. Genetic distance from potato as calculated by Geneious1. (DOCX) 15 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0201506 September 11, 2018 A plant mutualistic fungus, Periglandula species protecting Convolvulaceae from potato psyllid Acknowledgments We thank Deb Broers and Jen Stout for technical assistance. We thank Dan Johnson, Ju¨rgen Gross, Joe Munyaneza, and Kylie Swisher for reviewing an earlier draft of this manuscript. Psyllids from Weslaco TX (Central haplotype) were shipped to the ARS facility in Washington State under APHIS permit P526P-17-00366. Project administration: David R. Horton. Project administration: David R. Horton. Resources: William Rodney Cooper, Jennifer M. Duringer, Ismael E. Badillo-Vargas, Gabriela Esparza-Dı´az, Arash Rashed, David R. Horton. Supervision: Arash Rashed, David R. Horton. Supervision: Arash Rashed, David R. Horton. Writing – original draft: Navneet Kaur. Writing – original draft: Navneet Kaur. Writing – review & editing: David R. Horton. Writing – review & editing: David R. Horton. References 1. Munyaneza JE (2012) Zebra chip disease of potato: biology, epidemiology, and management. Am. J. Potato Res. 89: 329–350. 2. Teulon DA, Workman PJ, Thomas KL, Nielsen MC (2009) Bactericera cockerelli: incursion, dispersal and current distribution on vegetable crops in New Zealand. N.Z. Plant Prot. 62: 136–144. 3. Western Australia Agriculture and Food (2017) Tomato potato psyllid (TPP). https://www.agric.wa.gov. au/tomato-potato-psyllid-tpp. (Accessed September 2017). 4. Richards BL (1928) A new and destructive disease of the potato in Utah and its relation to the potato psylla. Phytopath. 18: 140–141. 5. Richards BL, Blood H (1933) Psyllid yellows of the potato. J. Agric. Res. 46: 189–216. 6. 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https://openalex.org/W4240508136	https://jurnal.syntaxtransformation.co.id/index.php/jst/article/download/279/406	Indonesian	null	Manajemen Pendidikan Karakter Dari Sudut Pandang Islam	Jurnal Syntax Transformation	2,021	cc-by-sa	5,101	ABSTRAK Manajemen adalah ilmu menggunakan bantuan orang lain untuk mencapai tujuan secara efektif, yang berarti bahwa bantuan dapat diberikan melalui bantuan orang lain. Dalam bentuk pikiran dan energi, bisa juga intuisi. Pendidikan pada hakikatnya adalah upaya untuk mendukung dan membimbing pemikiran dan fitrah setiap orang. Tujuannya adalah untuk memastikan bahwa pendidikan berkembang sesuai pendidikan karakter Nabi Muhammad untuk semaksimal mungkin mencapai tujuan yang diinginkan. Oleh karena itu, pendidikan Islam yang berkaitan dengan Islam merupakan ilmu yang dapat membimbing, mengarahkan dan berkembang. Metode yang digunakan dalam penelitian ini adalah metode kualitatif. Keberhasilan tentang pendidikan karakter sejak dini agar menjadi panutan. Untuk mengoptimalkan pengelolaan pendidikan karakter siswa, guru haruslah Fathonah, Shidiq, Amanah, dan Tabligh. Kata Kunci: manajemen; pendidikan; manajemen pendidikan Islam MANAJEMEN PENDIDIKAN KARAKTER DARI SUDUT PANDANG ISLAM Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani Universitas Muhammadiyah Malang (UMM) Malang Jawa Timur, Indonesia Email: Bayumaruf11@gmail.com, sabiqunkhoirot@gmail.com, dan muhmdfathielmadani@gmail.com Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani Universitas Muhammadiyah Malang (UMM) Malang Jawa Timur, Indonesia Email: Bayumaruf11@gmail.com, sabiqunkhoirot@gmail.com, dan muhmdfathielmadani@gmail.com INFO ARTIKEL ABSTRACT INFO ARTIKEL Diterima 5 Mei 2021 Direvisi 14 Mei 2021 Disetujui 20 Mei 2021 Management is the science of using other people's help to achieve goals effectively, which means that help can be given through the help of others. In the form of thoughts and energy, it can also be intuition. Education is essentially an effort to support and guide the thoughts and nature of each person. The aim is to ensure that education develops according to the character education of the Prophet Muhammad to achieve the desired goal. Therefore, Islamic education related to Islam is a science that can guide, direct and develop. The method used in this research is qualitative method. The success of character education from an early age is to become a role model. To optimize the management of student character education, teachers must be Fathonah, Shidiq, Amanah, and Tabligh. Keywords : management; education; management of Islamic education Vol. 2 No. 5, Mei 2021 Sosial Sains Vol. 2 No. 5, Mei 2021 Sosial Sains Jurnal Syntax Transformation y f p-ISSN : 2721-3854 e-ISSN : 2721-2769 p-ISSN : 2721-3854 e-ISSN : 2721-2769 How to cite: Qoustaulani, Bayu Ma’ruf. Sabiqun Khoirot dan M. Fathi El Madani (2021) Manajemen Pendidikan Karakter dari Sudut Pandang Islam. Jurnal Syntax Ttansformation 2(4). https://doi.org/ 10.46799/jurnalsyntaxtransformation.v2i5.279 E-ISSN: 2721-2769 Published by: Ridwan Institute Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manajemen Pendidikan Karakter dari Sudut Pandang Islam dalam ranah keagamaan, khususnya Islam itu sendiri. manajemen sebagai suatu usaha yang diberdaya gunakan agar goal yang di inginkan itu terwujud dengan sumber daya yang ada. Mengenai beberapa pandangan Mary Parker Follerr manajemen mengacu pada planing, pengolahan organisasi, memimpin serta mengupayakan keadaan terkendali dari setiap personel dan pasokan lainnya agar dapat secara efektif dan efisien mewujudkan goal seni organisasi. Sehingga dapat ditarik simpulan bahwa manajemen adalah ilmu yang mempelajari bagaimana menggunakan bantuan orang lain untuk mencapai tujuan secara efektif dan efektif, yang artinya bantuan dapat diberikan melalui bantuan orang lain. Berupa pikiran dan energi, bisa juga intuisinya. Dari perspektif manajemen, ada empat (empat) hal yang tidak boleh dilupakan, yaitu: pertama, kegiatan atau tugasyang nyata adanya: memanage semua konten yang akan digunakan di masa depan; kedua, Tujuan atau objek harus jelas agar dapat dinilai secara objektif (fisik bukan fisik) ketiga, proses: lanjutkan secara mendasar, terencana, sistematis dan teratur; keempat, Sasaran: agar dapat sampai pada goal yang diinginkan bersama. (Slamet, 2019) Beberapa pakar memperdebatkan pendidikan Islam itu sendiri. Menurut pakar Muhammad SA Ibrahim, Pendidikan Islam merupakan suatu sistem pendidikan dimana masyarakat dapat mengakses dengan mudah agar dapat menuntun dirinya pribadi menuju kehidupannya sesuai dengan cita-cita Islam sehingga masyarakat dapat memudahkan dirinya dalam menjalani pesatnya perkembangan hidup serta ilmu pengetahuan dan teknologi (iptek). Sudah ada dan dibagikan, sama hari ini. Kemudian Fadhil Al-Jamali yang juga ahli dalam memahami pendidikan Islam mengatakan bahwa pendidikan Islam merupakan salah satu bentuk pengembangan yang keras, menyemangati dan mengajak seseorang untuk maju sesuai dengan nilai dan norma yang tinggi serta kehidupan yang mulia, sehingga membentuk sejenis tidak peduli apakah itu terkait dengan tindakan, alasan dan perasaan Selain semua aspek kehidupan, mereka memiliki kepentingan pribadi yang lebih besar. Yang terakhir (Soedardi, 2019), Pendidikan Islam adalah sistematika usaha untuk mengubah perbuatan seseorang dalam kehidupan pribadinya, lingkungan serta orang-orang disekitarnya, sesuai mekanisme pengajaran yang dijadikan sebagai kegiatan dengan asasi dan profesionalitas diantara profesi-profesi asasi dalam masyarakat. Sehingga dapat ditarik sebuah simpulan bahwa Pendidikan Islam adalah sebuah suatu upaya yang sistematis, terarah, dan teratur dalam upaya membentuk jati diri setiap muslim agar kemampuan yang ada dalam dirinya itu berkembang dan menjadikan setiap hak dan kewajibannya sebagai Khalifah pemimpin di bumi Allah swt. dimuka bumi yang luas ini, baik kepada Tuhannya (Allah swt.), sesama manusia, dan sesama makhluk lainnya (baik hewan, tumbuhan dan makhluk lainnya). Pendahuluan Sebelum membahas apa itu manajemen pendidikan Islam, hal mendasar yang diketahui yakni tau apa itu manajemen dan pendidikan Islam itu sendiri. Istilah Manajemen Pendidikan Karakter dari Sudut Pandang Islam Pendidikan ini adalah pendidikan Kemudian membahas pendidikan Islam, perlu dipahami konsep-konsep umum yang berkaitan dengannya. Pendidikan menurut (Fadlali, 2017) merupakan upaya sadar setiap orang yang dikatakan dewasa untuk mengarahkan dan mengembangkan jati diri dan keterampilan yang mendasar dari setiap peserta didik dalam aturan pendidikan formil dan non formil. Sehingga, pendidikan pada hakikatnya adalah upaya menolong dan membimbing gagasan dan fitrah setiap manusia, tujuannya agar pendidikan berkembang semaksimal mungkin untuk mencapai tujuan yang ideal. Oleh karena itu, pendidikan Islam yang dihubungkan dengan Islam merupakan ilmu yang dapat membimbing, membimbing dan mengembangkan kepribadian peserta didik Syntax Transformation, Vol. 2 No. 5, Mei 2021 583 Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani yang didasarkan pada firman allah dan Hadist. meningkatkan kualitas pendidikan secara menyeluruh. Kemudian kebijakan tentang pesantren dilakukan secara berkesinambungan (berkelanjutan), serta menyempurnakan dan mendayagunakan setiap instruksi yang sudah terlaksana jauh sebelumnya. Ketika fase ini, sekolah agama (madrasah) tidak terlalu dianggap sebagai satu kesatuan Sistem Pendidikan Nasional, tetapi masih merupakan lembaga pendidikan yang langsung di bawahi oleh Menteri Agama, karena belum diatur oleh muatan agama dalam skala yang lebih luas, demikianlah adanya. belum menerapkan standar pendidikan yang diterapkan masa itu, oleh karena itu di era orde baru muncul berita tentang dualisme pendidikan di Indonesia di masyarakat. Sejarah pendidikan Islam merupakan peristiwa atau bagian dari masa lalu dalam pendidikan Islam itu sendiri, dalam sejarah pendidikan Islam diartikan juga sebagai proses menuju kemajuan dengan segala sesuatu dalam pendidikan Islam pada masa itu. Oleh karena itu, makna sejarah pendidikan Islam secara luas merupakan fenomena yang terjadi dengan berbagai kondisi dan keadaan. Sebelum membahas bagaimana mengelola pendidikan Islam, terlebih dahulu kita harus memahami situasi Islam di Indonesia, dengan kata lain, sejarah masuknya Islam ke Indonesia. Pendidikan Islam di Indonesia muncul dan terus berkembang seiring dengan kemunculan dan perkembangan Islam di Indonesia, serta telah memberikan banyak kontribusi. Kaji pula pembahasan ini, karena Islam Indonesia adalah mayoritas, oleh karena itu sesuai dengan peradaban yang dewasa dan efektif saat ini dalam masyarakat global, harus ada pengelolaan yang tepat sasaran, terarah, terarah, dan baik tanpa diskriminasi. Menentang agama lain. Sehingga, dapat diruntut cerita awal pendidikan Islam di Indonesia yang dibagi berdasarkan periode atau periode, dibagi menjadi tahapan sebagai berikut: pertama, masa munculnya Islam ke Indonesia; kedua, masa perkembangan proses adaptasi; ketiga, masa perkembangan kerajaan Islam; keempat, Masa penjajahan Belanda; kelima, masa penjajahan Jepang; keenam, masa orde lama; ketujuh, masa orde baru. Manajemen Pendidikan Karakter dari Sudut Pandang Islam Oleh karena itu, apabila kedua kata ini digabungkan, maka Manajemen Pendidikan Islam merupakan suatu upaya mengelola Lembaga pendidikan Islam bernuansa Islam. Mekanisme tersebut memiliki mekanisme yang dapat melewati sumber belajar yang ada dan hal-hal terkait lainnya, sehingga secara efektif mencapai tujuan Islam. pendidikan dan didasarkan pada Alquran dan Hadis. Makna tersebut kemudian memiliki makna yang saling terkait dan merupakan bidang pendidikan dasar agama Islam. Setelah Indonesia merdeka, Pendidikan Agama Islam di Indonesia mula mendapat perhatian dan ditangani dengan serius, baik di sekolah umum (SD, SMP, SMA) maupun di sekolah swasta(sekeolah berbayar). Upaya yang dilakukan antara lain memberikan bantuan kepada lembaga pendidikan sesuai dengan rekomendasi dari Komite Nasional Pusat (BPKNP) pada tanggal 23 Desember 1945. Kebijakan Pemerintah ini menitikberatkan pada pendidikan Islam di Madrasah di Indonesia yang positif dan konstruktif serta mendapat respon yang baik. Terutama dalam dua dekade terakhir dari tahun 1980-an hingga 1990-an. Pada masa Pemerintahan Orde Baru, sekolah Islam didirikan untuk pemerataan kesempatan pendidikan agama Islam dan untuk Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manajemen Pendidikan Karakter dari Sudut Pandang Islam berupa tulisan atau teks dari objek yang diamati, dan narasumber merupakan sumber data utama. perkembangan tersebut akan membawa setiap orang ke dalam dilema global yang tidak terkendali.Oleh karena itu, perlu diambil solusi-solusi baru dalam pendidikan Islam, dan harus segera diperhatikan bahwa perkembangan sosial saat ini sudah mulai dipengaruhi oleh permasalahan bangsa Indonesia. Keputusan, jadi tujuan ini harus segera dicapai. Dalam masyarakat, semakin banyak orang tidak bisa lagi meminimalkan kerusakan. Untuk itu diperlukan peran banyak partai politik agar pendidikan Islam berbasis Alquran dan Hadits dapat terlaksana dengan sebaik-baiknya tanpa ada kendala yang sangat mendasar. (Ibrahim et al., n.d.) Penelitian kualitatif sebagai tradisi khusus dalam ilmu sosial, yang pada dasarnya bertumpu pada observasi manusia dan terkait dengan bahasa dan terminologi mereka. Disebut penelitian kualitatif deskriptif karena data yang dianalisis tidak menerima atau menolak hipotesis (jika ada). Metode Penelitian Penelitian kualitatif adalah proses penelitian yang menghasilkan data deskriptif dari bahasa lisan atau tulisan orang dan perilaku yang diamati. Dalam metode penelitian deskriptif, data yang diperoleh Syntax Transformation, Vol. 2 No. 5, Mei 2021 584 Hasil dan Pembahasan Manajemen pendidikan selalu diupayakan untuk dapat menjalankan mekanisme secara terorganisir terkait dengan bidang pendidikan. Fungsi yang tercantum dalam manajemen pendidikan meliputi rencana atau rencana yang akan digunakan dalam kegiatan agar dapat berjalan lebih sistematis dan terstruktur. Kemudian atur atau atur agar bisa membagi dan mendistribusikan tugas yang ada. Kemudian dengan terwujudnya rencana dan organisasi yang telah dibuat dan ditata sejak pertama kali pelaksanaan kegiatan tersebut terdapat fungsi gerakan atau promosi. Kampanye tersebut bertujuan untuk mendorong masyarakat mencapai tujuan bersama yang disepakati oleh administrasi pendidikan yang ada. Secara umum, manajemen pendidikan adalah manajemen yang membutuhkan perencanaan sebelumnya. Manajemen juga merupakan organisasi untuk mencapai tujuan. Ini termasuk supervisi yang terarah dan terorganisir agar pengelolaan pendidikan tidak melenceng dari tujuan yang ingin dicapai sejak awal.(Maujud, 2018) Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani 3) Beritahu orang bahwa mereka adalah hamba yang diciptakan oleh Allah SWT, dalam hal ini hamba harus bertindak sesuai dengan nasihat Allah SWT. Jauhi segala bentuk larangan. Hal ini membuktikan dedikasi setiap hamba kepada penciptanya dan senantiasa beribadah sesuai kodratnya sebagai hamba. hanya menggunakan satu metode maka Guru Siswa akan merasa kesulitan untuk mengajar, dan siswa akan merasa bosan, bosan, dan pada akhirnya mempengaruhi nilai mata pelajaran yang mereka pelajari. Hal ini menuntut tenaga pendidik agar memperoleh kecerdasan intelektual, kreatif dan inovatif yang mendalam menghadapi segala hal yang berkaitan dengan daya tarik siswa dalam proses pembelajaran. Selain sangat kreatif dalam pengembangan pembelajaran di kelas, pendidik juga harus mampu membuat suasana kelas menjadi lebih damai, menarik, dan tentunya tidak membuat stres. Objek atau ruang lingkup pendidikan Islam sifatnya menyeluruh, sebab meliputi pendidikan Islam formal (lembaga pendidikan), pendidikan informasi Islam (pendidikan internal) dan pendidikan Islam informal (mengikuti majelis dan kajian islami). Perbedaan antara manajemen pendidikan dan manajemen pendidikan Islam sangat mendasar dan mendesak. Perbedaan ini menuntut pengelola untuk memiliki pengetahuan dan keterampilan yang komprehensif dan komprehensif guna menerapkan hasil dan proses mengatur diri dalam Pendidikan Islam. Pendidikan Islam bersumber dari ajaran Iqra’ yang berasal dari surat yang paling awal diterima oleh baginda Rsasulullah SAW yakni surah Al Alaq ayat 1- 5 tentang anjuran kita untuk membaca, sebab diriwayatkan bahwa menanggung kebodohan didunia itu sangatlah pedih hingga keakhirat. Sehingga kita ummat pengikut rasul hendaknya menuntut ilmu dengan bersungguh-sungguh khususnya ilmu agama. Dari pandangan sosialnya, ayat diatas memiliki 3 artian yang mendalam, yaitu perintah untuk dibacakan terhadap kaum mukmin, artian penting dan fundamental ilmu manusia, pemahaman terkait keberadaa pencipta, pentingnya keberadaan pencipta manusia, dan ilmu manusia. dari siapa yang datang dari. Ciptakan tempat bagi umat manusia. (Ridwan & Ulwiyah, 2020) Istilah coaching, mentoring and nurturing, dan teaching and training meliputi pengertian upaya mempengaruhi jiwa siswa melalui proses langkah demi langkah atau sistematis menuju suatu goal yang ingin dicapai, yakni membuat taqwa tertanam dalam diri dan moralitas serta menjunjung tinggi tindakan yang benar dalam kehidupan sehari-hari. jiwa santri menurut ajaran Islam Membentuk kepribadian dan kebajikan. Pendidikan Islam juga berarti pembinaan seseorang agar dapat berkembang dengan sebaik-baiknya sesuai dengan ajaran Islam, asal-usul dan konsep ajaran Islam bersumber dari Alquran dan Sunnah. selalu Pendidikan Islam adalah Muslim yang taat, penuh percaya kepada Allah SWT. Melalui keberkahan ajaran Islam, secara sadar dan mendasar kita akan membimbing dan membimbing tumbuh dan berkembangnya fitrah manusia, sehingga mencapai titik tertinggi pertumbuhan dan perkembangan di era yang semakin maju dan sulit saat ini. Oleh karena itu, tujuan yang ingin dicapai dan diwujudkan dalam pendidikan agama Islam meliputi tiga aspek sebagai berikut: 1) Sadar akan keberadaan manusia sebagai individu, yaitu manusia yang tidak dapat dipisahkan dari individu lain, harus mampu menjalankan semua fungsi dan tanggung jawabnya secara pribadi, tanpa harus bertindak sendiri; harus mampu bertindak sebagai SWT manusia yang diciptakan oleh Allah. Keyakinan, dan yang terpenting, juga berguna pada makhluk lain yang diciptakan Allah, makhluk ini tidak hanya hidup dan mati di bumi, tetapi juga berfungsi sebagai kehidupan di bumi. Menyebutkan tatanan pendidikan Islam yang memiliki kualitas dan kuantitas yang sesuai dan tidak meninggalkan konsep- konsep yang telah ada sebelumnya tetapi membutuhkan inovasi dan modifikasi baru, serta penerapan yang efektif dan efektif pada tatanan global, di mana banyak pertanyaan yang tidak diragukan lagi terungkap dan lengkap. Berbagai perkembangan, dan 2) Mengenali fungsi manusia sebagai makhluk sosial. Manusia sebagai makhluk sosial berarti manusia perlu bersosialisasi dan mengobrol dengan orang lain, dan makhluk itu tentunya membutuhkan orang lain untuk berkomunikasi, bersosialisasi dan hidup. Syntax Transformation, Vol. 2 No. 5, Mei 2021 585 Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manajemen Pendidikan Karakter dari Sudut Pandang Islam Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional disebut Pendidikan Agama Formal (Islam), seperti Pesantren / Madrasah Diniyah (Ula, Wustha, Ulya dan Ma 'Hadits). 'Ali). Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional disebut Pendidikan Agama Formal (Islam), seperti Pesantren / Madrasah Diniyah (Ula, Wustha, Ulya dan Ma 'Hadits). 'Ali). antusiasme siswa dengan cara yang stabil dan berkualitas tinggi. Hal ini merefleksikan metode pendidikan Islam adalah untuk membimbing keefektifan pembelajaran, memberikan solusi yang mudah bagi setiap siswa dalam belajar sesuai dengan kemauan dan bakatnya sendiri, serta memberikan dorongan terhadap siswa untuk tetap semngat dalam proses pembelajaran. (Zaini, 2018) 2) PAUD / RA, BA, TA, sekolah Islam dan perguruan tinggi seperti IAIN, STAN atau Universitas Islam Nasional di bawah Kementerian Agama. 3) Pendidikan anak usia dini, RA, BA, TA, sekolah / perguruan tinggi yang diselenggarakan di bawah naungan yayasan dan organisasi Islam. Menjalankan tugasnya, pengelola pendidikan Islam harus kooperatif dan partisipatif. Hal ini karena. Ada banyak alasan mengapa manajemen pendidikan Islam harus bekerjasama dan berpartisipasi, hal ini karena kita tidak bisa lepas dari batasan- batasan tertentu dalam kehidupan ini. Menurut (Sitepu, 2011), imitasi tersebut antara lain: 4) studi agama Islam yang dilaksanakan di sekolah / madrasah / universitas Islam sebagai mata pelajaran atau mata pelajaran, atau sebagai program studi; dan 4) studi agama Islam yang dilaksanakan di sekolah / madrasah / universitas Islam sebagai mata pelajaran atau mata pelajaran, atau sebagai program studi; dan 5) 5) dilingkungan internal dan di mesjid, TPA, dan / atau Forum Kajian Islam, para pemuka agama dan lembaga lain yang saat ini tengah marak dan diusahakan terus agar dapat melakukan pendidikan Islam, atau melalui jalur pendidikan formal dan nonformal (Islam). 1) Keterbatasan fisik (alamiah), misalnya agar dapat mencukupi kebutuhan pangan ia harus berkembang, hal ini biasanya dilakukan oleh orang lain atau dengan orang lain. 2) Batasan Psikologi (Psikologi). Manusia akan menghargai dan menghormatinya. Manajemen Pendidikan Islam (MPI) merupakan usaha dalam perencanaan, pelaksanaan, pengorganisasian serta evaluasi belajar yang akan dilakukan untuk meningkatkan kualitas lembaga pendidikan Islam. MPI setidaknya harus memperoleh segala hal untuk mengimplementasikannya dengan baik, diantaranya yakni : 3) Batasan sosiologi. Tanpa orang lain, manusia tidak akan bertahan. 4) Batasan biologis. Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani Menggunakan metode pendidikan Islam harus ditekankan bahwa sesuai terhadap pencapaian pendidikan Islam yaitu pembentukan individu, bagaimana pendidik (dalam hal ini guru) memahami hakikat metode pendidikan dan relevansinya serta setiap individu yang beriman, dan setiap insan yang sholeh, menjadikan selalu siap taat terhadap penciptanya Sebagai bentuk cinta. Pengharapan dari diadakannya proses seperti ini adalah agar PBM dan output dari pembelajaran yang diadakan berbasis pengajaran Islam lebih efisien dansesuai harapan, serta meningkatkan kepekaan siswa melalui teknik motivasi yang merangsang semangat belajar untuk mengamalkan ketentuan ajaran Islam. Kembangkan Penerapan metode pendidikan agama Islam pada awalnya tidak tetap atau acak. Hal ini guru pendidik harus menggunakan metode yang sesuai dengan kondisi dan keadaan yang pernah dialaminya di lapangan, karena jika Syntax Transformation, Vol. 2 No. 5, Mei 2021 586 Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manusia secara biologis termasuk makhluk termasuk yang lemah, oleh karena itu untuk meningkatkan kemampuan pertahanannya sendiri, manusia harus bekerjasama, saling memberi dan menerima, serta bersatu dengan manusia dan membentuk ikatan.Ayat al-Qur’an yang berkenaan dengan cooperative dan partisipatif ini anatara lain, surat al-Maidah ayat 2 (Sitepu, 2011) 1) Harus mempunyai epistemologi yang tersusun atas wahyu / rasionalitas- kenyataan. Pengelolaan lembaga pendidikan Islam mutlak didasarkan pada keberadaan firman Allah dan sabda Rasul (Al-Quran Hadits), dan kekuatan wahyu dipahami berdasarkan penalaran kontekstual (logika) (disesuaikan berdasar pada perkembangan IPTEK). Penyelenggaraan pendidikan Islam merupakan kegiatan pendidikan yang bertujuan untuk mewujudkan kaidah dan pembelajaran islam. Padahal, di negara ini pendidikan Islam setidaknya dapat dibedakan menjadi 5 jenis yaitu sebagai berikut : 2) Harus mempunyai rancangan ilmiah serta rancangan misionaris. Lembaga pendidikan Islam bukan hanya untuk menjalankan tugas keilmuannya, atau untukpengadakan sebuah ilmu, jati diri 1) Pondok Pesantren atau Madrasah Diniyah, menurut Undang-Undang Syntax Transformation, Vol. 2 No. 5, Mei 2021 587 Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani dan keterampilan, dan upaya yang dilakukan untuk membuat sesama Muslim dan non-Muslim memahami Islam. Inti dari penyebaran islam adalah memberikan pemahaman agar tidak salah paham tentang Islam, karena Islam sebenar- benarnya tanpa pamrih menyebarkan kasih sayang terhadap setiap insan. dan pelatihan (pelatihan) dan evaluasi kinerja pendidik. dan keterampilan, dan upaya yang dilakukan untuk membuat sesama Muslim dan non-Muslim memahami Islam. Inti dari penyebaran islam adalah memberikan pemahaman agar tidak salah paham tentang Islam, karena Islam sebenar- benarnya tanpa pamrih menyebarkan kasih sayang terhadap setiap insan. 5) Pengaturan biaya. 6) Pengaturan infrastruktur. 7) Pengaturan sumber daya. 8) Pengaturan hubungan masyarakat. 8) Pengaturan hubungan masyarakat. Secara umum pendidikan Islam memiliki 3 (tiga) metode manajemen dasar, yaitu: Alquran, Syariah Islam dan hukum yang berlaku di Indonesia. 3) Sasaran MPI meliputi: sumber daya manusia (SDM), sumber material dan sumber spiritual (hati / hati). 1) Alquran memiliki ayat yang melimpah didalam Alquran yang bisa dijadikan dasar pengelolaan pendidikan Islam. Setiap ayat ini dapat dimengerti setelah dipelajari dengan seksama. Setiap ayat Alquran merupakan dasar penyelenggaraan pendidikan Islam. 4) Pelatihan induksi dibagi menjadi dua dimensi: terkenal di dunia, pekerjaan ibadah, gaji dan remunerasi. Untuk orang-orang yang berperan dalam Lembaga Pendidikan harus mampu mensinergikan dan memadukan kedua dimensi tersebut guna mencapai keseimbangan hidup yang diinginkan. 2) As-Sunnah Rasulullah SAW adalah seorang pelatih, dia juga menekankan pada pendidikan dan menginspirasi umatnya untuk berpartisipasi dalam pendidikan dan pengajaran. Manajemen Pendidikan Karakter dari Sudut Pandang Islam Manajemen Pendidikan Karakter dari Sudut Pandang Islam kebutuhan manajemen, tentunya kita memperoleh gambaran pencapaian dari manajemenyang telah dirancang. Diketahui bersama bahwa managemen merupakan alat organisasi, keberadaan alat ini menjadi sangan diharapkan untuk memperoleh pencapaian yang mendasar. Sumber daya yang dapat diperbarui sesuai zaman serta dapat digabungkanagar sampai pada goal yang diinginkanterhadap pendidikan, termasuk 3M (sumber daya manusia, keuangan dan materi), dan semua sumber daya tersebut tidak terbatas pada sumber daya sekolah / madrasah sebagai pimpinan lembaga pendidikan atau perguruan tinggi Islam. Institusi pendidikan. Berkomunikasi pada orang yang bersangkutan, baik didalam ataupun diluar, sangat penting untuk meningkatkan dan mendorong kemajuan lembaga pendidikan yang dipimpinnya, inilah proses pengelolaan yang sebenarnya. (Chotimah, 2020) penelitian atau lembaga penelitian. dan alasan industri berharga lainnya. Lembaga dan organisasi non-pemerintah. Untuk merealisasikan atau merealisasikan semua aspek yang telah diuraikan di atas, tentunya tidak lepas dari berbagai kendala yang berkaitan dengan pengelolaan itu sendiri. Pengelolaannya sendiri sebenarnya sudah dijelaskan di dalam Alquran. Jika kita ingin memahami dan menganalisis manajemen untuk mengetahui kemana harus pergi, kendala apa yang akan dihadapinya, keuntungan apa yang harus diberikan dan bagaimana kita mengendalikan kapal pendidikan Islam agar penumpang lebih nyaman untuk memahami aspek, lokasi, di kapal tersebut. kami kontrol, Tidak akan mengancam penumpang. Untuk merealisasikan atau merealisasikan semua aspek yang telah diuraikan di atas, tentunya tidak lepas dari berbagai kendala yang berkaitan dengan pengelolaan itu sendiri. Pengelolaannya sendiri sebenarnya sudah dijelaskan di dalam Alquran. Jika kita ingin memahami dan menganalisis manajemen untuk mengetahui kemana harus pergi, kendala apa yang akan dihadapinya, keuntungan apa yang harus diberikan dan bagaimana kita mengendalikan kapal pendidikan Islam agar penumpang lebih nyaman untuk memahami aspek, lokasi, di kapal tersebut. kami kontrol, Tidak akan mengancam penumpang. Lembaga pendidikan Islam dapat digolongkan kedalam lembaga industri aristokrat (Nobel Prize Industries) sebab mempunyai dua harapan yaitu, kepentingan dan masyarakat. Misi laba adalah memperoleh laba, apabila efisiensi dan efektivitas dana mencukupi maka keuntungan dapat direalisasikan disini, sehingga pendapatan yang diperoleh hasil yang banyak dibanding biaya pengoperasian yang digunakan. Harapan sosiologis adalah mewarisi dan menginternalisasi nilai moral yang berbudi pekerti. Jika lembaga pendidikan Islam mempunyai kas awal, maka manusia dan modal sosial yang cukup, dan setiap keluaran berikutnya sangat efektif dan efisien, tugas kedua dapat diselesaikan secara maksimal. Manajemen Pendidikan Karakter dari Sudut Pandang Islam Rasulullah SAW berkata: Barangsiapa menyembunyikan ilmunya, Allah akan mengikatnya dengan tali bri yang berapi- api (HR. Ibn Majah). Berdasarkan Hadist di atas, Rasulullah SAW sangat mementingkan pendidikan. Selain itu, dia juga mengkhawatirkan manajemen. MPI harus dapat membuat perkembangan dan pengoptimalan segala hal yang dimiliki setiap orang. Setidaknya potensi tersebut terletak pada klasifikasi pembelajaran / pendidikan, yaitu kognisi (kecerdasan), emosi (kecerdasan kepribadian dan sikap) dan psikomotor (kecerdasan mekanik / otot). Manajemen pendidikan Islam memiliki beberapa jenis manajemen, dan setiap pengelola harus mengembangkannya tanpa terkecuali. Sertakan berikut ini: 3) 3) Legislasi Indonesia saat ini Dalam Undang-Undang Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional, disebutkan dalam Pasal 30 ayat 1: "Pendidikan agama diselenggarakan oleh pemerintah dan / atau komunitas pemeluk agama sesuai dengan peraturan perundang-undangan." 3) Legislasi Indonesia saat ini Dalam Undang-Undang Nomor 20 Tahun 2003 tentang Sistem Pendidikan Nasional, disebutkan dalam Pasal 30 ayat 1: "Pendidikan agama diselenggarakan oleh pemerintah dan / atau komunitas pemeluk agama sesuai dengan peraturan perundang-undangan." 1) Manajemen kurikulum: cara penyusunan bahan ajar layaknya rencana kurikulum, silabus dan analisis mata pelajaran yang berkaitan dengan proses pembelajaran. 2) Manajemen kemahasiswaan: Kelompok berdasarkan psikologi, kecerdasan dan manajemen kemahasiswaan harus dikelola dengan baik. Tentunya dalam menjalankan setiap kegiatan kita perlu melakukan upaya-upaya yang berefisiensi tinggi, efisien dan ekonomis, untuk itu kita perlu berpegang pada setiap sistem organisasi yang ada dan yang sekarang. Sehingga dapat diketahui ,bahwa tindakan penyelewengan dana dan biaya dapat diminimalkan melalui prinsip organisasi. Memahami identitas mereka dan 3) Manajemen sumber daya manusia: termasuk pendidik dan pendidik, dan bagaimana menggunakan infrastruktur yang memadai untuk proses belajar mengajar yang maksimal. 4) Manajemen personalia: Menekankan manajemen tiga hal: seleksi, pendidikan Syntax Transformation, Vol. 2 No. 5, Mei 2021 588 Manajemen Pendidikan Karakter dari Sudut Pandang Islam Cara terbaik adalah tidak menggunakan metode ini sesering mungkin karena psikologi dan psikologi anak target. 3) Menurut (Suseni et al., 2013) metode distribusi merupakan metode pengenalan materi pembelajaran dalam hal ini pendidik adalah guru yang memberikan tugas khusus dan terstruktur kepada siswasebagai usaha pembelajaran. Kemudian jika berdasar pada (Suparti, 2014), metode resitasi atau pekerjaan rumah merupakan metode penyampaian buku teks dengan memberikan pekerjaan rumah kepada siswa dalam kurun waktu tertentu, dan hasilnya harus dijelaskan di depan guru. Sebagai bentuk penguasaan tugas. 7) Metode hukuman, yaitu metode menghukum siswa. Hukuman adalah cara terburuk, tetapi harus digunakan dalam kondisi tertentu. Cara terbaik adalah tidak menggunakan metode ini sesering mungkin karena psikologi dan psikologi anak target. Manajemen Pendidikan Karakter dari Sudut Pandang Islam Oleh karena itu, pengelolaan lembaga pendidikan Islam bukan Cuma membutuhkan jiwa profesionalisme kuat, namun harus mempunyai harapan dengan harapan suci dan pola pikir yang kaya, serta pengelolaan dan pengembangan hospital, panti asuhan, yayasan sosial, lembaga Dalam pengembangan dan pengelolaan manajemen pendidikan Islam, beberapa metode pembelajaran dapat digunakan, seperti pengucapan, ceramah, pekerjaan rumah, pengajian, latihan, diskusi, tanya jawab, dongeng dan hukuman. Adapun pengertiannya masing-masing yaitu: Satu jenis. 1) Metode pelafalan adalah tiruan proses belajar, dan rantai informasinya bersumber dari seorang pendidik, siswa diharuskan dapat terus menerus Syntax Transformation, Vol. 2 No. 5, Mei 2021 589 Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani 6) Metode diskusi, yaitu sistem pembelajaran pemaparan materi, tanya jawab dan pengajaran secara aktif di dalam kelas.terkait dengan pelaksaannya yakni terdapat pemaparan materi atau konsep diskusi lalu dibuka sesi tanya jawab dengan boleh mengembangkan pertanyaan dan jawab yang sesuai dengan konsep yang dibahas. Jawaban yang mengandung berbagai kemungkinan perlu datang dari kegotong-royongan peserta diskusi agar jawaban yang disesi belakang dianggap paling benar atau paling baik. menyampaikannya kepada siswa lain, begitu pula sebaliknya. menyampaikannya kepada siswa lain, begitu pula sebaliknya. 2) Metode ceramah adalah isi dimana guru (guru lebih aktif) menggunakan alat atau media pembelajaran untuk memberikan ceramah di kelas, sehingga ceramahnya lebih jelas.Berdasarkan pada teladan yang baik , guru Pendidikan Agama Islam, guru luar mengetahui cara mendidik tersebut dimaknai oleh guru sebagai informasi dan penjelasan lisan di dalam kelas, sedangkan siswa menyimak dengan cermat dan menuliskan materi pokok yang disampaikan. 2) Metode ceramah adalah isi dimana guru (guru lebih aktif) menggunakan alat atau media pembelajaran untuk memberikan ceramah di kelas, sehingga ceramahnya lebih jelas.Berdasarkan pada teladan yang baik , guru Pendidikan Agama Islam, guru luar mengetahui cara mendidik tersebut dimaknai oleh guru sebagai informasi dan penjelasan lisan di dalam kelas, sedangkan siswa menyimak dengan cermat dan menuliskan materi pokok yang disampaikan. 3) Menurut (Suseni et al., 2013) metode distribusi merupakan metode pengenalan materi pembelajaran dalam hal ini pendidik adalah guru yang memberikan tugas khusus dan terstruktur kepada siswasebagai usaha pembelajaran. Kemudian jika berdasar pada (Suparti, 2014), metode resitasi atau pekerjaan rumah merupakan metode penyampaian buku teks dengan memberikan pekerjaan rumah kepada siswa dalam kurun waktu tertentu, dan hasilnya harus dijelaskan di depan guru. Sebagai bentuk penguasaan tugas. 7) Metode hukuman, yaitu metode menghukum siswa. Hukuman adalah cara terburuk, tetapi harus digunakan dalam kondisi tertentu. Manajemen Pendidikan Karakter dari Sudut Pandang Islam Azhar Indonesia Seri Pranata Sosial, 1(2), 83–91. Google Scholar meningkatkan kualitas lembaga pendidikan Islam. Manajemen pendidikan Islam memiliki beberapa jenis manajemen, dan setiap pengelola harus mengembangkannya tanpa terkecuali, diantaranya manajemen kurikulum, manajemen kemahasiswaan, Manajemen sumber daya manusia, Manajemen personalia, manajemen biaya, manajemen infrastruktur, manajemen sumber daya, dan manajemen pengaturan hubungan masyarakat. Slamet, S. (2019). Implementasi Manajemen Strategik Di Madrasah Tsanawiyah Muhammadiyah Baruamba Bumiayu Brebes. http://repository.iainpurwokerto.ac.id/61 76/. Google Scholar Soedardi, R. A. (2019). Does Religion Matter? Understanding Religion Subject for Formal Education. At-Tarbawi: Jurnal Kajian Kependidikan Islam, 4(2), 104. Google Scholar Kesimpulan Manajemen pendidikan selalu diupayakan untuk dapat menjalankan mekanisme secara terorganisir terkait dengan bidang pendidikan. Fungsi yang tercantum dalam manajemen pendidikan meliputi rencana atau rencana yang akan digunakan dalam kegiatan agar dapat berjalan lebih sistematis dan terstruktur. Objek atau ruang lingkup pendidikan Islam sifatnya menyeluruh, sebab meliputi pendidikan Islam formal (lembaga pendidikan), pendidikan informasi Islam (pendidikan internal) dan pendidikan Islam informal (mengikuti majelis dan kajian islami). Pendidikan Islam juga berarti pembinaan seseorang agar dapat berkembang dengan sebaik-baiknya sesuai dengan ajaran Islam, asal-usul dan konsep ajaran Islam bersumber dari Alquran dan Sunnah. 4) Metode menghafal didasarkan pada kata “hafal” berarti sudah berada di dalam memori dan dapat diutarakan di luar pikiran. Oleh karena itu, pengajian berarti berusaha belajar dengan menghafal sesuatu yang ada di dalam ingatan untuk menyimpan pengajian tersebut sehingga ingatan tersebut jelas. 5) Metode Praktik adalah salah satu jenis metode pengajaran, siswa dapat melaksanakan kegiatan pelatihan praktik di kelas dan di tempat agar siswa memiliki rasa percaya diri atau keterampilan yang lebih tinggi.Dalam praktik ini siswa dapat diajarkan etika, seperti etika bertanya, teman sebaya etika, dll. Manajemen Pendidikan Islam (MPI) merupakan usaha dalam perencanaan, pelaksanaan, pengorganisasian serta evaluasi belajar yang akan dilakukan untuk Syntax Transformation, Vol. 2 No. 5, Mei 2021 590 Manajemen Pendidikan Karakter dari Sudut Pandang Islam Bibliografi Suparti, S. (2014). Penggunaan Metode Penugasan atau Resitasi Untuk Meningkatkan Hasil Belajar Siswa Kelas III dalam Memahami Konsep Mengenal Pecahan Sederhana. PEDAGOGIA: Jurnal Pendidikan, 3(1), 54. Google Scholar Chotimah, H. (2020). Upaya Peningkatan Kemandirian Ekonomi Umat Melalui NU-Preneur. 1, 60–69. Google Scholar Fadlali, A. (2009). Fitrah Akliyah Dalam Pendidikan Islam. Forum Tarbiyah, 7(2), 167–180. Google Scholar Suseni, P., Koyan, I. W., & Sudatha, I. G. W. (2013). Penerapan metode penugasan melalui kegiatan melipat kertas untuk meningkatkan keterampilan motorik halus anak di tk satya ananda banjarasem. JUrnal Pendidikan Anak Usia Dini Undiksha, 1(1), 1–10. Google Scholar Ibrahim, S., Agama, I., Negeri, I., & Amai, S. (n.d.). Menata Pendidikan Islam Di Indonesia Sulaiman Ibrahim Institut Agama Islam Negeri Sultan Amai Gorontalo ABSTRAK. 103–116. Google Scholar Maujud, F. (2018). Implementasi Fungsi- Fungsi Manajemen dalam Lembaga Pendidikan Islam (Studi Kasus Pengelolaan Madrasah Ibtidaiyah Islahul Muta’allim Pagutan). Jurnal Penelitian Keislaman, 14(1), 31–51. Google Scholar Zaini, A. (2018). Metode-Metode Pendidikan Islam Bagi Anak Usia Dini. ThufuLA: Jurnal Inovasi Pendidikan Guru Raudhatul Athfal, 2(1), 25. Google Scholar Amrozi, Shoni Rahmatullah. 2020. Jurnal Ilmu Pendidikan Islam : Sejarah Pendidikan Islam Di Indonesia ; Perspektif Sejarah Kritis Ibnu Kholdun. Vol. 04. No. 01. Google Scholar Ridwan, I., & Ulwiyah, I. (2020). Sejarah Dan Kontribusi Majlis Ta’Lim Dalam Peningkatan Kualitas Pendidikan Di Indonesia. Jurnal Pendidikan Karakter JAWARA (Jujur …, 6, 17–42. http://jurnal.untirta.ac.id/index.php/JAW ARA/article/view/8299. 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Fathi El Madani Dan Kurikulum 2013. Vol. 1. No. 2. Google Scholar Copyright holder: Bayu Ma’ruf Qoustaulani, Sabiqun Khoirot dan M. Fathi El Madani (2021). First publication right: Journal Syntax Transformation This article is licensed under: 592 Syntax Transformation, Vol. 2 No. 5, Mei 2021
https://openalex.org/W3037757389	https://www.frontiersin.org/articles/10.3389/fnbot.2020.00043/pdf	English	null	Interactive Natural Language Grounding via Referring Expression Comprehension and Scene Graph Parsing	Frontiers in neurorobotics	2,020	cc-by	10,726	Interactive Natural Language Grounding via Referring Expression Comprehension and Scene Graph Parsing Natural language provides an intuitive and effective interaction interface between human beings and robots. Currently, multiple approaches are presented to address natural language visual grounding for human-robot interaction. However, most of the existing approaches handle the ambiguity of natural language queries and achieve target objects grounding via dialogue systems, which make the interactions cumbersome and time-consuming. In contrast, we address interactive natural language grounding without auxiliary information. Speciﬁcally, we ﬁrst propose a referring expression comprehension network to ground natural referring expressions. The referring expression comprehension network excavates the visual semantics via a visual semantic-aware network, and exploits the rich linguistic contexts in expressions by a language attention network. Furthermore, we combine the referring expression comprehension network with scene graph parsing to achieve unrestricted and complicated natural language grounding. Finally, we validate the performance of the referring expression comprehension network on three public datasets, and we also evaluate the effectiveness of the interactive natural language grounding architecture by conducting extensive natural language query groundings in different household scenarios. Edited by: Emanuele Menegatti, University of Padova, Italy Reviewed by: Xavier Hinaut, Inria Bordeaux - Sud-Ouest Research Centre, France Davide Marocco, University of Naples Federico II, Italy Correspondence: Song Tang tang@informatik.uni-hamburg.de Edited by: Emanuele Menegatti, University of Padova, Italy Reviewed by: Xavier Hinaut, Inria Bordeaux - Sud-Ouest Research Centre, France Davide Marocco, University of Naples Federico II, Italy Keywords: interactive natural language grounding, referring expression comprehension, scene graph, visual and textual semantics, human-robot interaction Keywords: interactive natural language grounding, referring expression comprehension, scene graph, visual and textual semantics, human-robot interaction 1. INTRODUCTION Natural language grounding aims to locate target objects within images given natural language queries, and grounding natural language queries in visual scenes can create a natural communication channel between human beings, physical environments, and intelligent agents. Moreover, natural language grounding is widely used in image retrieval (Gordo et al., 2016), visual question answering (Li et al., 2018), and robotics (Paul et al., 2018; Mi et al., 2019). Natural language grounding aims to locate target objects within images given natural language queries, and grounding natural language queries in visual scenes can create a natural communication channel between human beings, physical environments, and intelligent agents. Moreover, natural language grounding is widely used in image retrieval (Gordo et al., 2016), visual question answering (Li et al., 2018), and robotics (Paul et al., 2018; Mi et al., 2019). Received: 15 August 2020 Accepted: 27 May 2020 Published: 25 June 2020 ORIGINAL RESEARCH published: 25 June 2020 doi: 10.3389/fnbot.2020.00043 2. RELATED WORK 2.1. Natural Language Grounding for HRI Multiple approaches have been proposed to address natural language grounding for HRI. Schiﬀer et al. (2012) adopted decision-theoretic planning to interpret spoken language commands for natural language-based HRI in domestic service robotic applications. Steels et al. (2012) presented Fluid Construction Grammar (FCG) to understand natural language sentences, and FCG was suitable for real robot requires because of its robustness and ﬂexibility. Fasola and Matari´c (2014) proposed a probabilistic method for service robots to interpret spatial language instructions. Inspired by the role of referring expression comprehension, we propose an interactive natural language grounding architecture based on referring expression comprehension. Speciﬁcally, we propose a semantic-aware network for referring expression comprehension task. The proposed semantic-aware network is composed of a visual semantic-aware network, a language attention network, and a target localization module. The visual semantic-aware network highlights the visual semantics of regions by fully utilizing the characteristics of deep features extracted from a pretrained CNN (Convolutional Neural Network). The language attention network learns to assign diﬀerent weights to each word in expressions and parse expressions into phrases that embed information of target candidate, relation between objects, and spatial location, respectively. And the target localization module combines the visual and textual representations to locate target objects. We train the proposed network on three popular referring expression datasets: RefCOCO (Yu et al., 2016), RefCOCO+ (Yu et al., 2016), and RefCOCOg (Mao et al., 2016). g g Twiefel et al. (2016) combined an object classiﬁcation network, a language understanding module with a knowledge base to understand spoken commands. Paul et al. (2018) proposed a probabilistic model named adaptive distributed correspondence graph to understand abstract spatial concepts, and an approximate inference procedure to realize concrete constituents grounding. Patki et al. (2019) utilized distributed correspondence graph to infer the environment representation in a task-speciﬁc approach. Katsumata et al. (2019) introduced a statistical semantic mapping method that enables the robot to connect multiple words embedded in spoken utterance to a place in a semantic mapping processing. However, these models did not take into account the inherent vagueness of natural language. Our previous work (Mi et al., 2019) ﬁrst presented an object aﬀordances detection model, and then integrated the object aﬀordances detection with a semantic extraction module for grounding intention-related spoken language instructions. This model, however, was subject to limited categories of aﬀordances, so it can not ground unconstrained natural language. Citation: With applications of robots becoming omnipresent in varied human environments such as factories, hospitals, and homes, the demand for natural and eﬀective human-robot interaction (HRI) has become urgent. Natural language grounding-based HRI is also attracting considerable attention, and multiple approaches have been proposed (Schiﬀer et al., 2012; Steels et al., 2012; Twiefel et al., 2016; Ahn et al., 2018; Hatori et al., 2018; Paul et al., 2018; Shridhar and Hsu, 2018; Mi et al., 2019; Patki et al., 2019). Mi J, Lyu J, Tang S, Li Q and Zhang J (2020) Interactive Natural Language Grounding via Referring Expression Comprehension and Scene Graph Parsing. Front. Neurorobot. 14:43. doi: 10.3389/fnbot.2020.00043 June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org Interactive Natural Language Grounding Mi et al. Mi et al. Natural language grounding-based HRI requires a comprehensive understanding of natural language instructions and working scenarios, and the pivotal issue of is to locate the referred objects in working scenarios according to given instructions. Although the existing models achieve promising results, some of them either do not take the inherent ambiguity of natural language into consideration (Paul et al., 2018; Katsumata et al., 2019; Mi et al., 2019; Patki et al., 2019), or alleviate the ambiguity via drawing support from auxiliary information, such as dialogue system (Ahn et al., 2018; Hatori et al., 2018; Shridhar and Hsu, 2018) and gestures (Shridhar and Hsu, 2018). However, the dialogue-based disambiguation systems entail time cost and cumbersome interactions. evaluate the performance of the interactive natural language grounding architecture, we collect plenty of indoor working scenarios and diverse natural language queries. Experimental results demonstrate that the presented natural language grounding architecture can ground complicated queries without the support from auxiliary information. To sum up, our major contributions are two-fold. First, we propose a semantic-aware network for referring expression comprehension, in which we take full advantage of the characteristics of the deep features and exploit the rich contexts of referring expressions. Second, we present a novel interactive natural language grounding architecture by combining the referring expression comprehension network with scene graph parsing to ground complicated natural language queries. Tasks that utilize textual descriptions or questions to help human beings to understand or depict images and scenes are in agreement with the human desire to understand visual contents at a high semantic level. Frontiers in Neurorobotics \| www.frontiersin.org Citation: Examples of these tasks include dense captioning (Johnson et al., 2016), visual question answering (Antol et al., 2015), referring expression comprehension (Yu et al., 2016), etc. Referring expression comprehension imitates the role of a listener to locate target objects within images given referring expressions. Compared to other tasks, referring expression comprehension focuses on objects in visual images and locates speciﬁc targets via modeling the relationship between objects and referring expressions. June 2020 \| Volume 14 \| Article 43 2. RELATED WORK In real applications, natural language queries are complicated and ambiguous. While the expressions in the referring expression datasets are simple sentences and only indicate one target, so the complicated queries can not be grounded only by the trained referring expression comprehension model. Inspired by the role of scene graph which describes objects within visual images and the relationship between objects, we integrate the referring expression comprehension network with scene graph parsing (Johnson et al., 2015) to ground unconstrained and complicated natural language queries. Shridhar and Hsu (2018) adopted a pretrained captioning model, DenseCap (Johnson et al., 2016), to generate expressions for detected regions in uncluttered working scenarios, and through conducting K-means clustering to identify the relativeness of input instructions and the generated expressions. The expressions generated by DenseCap (Johnson et al., 2016) do not include the interaction information between objects, such as the relationship between objects. Therefore, the authors of work (Shridhar and Hsu, 2018) employed gestures and a dialogue system to disambiguate spoken instructions. Hatori et al. (2018) Moreover, we conduct extensive experiments on test sets of the three referring expression datasets to validate the proposed referring expression comprehension network. In order to June 2020 \| Volume 14 \| Article 43 2 Interactive Natural Language Grounding Mi et al. (2018a) extracted deep features from two diﬀerent convolutional layers to predict region attribute cues. However, these mentioned approaches neglected the rich information embedded in the extracted deep features. drew support from a referring expression comprehension model (Yu et al., 2017) to identify the target candidates, and tackled with the ambiguity of spoken instructions via conversation between human users and robots. Ahn et al. (2018) ﬁrst employed hourglass network (Newell et al., 2016) to generate position heatmap for working scenarios, and combined the generated heatmap with a question generation module to locate targets according to the answers for the generated questions. Thomason et al. (2019) translated the spoken instructions into discrete robot actions and improved objects grounding through clariﬁcation conversations with human users. Nevertheless, dialogue systems usually make HRI cumbersome and time-consuming. Attention mechanism was introduced for image captioning (Xu et al., 2015) and become an indispensable component in deep models to acquire superior results (Anderson et al., 2018; Yu et al., 2018a). 2. RELATED WORK Compared with image captioning and visual question answering, referring expression comprehension is widely used in image retrieval (Chen k. et al., 2017), video question answering (Gao et al., 2017), and natural language based HRI (Hatori et al., 2018; Shridhar and Hsu, 2018). 2. RELATED WORK Due to the excellent performance of attention mechanisms, they have also been utilized in referring expression comprehension (Hu et al., 2017; Deng et al., 2018; Yu et al., 2018a; Zhuang et al., 2018). Hu et al. (2017) parsed the referring expressions into a triplet (subject, relationship, object) by an external language parser, and computes the weight of each part of parsed expressions with soft attention. Deng et al. (2018) introduced an accumulated attention network that accumulated the attention information in image, objects, and referring expression to infer targets. Zhuang et al. (2018) argued that the image representation should be region-wise, and adopted a parallel attention network to ground target objects recurrently. Notwithstanding, these models processed expressions as holistic and ignored the rich context of expressions. Wang et al. (2019) introduced a graph-based attention mechanism to address the target candidates and the relationships between objects within images, while the visual semantic in images was neglected. Thomason et al. (2016) took into account visual, haptic, auditory, and proprioceptive data to predict the target objects, and the natural language grounding supervised by an interactive game. However, this model needs to gather language labels for objects to learn lexical semantics. Magassouba et al. (2018) presented a multi-modal classiﬁer generative adversarial network to enable robots to implement carry-and-place tasks, and disambiguates the natural language commands by utilizing the contexts of working environments and the states of the robots. By contrast, we disambiguate natural language queries by a referring expression comprehension network and achieve interactive natural language grounding without auxiliary information. To alleviate the ambiguity of natural language queries, we take into consideration the relations, the region visual appearance diﬀerence, and the spatial location information during the referring expression comprehension network training. Besides, we integrate the trained referring expression comprehension model with scene graph parsing to achieve unrestricted and complicated interactive natural language grounding. Unlike the above mentioned approaches, we address the visual semantics of regions by taking advantage of the inherent semantic attributes of deep features, i.e., channel-wise and spatial characteristics of extracted deep features. Additionally, we explore the textual semantics by adopting BERT to generate word representations and employ a language attention network to learn to decompose expressions into phrases to ground target objects. 2.2. Referring Expression Comprehension Referring expression comprehension aims to locate the most related objects in images according to given referring expressions. Frontiers in Neurorobotics \| www.frontiersin.org 3. ARCHITECTURE OVERVIEW Natural language provides a more intuitive interface to achieve natural and eﬀective HRI. For grounding unrestricted and complicated interactive natural language queries, we propose a novel architecture, as shown in Figure 1. We decompose the interactive natural language grounding into two subtasks: (1) parse the complicated natural language queries into scene graph legends by scene graph parsing. The scene graph legend is a data structure consisting of nodes that denote objects with attributes and edges that indicate the relations between objects; (2) ground the parsed natural language queries by the referring expression comprehension network. In terms of representations of image regions and natural language referring expressions, existing approaches for referring expression comprehension can be generalized into two categories: (1) visual representations un-enriched models, which directly extract deep features from a pretrained CNN as the visual representations of detected image regions (Mao et al., 2016; Yu et al., 2016, 2017; Hu et al., 2017; Deng et al., 2018; Zhang et al., 2018; Zhuang et al., 2018). (2) visual representations enriched models, which enhance the visual representations by adding external visual information for regions (Liu et al., 2017; Yu et al., 2018a,b). Liu et al. (2017) leveraged external knowledge acquired by an attributes learning model to enrich the information of regions. Yu et al. (2018b) trained an object detector on the Visual Genome dataset (Krishna et al., 2017) to generate diversiﬁed and discriminative proposals. Yu et al. In this work, we aim to locate the most related referents in working scenarios given interactive natural language expressions without auxiliary information. The inputs consist of a working scenario given as an RGB image and an interactive natural language instruction given as text, and the outputs are the bounding boxes of target objects. We parse the input natural language instructions into scene graph legends by scene graph parsing, and then we ground the acquired scene graph legends via the referring expression comprehension network. June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org 3 Mi et al. Interactive Natural Language Grounding FIGURE 1 \| The architectural diagram of the proposed interactive natural language grounding. We ﬁrst parse the interactive natural language queries into scene graph legends by the scene graph parsing. We then ground the generated scene graph legends via the referring expression comprehension network. The mark rectangle in bottom encompasses the scene graph parsing result for the input natural language query. 4.1. Language Attention Network We propose a language attention network to learn the diﬀerent weights of each word in referring expressions, and also to learn to parse the expressions into target candidate embedding rtar, relation embedding rrel, and spatial location embedding rloc, respectively. 3. ARCHITECTURE OVERVIEW The rounded rectangles with black dashed lines denote the parsed scene graph legends, color shaded rectangles represent referents, no color shaded rectangle is an object, ovals indicate objects attributes, rounded rectangles act for edges which indicate relations between target and other objects. The same color of the bounding boxes in the output image and the referents in the generated scene graph legends denotes a grounding. FIGURE 1 \| The architectural diagram of the proposed interactive natural language grounding. We ﬁrst parse the interactive natural language queries into scene graph legends by the scene graph parsing. We then ground the generated scene graph legends via the referring expression comprehension network. The mark rectangle in bottom encompasses the scene graph parsing result for the input natural language query. The rounded rectangles with black dashed lines denote the parsed scene graph legends, color shaded rectangles represent referents, no color shaded rectangle is an object, ovals indicate objects attributes, rounded rectangles act for edges which indicate relations between target and other objects. The same color of the bounding boxes in the output image and the referents in the generated scene graph legends denotes a grounding. We elaborate the details of the referring expression comprehension network in section 4, and we describe the scene graph parsing in section 5. Following this, we outline the experiments conducted to evaluate the referring expression comprehension network and the interactive natural language grounding architecture in section 6. components of the target localization module. Figure 2 illustrates the details of the proposed semantic-aware network for referring expression comprehension. Frontiers in Neurorobotics \| www.frontiersin.org 4. REFERRING EXPRESSION COMPREHENSION VIA SEMANTIC-AWARE NETWORK For an expression r, we employ BERT (Devlin et al., 2019) to tokenize and encode r into contextualized word embeddings Er = [e1, e2, ..., eM], where ei ∈R1×1024. We then feed Er into an one-layer BiLSTM: Given a referring expression r with M words r = {wi}M i=1 and an image I with N region of interests (RoIs) I = {oj}N j=1, we model the relation between wi and oj to locate the target object. In this study, we propose a referring expression comprehension network comprises: (1) a language attention network learns to assign diﬀerent weights to each word in expressions, and parse the expressions into phrases that denote target candidate, relation between target candidate and other objects, and location information; (2) a visual semantic-aware network generates semantic-aware visual representation, which is acquired by the channel-wise and the region-based spatial attention; (3) a target localization module achieves targets grounding by combining the outputs of the language attention network, the output of the visual semantic-aware network with the Lout = BiLSTM(Er) (1) (1) where Lout is the output of the BiLSTM. where Lout is the output of the BiLSTM. To acquire the diﬀerent weight of each word, we compute attention distribution over the expressions by: αl = softmax(F(Lout)) L = g X i αl,iLout,i (2) (2) June 2020 \| Volume 14 \| Article 43 Mi et al. Interactive Natural Language Grounding FIGURE 2 \| Semantic-Aware network for referring expression comprehension. We adopt the language attention network to compute the different weights for each word in expressions, and learn to parse the expressions into phrases that embed the information of target candidate, relation, and spatial location, respectively. We conduct both channel-wise and region-based spatial attention to generate semantic-aware region visual representation. We further combine the outputs of the visual semantic-aware network, the language attention network, and the relation and location representations to locate the target objects. In the ﬁgure, f ′ v denotes the projected deep features, VC represents the channel-wise weighted deep feature, VS is the spatial weighted feature, fS v is the generated semantic-aware visual representation by concatenating f ′ v and VS, the details are described in section 4.2. The relation representation urel, the location representation uloc, and the details of the target candidate module, the relation module, and the location module are introduced in section 4.3. 4. REFERRING EXPRESSION COMPREHENSION VIA SEMANTIC-AWARE NETWORK 9 denotes a channel-wise multiplication for f ′ v and the generated channel-wise attention weight σ, 8 represents element-wise multiplication for VC and the acquired spatial attention weight γ (Best viewed in color). 4.2. Visual Semantic-Aware Network We take full advantage of the characteristics of deep features extracted from a pretrained CNN model, and we conduct channel-wise and region-based spatial attention to generate semantic-aware visual representation for each detected region. This process can be deemed as visual representation enrichment for the detected regions. where αl denotes the calculated attention weight, and X m=1 αl = 1. In the implementation, F is modeled by two convolution layers. The generated expression representation L ∈Rd×2048, d is length of expressions in diﬀerent dataset. Expressions like “cup with printed red ﬂowers,” some words should be parsed to a phrase to represent speciﬁc information, e.g., “with printed red ﬂowers.” To this end, we employ a single perceptron layer and a softmax layer to learn to parse the expression into three module embeddings: 4.2.1. RoI Features Given an image, we adopt Faster R-CNN (Ren et al., 2015) to generate RoIs, and we extract deep feature fv ∈R7×7×2048 for each oj from the last convolutional layer of the 4th-stage of ResNet101 (He et al., 2016), where 7×7 denotes the size of the extracted deep feature, 2048 is the output dimension of the convolutional layer, i.e., the number of channels. We then project the deep feature fv into a 512-dimension subspace by a convolution operator with 1×1 kernel, i.e., the projected deep feature f ′ v ∈R7×7×512. L = ϕ(WtL + bt) [wtar, wloc, wrel] = softmax(L) (3) (3) where ϕ is a non-linear activation function, in this paper, we use hyperbolic tangent. Wt is a trainable weight matrix and bt represents a bias vector. wtar, wloc, wrel represent weights guided by target embedding rtar, relation embedding rrel, and spatial location embedding rloc, respectively. Frontiers in Neurorobotics \| www.frontiersin.org where D(·, ·) represents the consine distance measurement. where D(·, ·) represents the consine distance measurement. We put the weighted channel-wise deep features VC and the weighted expressions into an attention network similar to the channel-wise attention to calculate the spatial attention γ : 4.3. Target Localization Module In order to locate target objects for given expressions, we need to sort out the relevant candidates, the spatial location, and the appearance diﬀerence between the candidate and other objects. For instance, to understand the expression “the cow directly to the right of the largest cow,” we need to understand the spatial location “the right of,” and the appearance diﬀerence “largest” between the cows to identify the target “cow.” To this end, we deal with the relevant candidates, the relation and spatial location through a target candidate module, a relation module, and a spatial location module, respectively. Ac = ϕ((Wv,cV + bv,c) ⊗(Wt,cL + bt,c)) σ = softmax(Ac) (4) (4) where Wv,c and Wt,c are learnable weight matrices, bv,c and bt,c are bias vectors, Wv,c and bv,c are the parameters of the MLP for visual representation, while Wt,c and bt,c for textual representation. ⊗denotes outer product, σ ∈R1×512 is the learned channel-wise attention weight which encodes the semantic attributes of regions. In the following, Wv,. and bv,. represent the weight matrix and bias vector for visual representation, while Wt,. and bt,. denote the trainable parameters for textual representation. 4.3.1. Target Candidate Module We compute the target candidate phrase matching score by the target candidate module. For a given region semantic-aware representation f S v and target candidate phrase guided expression embedding rtar, we process f S v and rtar by L2-normalization and linear transform to compute the attention weights on each region: t = ϕ((Wvf S v + bv) ⊗(Wtrtar + bt)) β = softmax(t) (8) (8) 4.2.2. Channel-Wise Attention Essentially, deep features extracted from CNN are spatial, channel-wise, and multi-layer. Each channel of a deep feature correlates with a convolutional ﬁlter which performs as a pattern June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org 5 Mi et al. Interactive Natural Language Grounding weighted feature VC to obtain spatial weighted deep feature VS: weighted feature VC to obtain spatial weighted deep feature VS: detector (Chen L. et al., 2017). For example, the ﬁlters in lower layers detect visual clues such as color and edge, while the ﬁlters in higher layers capture abstract content such as object component or semantic attributes. Accordingly, performing channel-wise attention on higher-layer features can be deemed as a process of semantic attributes selection. VS = 8(VC, γ ) (7) (7) where 8 denotes element-wise multiplication for generated VC and the corresponding γ . We ﬁrst reshape the projected RoI deep feature fv′ to V=[v1, v2, ..., vdv], where vi ∈R7×7 is the i-th channel of the deep feature fv′, dv=512. We then perform average pooling on each channel to generate the channel-wise vector V = [v1, v2, ..., vdv], where V ∈ R1×512, vi represents the i-th pooled channel feature. Spatial weighted deep feature VS ∈R7×7×512 comprises the semantics guided by the channel-wise attention as well as the spatial weight of each region. Therefore, we deﬁne VS as semantic-aware deep feature. Finally, we concatenate VS with projected feature f ′ v to obtain semantic-aware visual representation for each region, i.e., f S v = [f ′ v ; VS], f S v ∈R7×7×1024, [· ; ·] denotes the concatenate operation. After the feature pooling, we ﬁrst utilize L2-normalization to process channel-wise vector V and expression representation L to generate more robust representations, we then perform channel-wise attention by a channel-wise attention network which is composed of an MLP (multi-layer perceptron) and a softmax layer. For the detected image region, the input of the attention network is average-pooled feature V and the weighted expression representation L. The channel-wise attention weight is acquired by: Frontiers in Neurorobotics \| www.frontiersin.org 4.2.3. Region-Based Spatial Attention The channel-wise attention attempts to address the semantic attributes of regions, while the region-based spatial attention is employed to attach more importance to the referring expressions related regions. To acquire region-based spatial attention weights, we ﬁrst combine the learned channel-wise attention weight σ with the projected deep feature f ′ v to generate channel-wise weighted deep feature VC. where β denotes the learned region-based attention weight. S We fuse β and f S v to obtain the target candidate phrase attended region visual representation utar, and we further compute the target candidate matching score star by: utar = β ⊗f S v utar = Wv,tarutar + bv,tar rtar = Wt,tarrtar + bt,tar star = D(utar, rtar) (9) VC = 9(fv′, σ) (5) (5) (9) where 9 is a channel-wise multiplication for deep feature channel and the corresponding channel weights, VC ∈R49×512. where D(·, ·) represents the consine distance measurement. 4.3.3. Spatial Location Module We calculate the location matching score through the location module. To deal with the spatial relation of candidates in images, following (Yu et al., 2016), we adopt a 5-dimensional spatial vector ul = [ xtl W , ytl H , xbr W , ybr H , w·h W·H ] to encode the top left position, bottom right position, and the relative size of the candidates in images. In order to address the relative position expression like “the right of,” “in the middle,” we adopt the relative location vector 1uij = [ [1xtl]ij wi , [1ytl]ij hi , [1xbr]ij wi , [1ybr]ij hi , wj·hj wi·hi ] which is obtained by comparing with ﬁve surrounding objects and concatenate with ul to generate candidate location representation uloc = [ul ; 1uij]. Considering the richness and diversity of natural language, and the relatively simple expressions in the three datasets, the trained referring expression comprehension model can not achieve complex natural language grounding. To this end, we combine scene graph with the referring expression comprehension network to ground unconstrained and sophisticated natural language. Scene graph was introduced in Johnson et al. (2015), in which the scene graph is used to describe the contents of a scene. Compared with dependency parsing, scene graph parsing generates less linguistic constituents. Given a natural language sentence, scene graph parsing aims to parse the natural language sentence into scene graph legends, which consist of nodes comprise objects with attributes and edges express the relations between target and objects. For instance, for the sentence “red apple next to the bottle,” the generated scene graph legend contains node (“red apple”) and node (“bottle”), and edge (“next to”). j Similar to the target candidate module, we process uloc and location phrase rloc, and then combine the transformed uloc and rloc to generate the location matching score sloc: uloc = Wv,loculoc + bv,loc rloc = Wt,locrloc + bt,loc sloc = D(uloc, rloc) (11) (11) Formally, a scene graph legend is deﬁned as a tuple G(S) = (N(S), E(S)), where N(S) = {N1(S), N2(S), ..., Nn(S)} is a set of nodes that encode objects with attributes, and E(S) = {E1(S), E2(S), ..., Em(S)} is a set of edges that express the relations between objects. Speciﬁcally, a node Ni(S) ⊆ni × Ai represents attribute Ai of an object ni (e.g., red apple). 4.3.3. Spatial Location Module An edge Ei(S) ⊆(no × R × ns) denotes the relation R between a subject no and an object ns, (e.g., next to). 4.3.2. Relation Module We adopt a relation module to obtain the matching score of a pair of candidates and relation embedding rrel. We use the average- pooled channel vector V as the appearance representation for each candidate. To tackle with the appearance diﬀerence between candidates, e.g., “the largest cow,” we calculate the visual appearance diﬀerence representation δvi= 1 n P j̸=i vi−vj \|\|vi−vj\|\| as (Yu et al., 2016), where n is the number of candidates chosen for As = ϕ((Wv,sVC + bv,s) ⊗(Wt,sL + bt,s)) γ = softmax(As) (6) (6) The acquired γ ∈R49×1 denotes the weight of each region related to the expressions. We further fuse the γ with channel-wise June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org 6 Interactive Natural Language Grounding Mi et al. caparison (in our implementation n = 5). We concatenate V and δvi as the candidates visual relation representation urel, i.e., urel = [V ; δvi]. We calculate the relation matching score by: caparison (in our implementation n = 5). We concatenate V and δvi as the candidates visual relation representation urel, i.e., urel = [V ; δvi]. We calculate the relation matching score by: and the average length of expressions in RefCOCO is 3.61, and the average number of words in RefCOCO+ expressions is 3.53. While RefCOCOg expressions were collected in a non- interactive way, therefore produces longer expressions and the average length is 8.43. From the perspective of expression length distribution, 97.16% expressions in RefCOCO contain less than 9 words, the proportion in RefCOCO+ is 97.06%, while 56.0% expressions in RefCOCOg comprise less than 9 words. Moreover, the expressions in the three datasets only indicate one referent, so the trained model cannot ground natural language instructions with multiple target objects. urel = Wv,relurel + bv,rel rrel = Wt,relrrel + bt,rel srel = D(urel, rrel) (10) (10) 4.4. Learning Objective Given a referring expression r and an image I with multiple RoIs pair, we calculate the target candidate score, the relation score, and the location score, through the three above introduced modules. We locate the target object by the ﬁnal grounding score: (12) G(oi\|r) = wtarstar + wrelsrel + wlocsloc (12) In general, a scene graph parser can be constructed on a corpus consisting of paired node-edge labels. However, no such dataset is released for interactive natural language grounding. In order to ensure the natural language is parsed correctly, we adopt a simple yet reliable rule, i.e., word-by-word match, to achieve scene graph alignment. Speciﬁcally, for a generated scene graph, we check the syntactic categories of each word in a node and an edge by part of speech. A parsed node should consist of a noun or an adjective, and an edge contains an adjective or an adverb. In practice, we adopt the language scene graph (Schuster et al., 2015) and the natural language toolkit (Perkins, 2010) to complete scene graph generation and alignment. In the implementation, we adopt a combined max-margin loss as the objective function: Lθ = X i [max(0, ξ −G(oi\|ri) + G(oi\|rj)) + max(0, ξ −G(oi\|ri) + G(ok\|ri))] (13) (13) where θ denotes the parameters of the model to be optimized, ξ is the margin between positive and negative samples. During training, we set ξ = 0.1. For each positive target and expression pair (oi, ri), we randomly select negative pairs (oi, rj) and (ok, ri), where rj is the expression for other objects, ok is the other object in the same image. Frontiers in Neurorobotics \| www.frontiersin.org 5. SCENE GRAPH PARSING 6.1. Referring Expression Comprehension Benchmark 6.1. Referring Expression Comprehension Benchmark The introduced referring expression comprehension network is trained on RefCOCO, RefCOCO+, and RefCOCOg. The referring expressions in RefCOCO and RefCOCO+ were collected by an interactive manner (Kazemzadeh et al., 2014), 6.1.3. Ablation Analysis We adopt diﬀerent combinations to validate the performance of each module, the results are shown in Table 1. According to (Yu et al., 2018b) and (Yu et al., 2018a), the models trained by the deep features extracted from VGG16 (Simonyan and Zisserman, 2014) generates lower accuracy than the features generated by ResNet101, so we do not train our model use VGG features. RefCOCO+ consists 141,564 expressions for 49,856 referents in 19,992 images. The split we use is same as (Yu et al., 2016). The training set consists of 120,191 expressions for 42,278 objects in 16,992 images, the validation partition contains 10,758 expressions for 3,805 objects in 1,500 images. TestA comprises 5,726 expressions for 1,975 objects in 750 images, and testB encompasses 4,889 expression for 1,798 objects in 750 images. Compared to RefCOCO, RefCOCO+ discards absolute location words and attaches more importance to appearance diﬀerentiators. First, we validate the performance of our model from the visual perspective. We concatenate the project feature fv′ and location representation uloc as the visual representation for each region, and adopt the output of the BiLSTM as the representation for expressions. We set this combination as the baseline, and the results are listed in Line 1. We then add relation representation urel to evaluate the beneﬁts of the relation module, and the results are listed in Line 2. RefCOCOg contains 95,010 expressions for 49,822 referents in 25,799 images. As they are collected in a non-interactive pattern, the length of referring expressions in RefCOCOg are longer than RefCOCO and RefCOCO+. RefCOCOg has two types of data splitting, (Mao et al., 2016) splits the dataset into train and validation, and no test set is published. Another data partition (Nagaraja et al., 2016) split the dataset as training, validation, and test sets. We run experiments on the second division, in which the training set contains 80,512 expressions for 42,226 objects in 21,899 images, the validation split includes 4,896 expressions for 2,573 objects in 1,300 images, and the test partition has 9,602 expressions for 5,023 objects in 2,600 images. Second, we test the eﬀectiveness of the visual semantic-aware network. We adopt the semantic-aware visual representation f S v combined with the location and relation representation, respectively. Compared to Line 1 and Line 2, the results listed in Line 3 and Line 4 show the beneﬁts of the visual semantic-aware network, and the accuracies are improved by nearly 2%. 6.1.3. Ablation Analysis Third, We employ two manners to evaluate the performance of the language attention network. We ﬁrst select fv′ as the visual representation for the target candidate, and combine the language attention network with the target localization module. It is clear that the results outperform than the results listed in Line 2. An interesting ﬁnding is that the results listed in Line 4 are close to Line 5, which also demonstrates the beneﬁts of the visual semantic-aware network. We then adopt f S v to represent the target candidate, and coalesce the language attention network with the other two modules. This combination acquires the best accuracies on the three datasets. 6.1.1. Datasets We train and validate the referring expression comprehension network on RefCOCO, RefCOCO+, and RefCOCOg. The images June 2020 \| Volume 14 \| Article 43 7 Mi et al. Interactive Natural Language Grounding ground truth bounding box, and select the IoU value larger than 0.5 as the correct visual grounding. of the three datasets were collected from MSCOCO dataset (Lin et al., 2014). of the three datasets were collected from MSCOCO dataset (Lin et al., 2014). RefCOCO comprises 142,210 expressions for 50,000 referents in 19,994 images. We adopt the same split with (Yu et al., 2016). The dataset is divided into training, validation, and test, respectively. The training set contains 120,624 expressions for 42,404 objects in 16,994 images, the validation set has 10,834 expressions for 3,811 objects in 1,500 images. The testing partition comprises two splits, testA and testB. TestA includes 5,657 expressions for 1,975 objects in 750 person-centric images, while testB owns 5,095 object-centric expressions for 1,810 objects in 750 images. We train our model with Adam optimizer with β1 = 0.9 and β2 = 0.999, we set the initial learning rate 0.0004 and decay every 5,000 iterations with weight decay 0.0001, and the total number of iterations is up to 30,000. 1https://github.com/huggingface/pytorch-pretrained-BERT 6.1.2. Experimental Setup eferring expression comprehension on test sets of RefCOCO, RefCOCO+, and RefCOCOg. Referring expressions are listed under th he red box represents the correct grounding, and the green bounding box denotes the ground truth. approach (Wang et al., 2019). (Wang et al., 2019) built the relationships between objects via a directed graph constructed over the detected objects within images. Based on the directed graph, this work identiﬁed the relevant target candidates by a node attention component and addressed the object relationships embedded in referring expressions via an edge attention module. This work focused on exploiting the rich linguistic compositions in referring expressions, while neglected the semantics embedded in visual images. In our proposed network, we address both the linguistic context in referring expressions and visual semantic in images. more appearance diﬀerence to depict objects. In addition, the expressions in RefCOCOg involve the descriptions of neighborhood objects of referents and frequently use the relation between objects to deﬁne the target objects. 6.1.2. Experimental Setup In practice, we set the length of the sentences to 10 for the expressions in RefCOCO and RefCOCO+, and pad with “pad” symbol to the expressions whose length is smaller than 10. We set the length of the sentences to 20 and adopt the same manner to process the expressions in RefCOCOg. Fourth, we compare the inﬂuence of diﬀerent word embeddings. We extract the embeddings from the last layer of BERT as the contextual representation for expressions and feed them into the language attention network, we denote this word embedding as LangAtten(I). Line 7 illustrates the obtained results. Compared with Line 6, the results show the advantage of the embeddings generated from the sum of the last four layers of BERT. We employ “bert-large-uncased” model1 to generate contextualized word embedding Er. According to Devlin et al. (2019), the word embedding from the sum of the last four layers acquire better results than the embedding extracted from the last layer. We select the embedding of the sum of the last four layers of BERT as Er. Therefore, the obtained expression representation q ∈R10×1024 for RefCOCO and RefCOCO+, and q ∈R20×1024 for RefCOCOg. Finally, we list some example results acquired by the referring expression comprehension network in Figure 3. According to the experimental results, the presented model is able to locate the target objects for complex referring expressions, as shown in the experiments on RefCOCOg. As shown in Table 1, compared with the results on RefCOCO+ and RefCOCOg, our model acquires better results on RefCOCO. We found the expressions in RefCOCO frequently utilize the attributes and location information to describe objects, while the expressions in RefCOCO+ abandon the location descriptions while utilize Given an image and referring expression pair, we utilize the ﬁnal ground score deﬁned in Equation 12 to compute the matching score for each object in the image, and pick the one with the highest matching score as the correct one. We calculate IoU (Intersection over Unit) between the selected region and the June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org 8 Interactive Natural Language Grounding Mi et al. TABLE 1 \| Ablation studies of our model using different module combinations. 6.1.2. Experimental Setup RefCOCO RefCOCO+ RefCOCOg val(%) testA(%) testB(%) val(%) testA(%) testB(%) val(%) test(%) 1 sub(ProjFeat)+loc 79.28 79.57 80.37 64.77 65.29 62.41 69.63 69.28 2 sub(ProjFeat)+loc+rel 79.99 80.24 80.82 64.89 66.00 63.57 70.14 69.96 3 sub(SemanAware)+loc 80.59 80.61 81.73 64.20 65.89 63.47 72.94 72.72 4 sub(SemanAware)+loc+rel 81.24 81.42 82.20 65.11 66.03 63.76 72.98 72.76 5 sub(ProjFeat)+loc+rel+LangAtten 81.83 82.10 82.20 66.42 67.46 63.84 73.33 72.81 6 sub(SemanAware)+loc+rel+LangAtten 83.51 83.74 83.18 68.16 69.66 64.66 76.00 74.81 7 sub(SemanAware)+loc+rel+LangAtten(I) 83.25 82.55 82.55 67.77 69.70 64.00 74.53 73.61 The bold values show the best grounding accuracy on each dataset split acquired by the proposed network. FIGURE 3 \| Example results of referring expression comprehension on test sets of RefCOCO, RefCOCO+, and RefCOCOg. Referring expressions are listed under the related images. In each image, the red box represents the correct grounding, and the green bounding box denotes the ground truth. TABLE 1 \| Ablation studies of our model using different module combinations. RefCOCO RefCOCO+ RefCOCOg val(%) testA(%) testB(%) val(%) testA(%) testB(%) val(%) test(%) 1 sub(ProjFeat)+loc 79.28 79.57 80.37 64.77 65.29 62.41 69.63 69.28 2 sub(ProjFeat)+loc+rel 79.99 80.24 80.82 64.89 66.00 63.57 70.14 69.96 3 sub(SemanAware)+loc 80.59 80.61 81.73 64.20 65.89 63.47 72.94 72.72 4 sub(SemanAware)+loc+rel 81.24 81.42 82.20 65.11 66.03 63.76 72.98 72.76 5 sub(ProjFeat)+loc+rel+LangAtten 81.83 82.10 82.20 66.42 67.46 63.84 73.33 72.81 6 sub(SemanAware)+loc+rel+LangAtten 83.51 83.74 83.18 68.16 69.66 64.66 76.00 74.81 7 sub(SemanAware)+loc+rel+LangAtten(I) 83.25 82.55 82.55 67.77 69.70 64.00 74.53 73.61 The bold values show the best grounding accuracy on each dataset split acquired by the proposed network. TABLE 1 \| Ablation studies of our model using different module combinations. FIGURE 3 \| Example results of referring expression comprehension on test sets of RefCOCO, RefCOCO+, and RefCOCOg. Referring expressions are listed under the related images. In each image, the red box represents the correct grounding, and the green bounding box denotes the ground truth. FIGURE 3 \| Example results of referring expression comprehension on test sets of RefCOCO, RefCOCO+, and RefCOCOg. Referring expressions are listed under the related images. In each image, the red box represents the correct grounding, and the green bounding box denotes the ground truth. GURE 3 \| Example results of referring expression comprehension on test sets of RefCOCO, RefCOCO+, and RefCOCOg. Referring ated images. In each image, the red box represents the correct grounding, and the green bounding box denotes the ground truth. Frontiers in Neurorobotics \| www.frontiersin.org 6.1.4. Comparison With State-of-the-Art expressions to depict two speciﬁc targets for each scenario, such as “the bottom row second donut from the left and the bottom rightmost mug.” For the self-collected scenarios, we ask the participants to give expressions with two or three referents for each image, for example, “move the red apple outside the box into the box and take the second water bottle from the right.” The collected working scenarios and expressions can be downloaded from the following link: https://drive.google.com/ open?id=1k4WgpHTGaYsIE9mMmDgE_kiloWnYSPAr. compared with the results on RefCOCO+ and RefCOCOg. The expressions in RefCOCO frequently utilize the location or other details to describe target objects, the expressions in RefCOCO+ abandon the location descriptions and adopt more appearance diﬀerence. While the expressions in RefCOCOg attach more importance to the relation between the target candidates and their neighborhood objects to depict the target objects. Finally, we show some failure cases on the three datasets in Figure 4. For complex expression, similar to “small table next to the chair,” our model generates closest weights for “table” and “chair.” Moreover, to locate the object with vague visual features, such as the target for “black sleeves” in the ﬁrst left image and “guy leg out” in the third image of the second row, our model frequently generates wrong predictions. For the long expression and image with the complex background, such as the two images in RefCOCOg, our model fails to generate correct predictions. In order to validate the performance of the proposed interactive natural language grounding architecture, we conduct grounding experiments on the collected indoor scenarios and natural language queries. We adopt the available scene graph parser source2 introduced (Schuster et al., 2015) to parse the complicated queries into scene graph legends (e.g., the parsing results listed in the rounded rectangles in the second row in Figure 5), and the trained referring expression comprehension model to locate target objects within given scenarios. 6.1.4. Comparison With State-of-the-Art Table 2 lists the results acquired by the proposed model and the state-of-the-art models. The table is split into two parts over the rows: the ﬁrst part lists the approaches without introducing the attention mechanism. The second illustrates the results acquired by attention integrated models. Second, through the experiments on the three datasets, the introduced model acquires better results on RefCOCO First, the proposed model outperforms the other approaches and acquire competitive results with the current state-of-the-art Frontiers in Neurorobotics \| www.frontiersin.org June 2020 \| Volume 14 \| Article 43 9 Interactive Natural Language Grounding Mi et al. TABLE 2 \| Comparison with the state-of-the-art approaches. RefCOCO RefCOCO+ RefCOCOg val(%) testA(%) testB(%) val(%) testA(%) testB(%) val(%) val(%) test(%) 1 visdif (Yu et al., 2016) - 67.57 71.19 - 52.44 47.51 59.25 - - 2 MMI (Mao et al., 2016) - 63.15 64.21 - 48.73 42.13 55.16 - - 3 attr+MMI+visdif (Liu et al., 2017) - 78.85 78.07 - 61.47 57.22 69.83 - - 4 Speaker (Yu et al., 2017) 79.56 78.95 80.22 62.26 64.60 59.62 72.63 71.65 71.92 5 Listener (Yu et al., 2017) 78.36 77.97 79.86 61.33 63.10 58.19 72.02 71.32 71.72 6 VC (Zhang et al., 2018) - 78.98 82.36 - 62.56 62.90 73.98 - - 7 DDPN+VGG16 (Yu et al., 2018b) 76.9 67.5 73.4 67.0 50.2 60.1 - - - 8 DDPN+ResNet101 (Yu et al., 2018b) 80.1 72.4 76.8 70.5 54.1 64.8 - - - 9 CMN (Hu et al., 2017) - - - - - - 69.30 - - 10 AccuAtten (Deng et al., 2018) 81.27 81.17 80.01 65.56 68.76 60.63 73.18 - - 11 PLAN (Zhuang et al., 2018) 81.67 80.81 81.32 64.18 66.31 61.46 69.47 - - 12 MAttNet+VGG16 (Yu et al., 2018a) 80.94 79.99 82.30 63.07 65.04 61.77 73.08 73.04 72.7 13 LGRANs (Wang et al., 2019) 82.0 81.2 84.0 66.6 67.6 65.5 - 75.4 74.7 14 VisSemanAware+LanAtten 83.51 83.74 83.18 68.16 69.96 64.66 - 76.00 74.81 The bold values show the best grounding accuracy on each dataset split. TABLE 2 \| Comparison with the state-of-the-art approaches. 2https://nlp.stanford.edu/software/scenegraph-parser.shtml Frontiers in Neurorobotics \| www.frontiersin.org 6.2. Interactive Natural Language Grounding Figure 5 lists some grounding results on the collected MSCOCO images. We adopt the referring expression comprehension network trained on the three datasets to ground the collected expressions, respectively. The accuracies of the collected expressions grounding for MSCOCO images acquired by the three models are RefCOCO 86.63%, RefCOCO+ 79.41%, and RefCOCOg 80.48%. Figure 6 shows the grounding example results on the self-collected scenarios. The grounding accuracies attained by the three models are RefCOCO 91.63%, RefCOCO+ 87.45%, and RefCOCOg 88.44%. From these experimental grounding results, it is clear that the trained referring expression comprehension models have superior robustness. We evaluate the eﬀectiveness of the presented interactive natural language grounding architecture in two diﬀerent manners. First, we collect 133 indoor scenarios from the test datasets of RefCOCO, RefCOCO+, and RefCOCOg, and collect 187 expressions that contain 2 referents for the selected images. These collected scenarios consist of the household objects that can be manipulated by robots. The average length of the expressions for MSCOCO images is 10.75. Second, we use a Kinect V2 camera to collect 30 images which are composed of the commonly used household objects and can be manipulated by robots. We collect 228 expressions, which contain 132 expressions with 2 referents and 96 expressions with 3 targets. The average number of words in these expressions is 14.31. Because of the properties of referring expressions in the RefCOCO, RefCOCO+, and RefCOCOg, the model trained on RefCOCO acquired the best results on the self-collected In order to collect diverse expressions for the collected images, we recruit 10 participants and show them diﬀerent scenarios. For the MSCOCO images, we ask the participants to give June 2020 \| Volume 14 \| Article 43 10 Mi et al. Interactive Natural Language Grounding Mi et al. Interactive Natural Language Grounding FIGURE 4 \| Examples of incorrect predictions. The red boxes show wrong visual groundings, and the green boxes denote the ground truth bounding boxes. FIGURE 5 \| Example results of interactive natural language grounding on MSCOCO images. The input natural language instructions are listed in the third row with rectangle, the scene graph parsing results are shown in the second row with rounded rectangle. FIGURE 4 \| Examples of incorrect predictions. The red boxes show wrong visual groundings, and the green boxes denote the ground truth bounding boxes. FIGURE 4 \| Examples of incorrect predictions. 6.2. Interactive Natural Language Grounding The red boxes show wrong visual groundings, and the green boxes denote the ground truth bounding boxes. FIGURE 4 \| Examples of incorrect predictions. The red boxes show wrong visual groundings, and the green boxes denote the gr FIGURE 5 \| Example results of interactive natural language grounding on MSCOCO images. The input natural language instructions are listed in the third row with rectangle, the scene graph parsing results are shown in the second row with rounded rectangle. FIGURE 5 \| Example results of interactive natural language grounding on MSCOCO images. The input natural language instructions are listed in the third row with rectangle, the scene graph parsing results are shown in the second row with rounded rectangle. within images, and the relation between targets and their neighborhood objects in the collected natural language queries. working scenarios. Instead of discarding spatial location words in expressions provided by RefCOCO+ expressions, and highlighting relationships between objects in RefCOCOg expressions, the collected expressions are more similar to the expressions in RefCOCO. Speciﬁcally, we take into consideration of descriptions of target attributes, spatial location of targets We also analyze the failure target object grounded working scenarios and related expressions, we found that the expressions with more “and” cannot be parsed correctly. For instance, the expression “take the apple between the bottle and the glass and June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org Frontiers in Neurorobotics \| www.frontiersin.org 11 Mi et al. Interactive Natural Language Grounding FIGURE 6 \| Example results of interactive natural language grounding on self-collected scenarios. The input natural language are listed in the rectangles, and the parsed scene graph legends are covered with related colors. FIGURE 6 \| Example results of interactive natural language grounding on self-collected scenarios. The input natural language are parsed scene graph legends are covered with related colors. FIGURE 6 \| Example results of interactive natural language grounding on self-collected scenarios. The input natural language are listed in the rectangles, and the parsed scene graph legends are covered with related colors. the red cup” will be parsed into four nodes “apple,” “bottle,” “glass,” and “red apple,” while the relation between “apple,” “bottle,” and “glass” is lost, which leads to a failure grounding. ground complicated natural language queries. 6.2. Interactive Natural Language Grounding Speciﬁcally, we ﬁrst parsed the complicated queries into scene graph legends, and then we fed the parsed scene graph legends into the trained referring expression comprehension network to achieve target objects grounding. We validated the performance of the presented interactive natural language grounding architecture by implementing extensive experiments on self-collected indoor working scenarios and natural language queries. Frontiers in Neurorobotics \| www.frontiersin.org REFERENCES He, K., Zhang, X., Ren, S., and Sun, J. 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A multimodal classiﬁer generative adversarial network for carry and place tasks from ambiguous language instructions. IEEE Robot. Autom. Lett. 3, 3113–3120. doi: 10.1109/LRA.2018.2849607 Gordo, A., Almazán, J., Revaud, J., and Larlus, D. (2016). “Deep image retrieval: learning global representations for image search,” in European Conference on Computer Vision (ECCV) (Amsterdam), 241–257. doi: 10.1007/978-3-319-46466-4_15 Mao, J., Huang, J., Toshev, A., Camburu, O., Yuille, A. L., and Murphy, K. (2016). 7. CONCLUSION We proposed an interactive natural language grounding architecture to ground unrestricted and complicated natural language queries. Unlike the existing methods for interactive natural language grounding, our approach achieved natural language grounding and queries disambiguation without the support from auxiliary information. Speciﬁcally, we ﬁrst presented a semantic-aware network for referring expression comprehension which is trained on three commonly used datasets in referring expressions. Considering the rich semantics in images and natural referring expressions, we addressed both visual semantic and textual contexts in the presented referring expression comprehension network. Moreover, we conducted multiple experiments on the three datasets to evaluate the performance of the proposed referring expression comprehension network. g g q Compared to the existing work for interactive natural language grounding, the proposed architecture is akin to an end-to-end approach to ground complicated natural language queries, instead of drawing support from auxiliary information. And the proposed architecture does not entail time cost as the dialogue-based disambiguation approaches. Afterward, we will improve the performance of the introduced referring expression comprehension network by exploiting the rich linguistic compositions in natural referring expressions and exploring more semantics from visual images. Moreover, the scene graph parsing module performs poorly when parsing complex natural language queries, such as sentences with more “and,” we will focus on improve the performance of the scene graph parsing. Additionally, we will exploit more Furthermore, we integrated the referring expression comprehension network with scene graph parsing to June 2020 \| Volume 14 \| Article 43 12 Interactive Natural Language Grounding Mi et al. the referring expression comprehension experiments, and designed interactive natural language architecture validation experiments. JL, ST, and QL provided critical revise advices for the manuscript. All authors contributed to the ﬁnal paper revision. eﬀective methods to ground more complicated natural language queries and conduct target manipulation experiments on a robotic platform. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. AUTHOR CONTRIBUTIONS This work was partly funded by the German Research Foundation (DFG) and National Science Foundation (NSFC) in project Crossmodal Learning under contract Sonderforschungsbereich Transregio 169, and the DAAD German Academic Exchange Service under CASY project. 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Frontiers in Neurorobotics \| www.frontiersin.org June 2020 \| Volume 14 \| Article 43 REFERENCES doi: 10.1007/978-1-4614-3064-3_10 Thomason, J., Padmakumar, A., Sinapov, J., Walker, N., Jiang, Y., Yedidsion, H., et al. (2019). “Improving grounded natural language understanding through human-robot dialog,” in IEEE International Conference on Robotics and Automation (ICRA) (Montreal, QC), 6934–6941. doi: 10.1109/ICRA.2019.8794287 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. Copyright © 2020 Mi, Lyu, Tang, Li and Zhang. 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. Thomason, J., Sinapov, J., Svetlik, M., Stone, P., and Mooney, R. J. (2016). “Learning multi-modal grounded linguistic semantics by playing “i spy,”” in Proceedings of the Twenty-Fifth International Joint Conference on Artiﬁcial Intelligence (IJCAI) (New York, NY), 3477–3483. Twiefel, J., Hinaut, X., Borghetti, M., Strahl, E., and Wermter, S. (2016). “Using natural language feedback in a neuro-inspired integrated multimodal June 2020 \| Volume 14 \| Article 43 Frontiers in Neurorobotics \| www.frontiersin.org 14
https://openalex.org/W3095235191	https://link.springer.com/content/pdf/10.1007/s42399-020-00597-2.pdf	English	null	Management of a Hospital-Wide COVID-19 Outbreak Affecting Patients and Healthcare Workers	SN Comprehensive Clinical Medicine	2,020	cc-by	4,393	Abstract To the best of our knowledge, here, we describe the first hospital-wide outbreak of SARS-CoV-2 that occurred in Germany in April 2020. We aim to share our experience in order to facilitate the management of nosocomial COVID-19 outbreaks in healthcare facilities. All patients and hospital workers were screened for SARS-CoV-2 repeatedly. An infection control team on the side was installed. Strict spatial separation of patients and intensified hygiene training of healthcare workers (HCW) were initiated. By the time of reporting, 26 patients and 21 hospital workers were infected with a cluster of cases in the geriatric department. Fourteen patients developed COVID-19 consistent symptoms and five patients with severe pre-existing medical conditions died. The outbreak was successfully contained after intensified infection control measures were implemented and no further cases among patients were detected over a period of 14 days. Strict application of standard infection control measures proved to be successful in the management of nosocomial SARS-CoV-2 outbreaks. Keywords COVID-19 . Infection control . SARS-CoV-2 . Nosocomial outbreak Keywords COVID-19 . Infection control . SARS-CoV-2 . Nosocomial outbreak * Steffen Höring shoering@ukaachen.de * Steffen Höring shoering@ukaachen.de 1 Division of Infection Control and Infectious Diseases, Medical Faculty, RWTH Aachen University Hospital, Aachen, Germany 2 Laboratory Diagnostic Center, RWTH Aachen University Hospital, Aachen, Germany 3 Department of Geriatric Medicine, RWTH Aachen University Hospital, Aachen, Germany 4 University Medical Center for Occupational Medicine, RWTH University, Aachen, Germany Management of a Hospital-Wide COVID-19 Outbreak Affecting Patients and Healthcare Workers Steffen Höring1 & René Fussen1 & Johannes Neusser1 & Michael Kleines2 & Thea Laurentius3 & Leo Cornelius Bollheimer3 & Doris Keller4 & Sebastian Lemmen1 Steffen Höring1 & René Fussen1 & Johannes Neusser1 & Michael Kleines2 & Thea Laurentius3 & Leo Cornelius Bollheimer3 & Doris Keller4 & Sebastian Lemmen1 Accepted: 15 October 2020 # The Author(s) 2020 /Published online: 26 October 2020 Accepted: 15 October 2020 # The Author(s) 2020 /Published online: 26 October 2020 https://doi.org/10.1007/s42399-020-00597-2 SN Comprehensive Clinical Medicine (2020) 2:2540–2545 COVID-19 COVID-19 4 University Medical Center for Occupational Medicine, RWTH University, Aachen, Germany Introduction a large nosocomial outbreak of SARS-CoV-2 that occurred at a satellite hospital of the University Hospital Aachen, Germany, with 26 patients and 21 healthcare workers infect- ed. The hospital, a formerly church-run facility, was integrated to the University Hospital in January 2020, hosting a geriatric department, a dermatological ward, and a mixed ward for multiple surgical disciplines with 170 beds in total. Located in Aachen, the hospital is situated in close proximity to the district of Heinsberg, the region where community transmis- sion of SARS-CoV-2 was first observed in Germany and where the cumulative incidence of SARS-CoV-2 is still among the highest in Germany [2]. Since the beginning of the novel coronavirus disease pandem- ic (COVID-19), inadvertent exposure of hospitalized patients and HCW to severe respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a major concern [1]. Here, we report Steffen Höring and René Fussen contributed equally to this work. This article is part of the Topical Collection on COVID-19 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s42399-020-00597-2) contains supplementary material, which is available to authorized users. Our report provides a narrative description of a nosocomial COVID-19 outbreak. Furthermore, we present the infection control measures implemented to contain the outbreak and describe potential sources of the outbreak. * Steffen Höring shoering@ukaachen.de Outbreak Description Against the background of the ongoing COVID-19 pandemic, the hospital’s policy and clinical processes were already adapted to prevent nosocomial transmission of SARS-CoV- 2. All HCW were obliged to wear a surgical face mask 4 University Medical Center for Occupational Medicine, RWTH University, Aachen, Germany SN Compr. Clin. Med. (2020) 2:2540–2545 2541 April 27/28, revealed no further cases among patients or hospital workers. throughout their working hours and visitors were no longer permitted. Furthermore, the University Hospital’s peripheral and intensive care capacities were steadily increased, for in- stance by postponing elective surgeries, in expectation of a rise in COVID-19 case numbers. In total, 150 PCR analyses were conducted on 50 patients and 701 PCR analyses were conducted on 270 hospital workers during the observational period. By the time of reporting (May 5), 26 out of 50 patients and 21 out of 270 tested HCW were infected, resulting in an attack rate of 52% and 7.8%, respectively. The median age of patients affected was 85 years and all patients had at least one or even several underlying diseases. Twenty-two of the 26 affected patients were patients of the geriatric department; the remaining four were dermatological and orthopedic patients. In this context, the first SARS-CoV-2-infected patient was revealed in the geriatric department on April 5. Although the patient, a 82-year-old man, showed no signs of infection, po- lymerase chain reaction (PCR) testing was performed since public health regulations demanded SARS-CoV-2 testing pri- or to his admission to a long-term care facility. As the hospital’s pandemic policies required a reduced op- eration mode, only 50 patients were present at the hospital at the time the first patient was detected. Hence, the potential index patient and all contact patients could be transferred im- mediately to single rooms. In addition, all patients of the ge- riatric department were screened for SARS-CoV-2 by naso- pharyngeal swabs and PCR analysis (Realstar® SARS-CoV-2 RT-PCR Kit, Altona Diagnostics, Germany) on April 7. The screening revealed ten more oligosymptomatic SARS-CoV-2- infected patients on two spatially separated geriatric wards. Figure 1 demonstrates the occurrence of cases among pa- tients and HCW during the outbreak period. While twelve patients were asymptomatic, nine patients developed COVID-19 with mild to moderate symptoms. Outbreak Description However, five elderly patients (mean age 82.2 years; range: 64–94 years) with severe pre-existing conditions (mean Charlson comor- bidity index 5.6; range: 4–7) succumbed to the infection; ac- cording to their wishes, no life-supporting measures were un- dertaken. Thus, a case fatality rate of 19.2% (5 out of 26 patients) among infected patients was observed. Among healthcare workers, one nurse was hospitalized due to wors- ening of her general condition and respiratory distress. By the time of reporting, she had been discharged and has recovered from the infection. Facing this high detection rate, cohorting of patients on a separated isolation ward was initiated by the infection control department on the same day. In parallel, on April 6, a 47-year-old nurse working in the geriatric department presented at the University Hospital’s COVID-19 screening center. During the absence from work, she had developed mild symptoms with fever, dry cough, and myalgia outside and was subsequently tested positive for SARS-CoV-2. The nurse belonged to a religious community of seven nuns, all of them working as nurses in the hospital. The remaining six nurses presented the next day at the screen- ing center reporting headache and faintness. In retrospect, the mild symptoms were considered COVID-19-consistent. PCR testing revealed five of them as SARS-CoV-2-positive. Infection Control Measures On April 7, an isolation ward with 40-bed capacity was established for confirmed SARS-CoV-2 cases. On this ward, intensified clinical monitoring was conducted by measuring vital signs and oxygen saturation at least six times a day. Infection control personnel (ICP) and an infectious diseases doctor were present during ward rounds to observe working processes and to support clinical decision-making on a daily basis. The ward facilities were partially restructured with a sluice area and a changing room at the entrance. In the time before the outbreak, it was common practice for nursing staff to work on different wards and to switch their deployments from day to day. With the implementation of the isolation ward, medical staff exclusively worked on one ward without intrahospital fluctuation. Considering the numerous COVID-19 cases among pa- tients and HCW, a hospital-wide screening was initiated on April 8 for all remaining SARS-CoV-2-negative patients and entire hospital staff. This hospital-wide screening revealed five more cases among patients as well as six nurses, one physiotherapist, and one resident of the dermatology depart- ment. While the cases were distributed in departments all over the hospital, there was a cluster among patients and HCW in the geriatric department. Follow-up screening of all SARS- CoV-2-negative patients and hospital staff was conducted repeatedly. A permanent on-site outbreak team was installed on April 8. The team met daily and consisted of members of the infec- tious disease and infection control department, the geriatric department, head nurses, and the hospital’s managing director. The first follow-up screening between April 11 and April 16 revealed ten more infected patients, four more cases among nursing staff, and two infected occupational therapists. The second follow-up screening between April 20 and 21 yielded one more infected hospital employee, a pastoral worker, but no new cases among patients. The last screening sessions, conducted on April 23/24 and Throughout the outbreak, all hospital workers wore a sur- gical face mask during their working hours. When caring for SARS-CoV-2-positive patients, gloves, goggles, and a protec- tive gown were worn additionally and an FFP-2 face mask SN Compr. Clin. Med. (2020) 2:2540–2545 2542 0 2 4 6 8 10 12 health care worker paents Fig. 1 SARS-CoV-2-positive PCR results in the course of the outbreak Fig. 1 SARS-CoV-2-positive PCR results in the course of the outbreak health care worker paents weeks. Infection Control Measures Due to colonization with multi-resistant bacteria, single-room isolation precautions were already performed; thus, no patient-to-patient contact occurred during the hospital stay. Following the hospitals’ pandemic regulations, no visi- tors were allowed in the preceding weeks. Therefore, we con- clude a nosocomial infection transmitted via an infected HCW. was used whenever aerosol-producing procedures were ex- pected. In order to ensure safe handling of the personal pro- tection equipment, i.e., preventing self-inoculation during ungloving, gowning, and masking, the medical staff was trained by ICP repeatedly. Since April 8, the geriatric clinic was closed for new ad- missions, and from April 17 on, this regulation was applied for the entire hospital. Spatial distancing was additionally ensured by keeping all SARS-CoV-2-negative patients in single rooms outside the isolation ward. Considering the date of admission and onset of symptoms, a further 21 cases among patients are categorized as nosoco- mial infections. However, in one case, SARS-CoV-2 virus was detected only 2 h after hospital admission pointing to a community-acquired infection. In three other cases, the date of admission was within the assumed incubation period; thereby, no definite mode of acquisition can be stated for these patients. As outlined above, SARS-CoV-2 screening of patients and hospital staff was performed twice weekly by nasopharyngeal swabs and PCR analysis. Examination of hospital workers, including HCW as well as administrational or technical per- sonnel, was conducted as a voluntary mass screening during working hours. SARS-CoV-2-positive staff were released from work and put under domestic quarantine until symptoms entirely for at least 48 h and PCR testing proved negative twice in a row. On the other hand, we analyzed the first cases among hos- pital staff, starting with the potential index nurse tested posi- tive for SARS-CoV-2 on the 6th of April. Low Ct values and worsening of symptoms in the days after diagnosis suggest a recently acquired COVID-19 infection with a high potential for viral spreading. Five of her household members, all of them nuns living together in a religious community, were infected showing lower viral loads but a simultaneous onset of symptoms. Contacts to patients and other hospital workers could not be traced back reliably since all affected nurses shifted teams and wards frequently within the hospital on a needs basis. No definite index case or source of infection can be determined for this cluster. Investigation of the Outbreak Source Our analysis of the intrahospital SARS-CoV-2 transmission dynamics is based on time of diagnosis, time of admission, time of onset of symptoms, viral load at initial PCR testing, and reported contacts of persons infected (see supplementary Figs. 1 and 2). PCR-derived threshold cycle (Ct) values served as a surrogate for viral load with low Ct values below 20 indicating a high viral load and Ct values above 30 representing a low viral load. Discussion On the one hand, there was the first case among patients, detected on April 5. The high viral load (Ct: 16) at the time of diagnosis and the fact that symptoms worsened after diagnosis point to a recently acquired COVID-19 infection. At the time of diagnosis, the patient was already hospitalized for several This report presents our first experience in managing a noso- comial COVID-19 outbreak. In total, 26 patients and 21 HCW were infected with SARS-CoV-2. Since mainly elderly pa- tients with severe pre-existing medical conditions were SN Compr. Clin. Med. (2020) 2:2540–2545 2543 avoiding close face-to-face contact with infected patients, might have altered temporarily. On the other hand, the viral spread might also occur between HCW. In our case, for in- stance, HCW occasionally reported not to have worn facemasks during breaks although spatial distance could not be kept in these situations. Both routes of transmission, HCW to patients and HCW to HCW, were successfully addressed in infection control training sessions in which HCW were instructed in the correct handling of personal protective equip- ment and in social distancing measures. Several reports point out the importance of verbal training sessions demonstrating that HCW education does not only improve the handling of PPE but also reduces anxiety and increases the sense of pre- paredness [6, 7]. affected, a high case fatality rate of 19% was observed during the outbreak. Nevertheless, intensified infection control mea- sures eventually led to successful containment. The outbreak occurred during the onset of the COVID-19 pandemic in Germany. By the time the outbreak emerged, the hospital policy already comprised preemptive infection con- trol measures in order to prevent intrahospital spread of SARS-CoV-2. Nevertheless, the outbreak could only be contained after all potential routes of intrahospital virus trans- mission were addressed by additional infection control mea- sures (Table 1). & First, the patient-to-patient transmission of SARS-CoV-2 had to be prevented. The reduction of contacts between geriatric patients was partially challenging since several patients suffered from cognitive impairment and did not follow social distancing recommendations or single-room isolation. Thus, cohorting of infected patients on an isola- tion ward proved to be an ideal solution in this scenario. Patients could move freely within the limits of the isola- tion ward and social contacts between patients were per- mitted without putting non-infected patients at risk. Table 1 Outbreak control measures Discussion Last, our measures aimed to prevent the introduction of new COVID-19 cases into the hospital. Thus, the hospital was closed for new admissions during the ongoing outbreak. In the post-outbreak period, we have continued to screen all patients on their day of admission and all geriatric inpatients once weekly for SARS-CoV-2 in order to detect new cases timely. Serial screening proved necessary since detection of a newly acquired COVID-19 infection cannot reliably be achieved by a single PCR test. Viral RNA shedding starts approximately a day before the onset of symptoms and peaks in the first week of the disease with no detectable RNA in the first days post infection [8–10]. This explains why, in our case, seven HCW were initially tested negative, while follow-up examinations revealed a COVID-19 infection. The second route of transmission addressed by our mea- sures was infected HCW, who potentially spread SARS-CoV- 2 to patients as well as to their co-workers. On the one hand, geriatric care requires close physical contacts thereby facilitat- ing viral spreading from HCW to patients. Consistently, first reports on outbreaks in nursing homes and geriatric wards show high attack rates and transmission dynamics comparable to our outbreak scenario [3–5]. We assume that by identifying asymptomatic SARS-CoV-2-infected patients and officially declaring the circumstances a nosocomial outbreak, not only personal protective equipment (PPE) was used more conse- quently but also HCWs’ practice of care, for example Eventually, the infection control measures undertaken in response to the outbreak (Table 1) turned out to be effective since no further case among patients was detected after April 15 and only one last case among healthcare workers occurred on April 20. This report emphasizes the necessity of an infec- tion control team on-site in an outbreak situation. Unlike many German hospitals, where infection control specialists are not present permanently, a well-established infection con- trol infrastructure with sufficient manpower was available in our case. Concerning the source of this outbreak, patient-to-patient contacts did not seem to be the main factor. Most affected patients were bedridden and had no contact with other pa- tients. Nevertheless, three patients suffering from dementia showed a tendency to wander around on the wards and entered other patients’ rooms without permission. This behavior might have led to SARS-CoV-2 transmissions in singular cases in the weeks preceding the outbreak. Discussion • Establishment of a multidisciplinary outbreak team • Establishment of an isolation ward • Single room placement of uninfected patients • Rejection of new admissions • Intensified clinical monitoring of COVID-19 patients • Serial PCR screening of patients and hospital employees • Employment leave for SARS-CoV-2-infected staff • Training of healthcare workers • Outbreak investigation • Establishment of a multidisciplinary outbreak team • Establishment of an isolation ward • Single room placement of uninfected patients • Rejection of new admissions • Intensified clinical monitoring of COVID-19 patients • Serial PCR screening of patients and hospital employees • Employment leave for SARS-CoV-2-infected staff • Training of healthcare workers • Outbreak investigation • Establishment of an isolation ward • Single room placement of uninfected patients A greater contribution to outbreak dynamics might have been made by infected, asymptomatic or paucisymptomatic HCWs. Although we have not revealed any erroneous infec- tion control behavior on the side of HCWs, we assume they played a crucial role in introducing and spreading SARS- CoV-2 in the hospital. The cluster observed among HCWs in the religious community serves as a good example of this • Serial PCR screening of patients and hospital employees • Employment leave for SARS-CoV-2-infected staff • Training of healthcare workers • Outbreak investigation SN Compr. Clin. Med. (2020) 2:2540–2545 2544 outbreak. Nevertheless, routine diagnostics and standard infec- tion control measures, e.g., contact precautions and screening of patients and HCW, proved to be efficient when applied to this novel pathogen and allowed successful outbreak management. assumption. The order of nuns lived under circumstances that clearly fostered viral transmission. They stayed together in a dormitory on the hospital’s premises, shared a household, and attended service together. All of them simultaneously devel- oped symptoms pointing to a commonly acquired infection, most likely outside their working hours. However, all of them were still employed in patient care during their assumed incu- bation period and therefore could have introduced and spread the virus on the wards as asymptomatic carriers. We therefore hypothesize that SARS-CoV-2 might have initially spread and incubated among HCWs and was subsequently transmitted to patients and further co-workers. Author Contribution SH and RF contributed equally to the study by writing the first draft and editing further versions of the manuscript. The conceptual idea was developed by SH, RF, SL, and CB. PCR testing was performed by MK. Discussion SH, RF, JN, TL, and DK collected and analyzed data. All authors critically reviewed the manuscript. All authors read and ap- proved the final manuscript. Funding Open Access funding enabled and organized by Projekt DEAL. Nevertheless, we must discuss alternative ways of SARS- CoV-2 introduction to the hospital. In one patient for example, a community-acquired infection was clearly given. Therefore, we assume that the outbreak was based on multiple routes of introduction and transmission. Eventually, a definite single outbreak source could not be determined. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Ethics Statement This study was approved by the ethics committee of the Aachen University Hospital (EK 140/20) Ethics Statement This study was approved by the ethics committee of the Aachen University Hospital (EK 140/20) Further molecular investigation, e.g., next-generation se- quencing (NGS), might have been useful to clarify infection chains retrospectively. Nevertheless, we state that no additional benefits for the actual outbreak management would have derived from further molecular diagnostics since the outbreak dynamics obviously suggested a nosocomial spread of SARS-CoV-2 in our case. We therefore claim that our report emphasizes the sufficiency of standard diagnostic methods under the exceptional circumstances of a nosocomial SARS-CoV-2 outbreak. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. There are exceptional infrastructural aspects that clearly facilitated the outbreak management in our case. Although the hospital affected was small with a bed capacity of 170 beds only, as a satellite hospital, it could fall back on the infrastructure and the financial power of a large tertiary care university hospital. This ideal setting not only allowed the closure of the entire facility to new admissions but also pro- vided fast diagnostic processes with same day PCR results and an infection control team on site. We are aware of the fact that these settings are not a common standard and therefore, the management of SARS-CoV-2-outbreaks can be more difficult for other hospitals and healthcare facilities. References 1. Wang D, Hu B, Hu C, et al. Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan. China JAMA. 2020;323(11):1061–9. https://doi.org/10.1001/jama.2020.1585. 2. Robert K-I. Archiv der Situationsberichte des Robert Koch-Instituts zu COVID-19 (ab 4.3.2020). 2020. Available from: https://www. rki.de/DE/Content/InfAZ/N/Neuartiges_Coronavirus/ Situationsberichte/Archiv.html. Finally, we plead for a frank and detailed communication of nosocomial SARS-CoV-2 outbreaks during the ongoing COVID-19 pandemic. Despite concerns of negative publicity, reporting of nosocomial outbreaks is essential to allow all parties in the healthcare sector to benefit from each other’s experience. 3. 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Publisher’s Note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional affiliations. 10. Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS- CoV-2 viral load in upper respiratory specimens of infected pa- tients. N Engl J Med. 2020;382(12):1177–9. Conclusion 5. McMichael TM, Currie DW, Clark S, Pogosjans S, Kay M, Schwartz NG, et al. Epidemiology of Covid-19 in a long-term care facility in King County, Washington. N Engl J Med. 2020;382: 2005–11. 5. McMichael TM, Currie DW, Clark S, Pogosjans S, Kay M, Schwartz NG, et al. Epidemiology of Covid-19 in a long-term care facility in King County, Washington. N Engl J Med. 2020;382: 2005–11. Our report demonstrates a successful containment strategy for nosocomial COVID-19 outbreaks. Multiple routes of transmis- sion and delayed PCR-based diagnosis of early-stage infections presented pitfalls and hampered the definite investigation of the 6. 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https://openalex.org/W4379878439	https://www.researchsquare.com/article/rs-2699813/latest.pdf	English	null	Distribution of Hemoglobinopathies among Premarital Couples in Al Majmaah, Saudi Arabia	Research Square (Research Square)	2,023	cc-by	157	Keywords: Posted Date: June 8th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2699813/v2 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. 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 Abstract The full text of this preprint has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Full Text The authors have withdrawn this preprint from Research Square. Page 2/2
W3155594216.txt	https://amc-journal.eu/index.php/amc/article/download/2478/1701	en		Enumerating symmetric peaks in non-decreasing Dyck paths	Ars mathematica contemporanea	2,021	cc-by	10,034	ISSN 1855-3966 (printed edn.), ISSN 1855-3974 (electronic edn.) ARS MATHEMATICA CONTEMPORANEA 21 (2021) #P2.04 https://doi.org/10.26493/1855-3974.2478.d1b (Also available at http://amc-journal.eu) Enumerating symmetric peaks in non-decreasing Dyck paths* Sergi Elizalde Department of Mathematics, Dartmouth College, Hanover, NH, U.S.A. Rigoberto Flórez † Department of Mathematical Sciences, The Citadel, Charleston, SC, U.S.A. José Luis Ramı́rez ‡ Departamento de Matemáticas, Universidad Nacional de Colombia, Bogotá, Colombia Received 7 November 2020, accepted 25 February 2021, published online 30 September 2021 Abstract Local maxima and minima of a Dyck path are called peaks and valleys, respectively. A Dyck path is non-decreasing if the heights (y-coordinates) of its valleys increase from left to right. A peak is symmetric if it is surrounded by two valleys (or endpoints of the path) at the same height. In this paper we give multivariate generating functions, recurrence relations, and closed formulas to count the number of symmetric and asymmetric peaks in non-decreasing Dyck paths. Finally, we use Riordan arrays to study weakly symmetric peaks, namely those for which the valley preceding the peak is at least as high as the valley following it. Keywords: Non-decreasing Dyck path, symmetric peak, generating function, Riordan array, Fibonacci number. Math. Subj. Class. (2020): 05A15, 05A19 * The authors are grateful to an anonymous referee for helpful comments. † Corresponding author. The author was partially supported by the Citadel Foundation, Charleston, SC. author was partially supported by Universidad Nacional de Colombia, Project No. 46240. E-mail addresses: sergi.elizalde@dartmouth.edu (Sergi Elizalde), rigo.florez@citadel.edu (Rigoberto Flórez), jlramirezr@unal.edu.co (José Luis Ramı́rez) ‡ The c b This work is licensed under https://creativecommons.org/licenses/by/4.0/ 2 1 Ars Math. Contemp. 21 (2021) #P2.04 Introduction A Dyck path is a lattice path in the first quadrant of the xy-plane that starts at the origin, ends on the x-axis, and consists of (the same number of) up-steps X = (1, 1) and downsteps Y = (1, −1). A peak is a subpath of the form XY , and a valley is a subpath of the form Y X. The height of a valley is the y-coordinate of its lowest point. A Dyck path is called non-decreasing if the heights of its valleys form a non-decreasing sequence from left to right (see Figure 1 for an example). Non-decreasing Dyck paths have been extensively studied in the literature, see [2, 5, 6, 8, 13, 15, 17, 20]. All the Dyck paths considered in this paper will be non-decreasing. Following the notation from [5, 6, 13, 14], we denote by D the set of all non-decreasing Dyck paths, and by Dn the set of all non-decreasing Dyck paths of length 2n, where the length is defined as the number of steps. For P ∈ Dn , we write \|P \|= n to denote its semilength. A pyramid of semilength h ≥ 1 is a subpath of the form X h Y h ; it is maximal if it can not be extended to a pyramid X h+1 Y h+1 . Flórez and Ramı́rez [16] introduced the concept of symmetric and asymmetric peaks in Dyck paths, see also recent follow-up work by Elizalde [11] and Flórez et al. [14]. This concept was motivated in part by Asakly’s [1] study of symmetric and asymmetric peaks in k-ary words. The concept of symmetric peaks is different from the notion of degree of symmetry, which has been considered by Elizalde [9, 10] as a measure of how symmetric a Dyck path is. In this paper we study symmetric peaks and asymmetric peaks in non-decreasing Dyck paths. A peak is symmetric if the maximal pyramid containing the peak is not preceded by an X and is not followed by a Y . A peak is weakly symmetric if the maximal pyramid containing the peak is not preceded by an X. A peak is asymmetric if the maximal pyramid containing the peak is either preceded by an X or followed by a Y . Geometrically, a peak is symmetric if the maximal pyramid containing the peak is either at ground level or bounded by two valleys at the same height, and it is asymmetric otherwise. For example, in the nondecreasing Dyck path in Figure 1, the first, third, fourth, and sixth peaks are symmetric. The weakly symmetric peaks are the symmetric ones along with the seventh peak. Finally, the second, fifth, and the seventh peaks are asymmetric. We are also interested in the size of the maximal pyramid containing a peak. We define the weight of a pyramid X h Y h to be equal to h. In [5, 7], the authors refer to this parameter as the height, but we will use the term weight to suggest that it is not affected by the location of the pyramid. We define the weight of a peak to be the weight of the maximal pyramid that contains it. The symmetric weight of a path is the sum of the weights of its symmetric peaks. Similarly, the asymmetric weight of a path is the sum of the weights of its asymmetric peaks. For example, the weights of the symmetric peaks in the path depicted in Figure 1 are 4, 3, 3, 2 from left to right, and so the symmetric weight of the path is 12. The weights of asymmetric peaks are 1, 3, and 1, and the asymmetric weight of the path is 5. Figure 1: A non-decreasing Dyck path of length 38. S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 3 The generating functions that we present throughout the paper, are given using the symbolic method (cf. [12]). In Section 2, we give generating functions, recurrence relations, and closed formulas enumerating symmetric peaks and asymmetric peaks in nondecreasing Dyck paths. In Section 3, we focus on the enumeration of peaks with respect to their weight, and we give a connection to directed column-convex polyominoes. In Section 4, we study weakly symmetric peaks, and we synthesize the results using Riordan arrays. A summary of notation used throughout the paper appears in Tables 1 and 2 in the appendix. 2 Counting symmetric peaks In this section we study the distribution of the number of symmetric peaks in Dn . We give recurrences, generating functions and closed formulas (in terms of Fibonacci numbers) that enumerate these statistics in non-decreasing Dyck paths. Throughout the paper we will use Fn and Ln to denote the nth Fibonacci number and the nth Lucas number, respectively. The set Dn can be partitioned into two disjoint sets An and Bn , where An consists of the paths that have at least one valley of ground level (height 0), and Bn = Dn \ An . Note that n−1 [ Dn = An ∪· Bn and An = · Cn,i , (2.1) i=1 where Cn,i consists of those paths whose first valley touches the x-axis at (2i, 0), and ∪· denotes disjoint union. There is a natural bijection Cn,i P → Dn−i 7 → P \ ∆i , (2.2) obtained by removing the first pyramid ∆i = X i Y i of each P ∈ Cn,i . Similarly, there is a bijection from Bn to Dn−1 obtained by removing the first up-step and last down-step from each path. From (2.1), a path Q ∈ D is either empty or has one of these two forms: Q = XP Y or Q = X k Y k P , where k ≥ 1 and P ∈ D is non-empty. This P decomposition P gives rise to the following equation for the generating function D(x) = P ∈D x\|P \| = n≥0 \|Dn \|xn : D(x) = 1 + xD(x) + x (D(x) − 1). 1−x (2.3) Solving this equation and removing the empty path, we obtain the generating function for non-decreasing Dyck paths with respect to their semilength: D(x) = ∞ X x(1 − x) = F2n−1 xn . 1 − 3x + x2 n=1 Therefore, \|Dn \|= F2n−1 . Other derivations of this generating function appear in [2, 13]. (2.4) 4 2.1 Ars Math. Contemp. 21 (2021) #P2.04 A generating function for the number of symmetric and asymmetric peaks In this section we give a multivariate generating function enumerating symmetric peaks and the number of asymmetric peaks in non-decreasing Dyck paths. We start by introducing some terminology. We define the insertion vertices of a path to be the lowest point of each valley Y X, the initial point of the path, and, if the path contains no valleys at positive height, the final point of the path. For a path P ∈ D, we use τ (P ), σ(P ), σ(P ), ν(P ), and ι(P ) to denote the number of peaks, the number of symmetric peaks, the number of asymmetric peaks, the number of valleys, and the number of insertion points of P , respectively. We are interested in the generating function X Dσ,σ (t, r, x) = tσ(P ) rσ(P ) x\|P \| . P ∈D i j n The coefficient of t r x in Dσ,σ (t, r, x) is the number of paths of length 2n with i symmetric peaks and j asymmetric peaks. Theorem 2.1. The generating function for non-decreasing Dyck paths with respect to the number of symmetric peaks and the number of asymmetric peaks is Dσ,σ (t, r, x) = 1 − (3 + t)x + (3 + 2t − r)x2 − (1 + t − r − r2 )x3 . (1 − (1 + t)x)(1 − (t + 2)x + (1 + t − r)x2 ) Proof. In order to obtain an expression for Dσ,σ (t, r, x), we show that non-decreasing Dyck paths where some of their symmetric peaks have been marked can be constructed by inserting marked symmetric peaks in certain positions of smaller non-decreasing Dyck paths. First, we refine Equation (2.3) by introducing a variable v that keeps track of the number P of valleys in the path. Letting Dν (v, x) = P ∈D v ν(P ) x\|P \| , the same decomposition gives vx Dν (v, x) = 1 + xDν (v, x) + (Dν (v, x) − 1), 1−x from where 1 − (1 + v)x Dν (v, x) = . 1 − (2 + v)x + x2 Next we introduce another refinement. Let D∆ ⊆ D denote the set of paths that consist of a non-empty sequence of pyramids, that is, paths of the formP X k1 Y k1 · · · X kj Y kj , where ki ≥ 1 for 1 ≤ i ≤ j, for some j ≥ 1. Let Dτ,ι (p, q, x) = P ∈D pτ (P ) q ι(P ) x\|P \| be the generating function with respect to the number of peaks and the number of insertion vertices. Recall that insertion vertices of P are the bottoms of the valleys, the initial point of P , and, in the case that P ∈ D∆ , the final point of P . Thus, ι(P ) = ν(P ) + 2 if P ∈ D∆ , and ι(P ) = ν(P ) + 1 otherwise. On the other hand, τ (P ) = ν(P ) + 1 unless P is empty, in which case τ (P ) = 0. Using that X x x/(1 − x) = , Dν∆ (v, x) = v ν(P ) x\|P \| = 1 − vx/(1 − x) 1 − x − vx ∆ P ∈D it follows that Dτ,ι (p, q, x) = q + pq(Dν (pq, x) − Dν∆ (pq, x) − 1) + pq 2 Dν∆ (pq, x) =q+ pq 2 x(1 − (2 + pq)x + (1 + p)x2 ) . (1 − x + pqx)(1 − (2 + pq)x + x2 ) (2.5) S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 5 By construction, the insertion vertices of P are those vertices where the insertion of a pyramid X k Y k creates a symmetric peak and results in another non-decreasing Dyck path. Our next step is to enumerate non-decreasing Dyck paths where some of its symmetric peaks have been marked. Formally, we are enumerating pairs (P, M ) where P ∈ D and M is a subset of the symmetric peaks of P . Let D∗ be the set of such pairs (P, M ), which we referPto as non-decreasing Dyck paths with marked symmetric peaks, and let Dτ∗ (p, u, x) = (P,M )∈D∗ pτ (P ) u\|M \| x\|P \| . The key observation is that elements of D∗ can be uniquely obtained from paths in D by inserting a possibly empty sequence of marked pyramids (that is, pyramids whose symmetric peak is marked) at each insertion vertex. Since replacing each insertion vertex with a sequence of marked pyramids corresponds to the substitution 1 , q= 1 − upx/(1 − x) we get 1 Dτ∗ (p, u, x) = Dτ,ι p, ,x . 1 − upx/(1 − x) In order to have a variable t that keeps track of the total number of symmetric peaks, as opposed to marked symmetric peaks, we make the substitution u = t − 1. Note that, if Σ(P ) is the set of symmetric peaks of a path P ∈ D, then X (t − 1)\|M \| = ((t − 1) + 1)\|Σ(P )\| = tσ(P ) . (2.6) M ⊆Σ(P ) It follows that Dτ,σ (p, t, x) = X pτ (P ) tσ(P ) x\|P \| = P ∈D X X pτ (P ) (t−1)\|M \| x\|P \| = Dτ∗ (p, t−1, x). P ∈D M ⊆Σ(P ) Finally, since σ(P ) = τ (P ) − σ(P ), we have Dσ,σ (t, r, x) = Dτ,σ (r, t/r, x) = Dτ,ι r, 1 ,x , 1 − (t − r)x/(1 − x) and the formula in the statement follows now from Equation (2.5). Corollary 2.2. The generating functions for the total number of symmetric peaks and the total number of asymmetric peaks in non-decreasing Dyck paths are, respectively, S(x) := X σ(P )x\|P \| = P ∈D X P ∈D 2.2 σ(P )x\|P \| = ∂ Dσ,σ (t, 1, x) ∂t ∂ Dσ,σ (1, r, x) ∂r = x(1 − 5x + 7x2 − x3 − x4 ) , (1 − 2x)(1 − 3x + x2 )2 = x3 (2 − 6x + 3x2 ) . (1 − 2x)(1 − 3x + x2 )2 t=1 r=1 (2.7) Recurrence relations and Fibonacci numbers P Let sn = P ∈Dn σ(P ), that is, the total number ofP symmetric peaks in all non-decreasing n Dyck paths of semilength n. Note that S(x) = n≥1 sn x is the generating function in Equation (2.7). Next we give a recurrence for sn that involves the Fibonacci numbers. Define the level of a pyramid to be the height of the base of the pyramid. 6 Ars Math. Contemp. 21 (2021) #P2.04 Theorem 2.3. The sequence sn satisfies the recurrence relation sn = 3sn−1 − sn−2 + F2(n−2) − 2n−3 for n ≥ 3, with initial values s1 = 1 and s2 = 3. Proof. Recall the decomposition given in (2.1). It is clear from the definition of nondecreasing Dyck paths that the first pyramid in every path in Cn,i has a symmetric peak. Applying the bijection Cn,i → Dn−i from Equation (2.2) to all paths in Cn,i removes a total of \|Dn−i \|= F2(n−i)−1 pyramids (using Equation (2.4)), each having a symmetric peak. This implies that the number of symmetric peaks in Cn,i equals F2(n−i)−1 plus the number of symmetric peaks in Dn−i . So, the total number of symmetric peaks in An is given by n−1 n−1 n−1 X X X si + F2(n−1) . (2.8) F2(n−i)−1 = sn−i + i=1 i=1 i=1 We now count the total number of symmetric peaks in Bn , using the fact that Bn maps bijectively into Dn−1 by deleting the first X and the last Y . Note, however, that the first and the last peak of paths in Bn are not symmetric (unless the path is a pyramid), but they may become symmetric after the first X and the last Y are deleted. This happens when the associated path in Dn−1 starts or ends with a pyramid at ground level, without the path being itself the pyramid ∆n−1 = X n−1 Y n−1 , resulting in more symmetric peaks in Dn−1 than in Bn . Therefore, to count the number of symmetric peaks in Bn , we take the number of symmetric peaks in Dn−1 , which is sn−1 , and subtract the total number of first and last pyramids at ground level of paths in Dn−1 \ {∆n−1 }. First of all, we want to know the total number of pyramids at ground level that occur at the end of the paths in Dn−1 \ {∆n−1 }. Note that if the last pyramid of a non-decreasing Dyck path is at ground level, then the path consists of a sequence of pyramids at ground level. From [13, Corollary 6.3], we deduce that the number of paths in Dn−1 ending with a pyramid ∆i = X i Y i at ground level, for 1 ≤ i ≤ n − 2, is 2(n−1−i)−1 . This implies that Pn−3 the total number of last pyramids at ground level in Dn−1 \{∆n−1 } is i=0 2i = 2n−2 −1. From a similar analysis as in the first paragraph of this proof, we have that the total number Pn−2 of first pyramids at ground level in Dn−1 \ {∆n−1 } is i=1 F2i−1 = F2(n−2) . So, the total number of symmetric peaks in Bn is given by sn−1 − F2(n−2) − 2n−2 + 1. Adding this to (2.8), we get ! n−1 X sn = si + F2(n−1) + sn−1 − F2(n−2) − 2n−2 + 1 , i=1 with s1 = 1, and s2 = 3. We can simplify the recurrence by computing sn+1 − sn = 2sn − sn−1 + F2(n−1) − 2n−2 . Therefore, sn+1 = 3sn−1 − sn−2 + F2(n−2) − 2n−3 . The first few values of the sequence sn for n ≥ 1 are 1, 3, 8, 22, 62, 177, 508, 1459, 4182, 11946, .... For example, Figure 2 shows the non-decreasing Dyck paths of length 6, where the total number of symmetric peaks is s3 = 8. S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 7 Figure 2: Non-decreasing Dyck paths of length 6. Next we give another expression for sn in terms of the Fibonacci and the Lucas numbers. Theorem 2.4. The sequence sn satisfies sn = F2n + n X (F2`−2 − 2`−2 )F2(n−`) = `=3 2F2n−2 + (n − 1)L2n−2 + 2n−1 . 5 Proof. We first consider the generating function of the bisection of the Fibonacci sequence X x F (x) = F2n xn = . 1 − 3x + x2 n≥0 By Equation (2.7), the generating function S(x) can be decomposed as x2 x2 1 − 5x + 7x2 − x3 − x4 = F (x) 1 + − S(x) = F (x) (1 − 2x)(1 − 3x + x2 ) 1 − 3x + x2 1 − 2x 2 x = F (x) 1 + xF (x) − . 1 − 2x Using the Cauchy product of series we obtain the desired result. The second equality follows from the recurrence relation given in Theorem 2.3. In [6, Theorem 2], the authors prove that the total number of peaks in Dn is tn = (2n − 1)F2n − (n − 5)F2n−1 . 5 (2.9) The next corollary is a direct consequence of Theorem 2.4 and Equation (2.9). Corollary 2.5. Let sn be the total number of asymmetric peaks in Dn . Then, for n ≥ 2, sn = 2F2n+1 + (n − 2)L2n−3 − 2n−1 . 5 The first few values of the sequence sn for n ≥ 1 are 0, 0, 2, 10, 37, 122, 379, 1136, 3326, 9580, .... From the identities in Theorem 2.4 and Corollary 2.5, we obtain some asymptotic results about the proportion of peaks in non-decreasing Dyck paths that are symmetric. Theorem 2.6. Among all peaks of non-decreasing Dyck paths, the proportion of those that are symmetric is asymptotically √ sn −1 + 5 lim = ≈ 0.618034. n→∞ tn 2 8 Ars Math. Contemp. 21 (2021) #P2.04 Proof. From the well-known limits √ Fn+1 1+ 5 lim =φ= n→∞ Fn 2 and √ Ln = 5, n→∞ Fn lim we have sn (2F2n−2 + (n − 1)L2n−2 )/5 + 2n−1 = lim n→∞ tn n→∞ ((2n − 1)F2n − (n − 5)F2n−1 )/5 2 + (n − 1)L2n−2 /F2n−2 + 5 · 2n−1 /F2n−2 = lim n→∞ (2n − 1)F2n /F2n−2 − (n − 5)F2n−1 /F2n−2 √ √ 5 −1 + 5 = 2 = . 2φ − φ 2 lim Corollary 2.7. Among all peaks of non-decreasing Dyck paths, the proportion of those that are asymmetric is asymptotically √ sn 3− 5 lim ≈ 0.381966. = n→∞ tn 2 We say that a symmetric peak is low if the y-coordinate of its top vertex is one, and that it is high if this coordinate is greater than 1. Note that every low peak is symmetric. By [6, Corollary 6], the total number of high peaks in Dn is ((2n − 1)F2n − nF2n−1 )/5. Together with Corollary 2.5, this implies the following. Corollary 2.8. The total number of high symmetric peaks in Dn is 1 (F2n−3 + (n − 4)L2n−2 ) + 2n−1 . 5 3 Symmetric weight and symmetric height Recall that the weight of a pyramid X h Y h is equal to h and that the weight of a peak is the weight of the maximal pyramid that contains it. In this section we give a multivariate generating function for non-decreasing Dyck paths with respect to the weight of their symmetric peaks, as well a recurrence relation for the total symmetric weight over Dn . We also give a recurrence relation for the total sum of the heights of symmetric peaks over Dn . At the end of the section we describe a connection with polyominoes. 3.1 A generating function for symmetric weight We introduce an infinite family of variables t = (t1 , t2 , . . . ) in order to keep track of symmetric peaks of a given weight. For P ∈ D and i ≥ 1, let ωi (P ) be the number of symmetric peaks of weight i in P . Let ω(P ) = (ω1 (P ), ω2 (P ), . . . ), and let tω(P ) = Q ωi (P ) . We are interested in the generating function i≥1 ti Dω (t, x) = X P ∈D tω(P ) x\|P \| . S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 9 P Theorem 3.1. Let P (t, x) = i≥1 ti xi . The generating function for non-decreasing Dyck paths with respect to the weights of their symmetric peaks is Dω (t, x) = 1 − 3x + 2x2 + x3 − (1 − x)3 P (t, x) . (1 − x)(1 − P (t, x))(1 − 2x − (1 − x)2 P (t, x)) Proof. We modify the proof of Theorem 2.1 in order to keep track of the weight of the inserted marked symmetric peaks. Replacing insertion vertices in non-decreasing Dyck paths with sequences of marked pyramids, with variable ui keeping track of marked pyramids of the form X i Y i for each i ≥ 1, corresponds to the substitution q= 1− 1 X ui xi i≥1 in Dτ,ι (1, q, x). A variant of Equation (2.6), where we replace Σ(P ) with the set of symmetric peaks of weight i, shows that the substitutions ui = ti − 1 yield the generating function where ti keeps track of the total number of symmetric peaks of weight i in nondecreasing Dyck paths. It follows that   !   1 1 X Dω (t, x) = Dτ,ι  , x 1,  = Dτ,ι 1, 1 − P (t, x) , x , 1− (ti − 1)xi 1−x i≥1 and the formula is now obtained from Equation (2.5). The symmetric weight of a path P ∈ D isP defined as the sum of the weights of its symmetric peaks, and it is denoted by ω(P ) = i≥1 ωi (P ). From Theorem 3.1, one can easily obtain a generating function for this statistic. Let X Dσ,ω (t, w, x) = tσ(P ) wω(P ) x\|P \| P ∈D be the generating function for non-decreasing Dyck paths with respect to the number of symmetric peaks and the symmetric weight of the path. Corollary 3.2. The generating function Dσ,ω (t, w, x) is equal to (1 − wx) 1 − (3 + w + tw)x + (2 + 3w + 3tw)x2 + (1 − 2w − 3tw)x3 − (1 − t)wx4 . (1 − x) (1 − (t + 1)wx) (1 − (2 + w + tw)x + 2(t + 1)wx2 − twx3 ) Proof. By definition, Dσ,ω (t, w, x) is obtained from Dω (t, x) by making the P substitution ti = twi for all i ≥ 1. When applied to P (t, x), this substitution yields i≥1 twi xi = twx/(1 − wx), and so the formula follows immediately from Theorem 3.1. Corollary 3.3. The generating function for the total symmetric weight in non-decreasing Dyck paths is W (x) := X P ∈D ω(P )x\|P \| = ∂ Dσ,ω (1, w, x) ∂w = w=1 x(1 − 5x + 7x2 − x3 − x4 ) . (1 − x)(1 − 2x)(1 − 3x + x2 )2 10 Ars Math. Contemp. 21 (2021) #P2.04 Comparing this formula with Equation (2.7), we see that W (x) = S(x) . 1−x Taking the coefficients of xn on both sides, and letting wn = total symmetric weight of Dn , we get wn = n X P P ∈Dn ω(P ) denote the sk , (3.1) k=1 that is, the total symmetric weight of paths in Dn equals the total number of symmetric Sn peaks of paths in k=1 Dk . Next we give a bijective proof of this equality.S n The right-hand side of (3.1) can be interpreted as counting paths in k=1 Dk with a distinguished symmetric peak. Indeed, for each k, the number of ways to choose path in Dk and select a symmetric peak of such path equals the total number of symmetric peaks of paths in Dk , namely sk . Similarly, the left-hand side of (3.1) can be interpreted as counting pairs (P̂ , i), where P̂ is a path in Dn with a distinguished symmetric peak, and i is an integer between 1 and the weight of the distinguished peak of P̂ . This is because, for a given path P ∈ Dn , the number of ways to choose a symmetric peak of P and then an integer i between 1 and the weight of that peak equals the sum of the weights of the symmetric peaks of P , which is ω(P ). Let us describe a bijection between the sets counted by both sides of (3.1). Given a path in Dk (for some k ≤ n) with a distinguished symmetric peak, insert a pyramid X n−k Y n−k at the top of the distinguished peak to obtain a pair (P̂ , i), where P̂ is a path in Dn with a distinguished symmetric peak (the same distinguished peak where the pyramid was inserted), and i = n−k. Conversely, given such a pair (P̂ , i), delete the pyramid X i Y i around the distinguished peak, to obtain a path in Dn−i with a distinguished symmetric peak (the same distinguished peak from where the pyramid was removed). 3.2 Recurrence relations and Fibonacci numbers Recall that wn denotes the sum of the symmetric weights of all paths in Dn . Similarly, let wn denote the sum of the asymmetric weights of all paths in Dn . For example, the paths in Figure 2 give w3 = 3 + 0 + 3 + 3 + 3 = 12 and w3 = 2. The next theorem follows immediately by applying Equation (3.1) to Theorem 2.3. Theorem 3.4. The sequence wn satisfies the recurrence relation wn = 3wn−1 − wn−2 + F2n−3 − 2n−2 + 1 for n ≥ 3, with initial values w1 = 1 and w2 = 4. The first few values of the sequence wn for n ≥ 1 are 1, 4, 12, 34, 96, 273, 781, 2240, 6422, 18368, .... From the expression for W (x) in Corollary 3.3, we obtain the following corollary. S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 11 Corollary 3.5. We have wn = F2n + n X (F2`−1 − 2`−1 + 1)F2(n−`) `=1 and wn = 1 (nL2n−1 − F2n ) + 2n − 1. 5 In [5, Theorem 8] the authors prove that the sum of the weights of all peaks in Dn is 2nF2n+1 + (2 − n)F2n . 5 As a direct application of Corollary 3.5, we obtain the following formula for the sum of the asymmetric weights of all paths in Dn . Corollary 3.6. We have wn = 3.3 1 (3F2n + nL2n−2 ) − 2n + 1. 5 Symmetric height The height of a peak is the y-coordinate of the vertex at the top of the peak. Denote by hn the total sum of the heights of all symmetric peaks of paths in Dn . For example, from the paths in Figure 2, we see that h3 = 12. Theorem 3.7. The sequence hn satisfies the recurrence relation hn = 3hn−1 − hn−2 + nL2n−5 + 7F2n−5 − 2n−2 + 1 5 for n ≥ 3, with initial values h1 = 1 and h2 = 4. Proof. We will find the total sum of the heights of all symmetric peaks of paths in Dn = An ∪· Bn by adding the total sum of the heights of all symmetric peaks in An and the total sum of the heights of all symmetric peaks in Bn . Recall that An = ∪·n−1 i=1 Cn,i , and that the first peak of every path in Cn,i is symmetric. From (2.2) we know that every path P ∈ Cn,i is a concatenation of the pyramid ∆i = X i Y i with a path Q ∈ Dn−i . So, the total sum of the heights of all symmetric peaks in P is given by the hight of ∆i (which is equal to i) plus the total sum of the heights of all symmetric peaks in Q. Summing over all paths P ∈ Cn,i , we deduce that the total sum of the heights of all symmetric peaks of Cn,i is i\|Dn−i \|+hn−i = iF2(n−i)−1 + hn−i (using that \|Dn−i \|= F2(n−i)−1 , see (2.4)). Therefore, the total sum of the heights of all symmetric peaks in An is given by n−1 X i=1 hn−i + n−1 X i=1 iF2(n−i)−1 = n−1 X hi + F2n−1 − 1. (3.2) i=1 We now count the sum of the heights of all symmetric peaks in Bn , using the fact that Bn is in bijection with Dn−1 , for which the sum of the heights of all symmetric peaks is hn−1 . The bijection is given by removing the first and the last step of the path. Let 12 Ars Math. Contemp. 21 (2021) #P2.04 us carefully analyze how the sum of the heights of the symmetric peaks is changed by this bijection. On the one hand, removing the first and last step of the path decreases the heights of the peaks by one. On the other hand, for paths in Dn−1 that begin or end with a pyramid at ground level, those pyramids contain a symmetric peak that does not give a symmetric peak in the corresponding path in Bn . To account for these cases, we subtract, from the total sum of heights of symmetric peaks in Dn−1 , the heights of the first and last peaks belonging to pyramids at ground level, and then we add one for each symmetric peak whose height has increased. We recall that the paths in Dn−1 \ {∆n−1 }, whose first pyramid is at ground level have the form ∆i Pn−1−i , where Pn−1−i ∈ Dn−1−i and 1 ≤ i ≤ n − 2. For fixed i, the height of all first pyramids in all such paths is given by i \|Dn−1−i \|= i F2(n−1−i)−1 . So, the total height of all first pyramids at ground level of paths in Dn−1 \ {∆n−1 } is given by n−2 X (n − 1 − i)F2i−1 = F2n−3 − 1. (3.3) i=1 We count the total height of pyramids at ground level that occur at the end of the paths in Dn−1 \ {∆n−1 }. If the last pyramid of a non-decreasing Dyck path is at ground level, then the whole path consists of a sequence of pyramids at ground level. From [13, Corollary 6.3], we deduce that the number of paths in Dn−1 ending with a pyramid ∆i at ground level, for 1 ≤ i ≤ n − 2, is 2(n−1−i)−1 . So, the total height of all last pyramids at ground level of paths in Dn−1 \ {∆n−1 } is given by n−2 X i 2n−i−2 = 2n−1 − n. (3.4) i=1 Now, —to account for the increase by one of peak heights caused by the addition of the initial X and the final Y to paths in Dn−1 — we add the total number of symmetric peaks in Dn−1 , which equals sn−1 (see Theorem 2.4). But this results in some over-counting due to the first and last pyramids at ground level of the paths in Dn−1 , so we have to subtract F2n−4 and 2n−2 − 1. All in all, the term that needs to be added to account for the increase in peak heights is 2F2n−4 + (n − 2)L2n−4 n−2 +2 − F2n−4 − 2n−2 + 1. (3.5) 5 Adding (3.2), hn−1 , and (3.5), and subtracting (3.3) and (3.4), we get the recurrence relation hn = n−1 X hi + F2n−1 − 1 + hn−1 + i=1 2F2n−4 + (n − 2)L2n−4 n−2 n−2 +2 − F2n−4 − 2 + 1 − F2n−3 − 1 + 2n−1 − n . 5 Simplifying, we have that hn = n−1 X i=1 hi + hn−1 + F2n−1 + nL2n−4 + L2n−5 − 2n−1 + n + 1, 5 S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 13 where h1 = 1, and h2 = 4. Now it is easy to see that hn+1 − hn = 2hn − hn−1 + F2n + nL2n−3 + 5F2n−3 − 2n−1 + 1. 5 Therefore, hn = 3hn−1 − hn−2 + nL2n−5 + 7F2n−5 − 2n−2 + 1. 5 The first few values of the sequence hn for n ≥ 1 are 1, 3.4 4, 12, 35, 104, 315, 964, 2957, 9044, 27502, .... Connections with dccp-polyominoes Non-decreasing Dyck paths are in bijection with a family of polyominoes called directed column-convex polyominoes (dccp). A polyomino is directed if each of its cells can be reached from its bottom left-hand corner by a path which is contained in the polyomino and uses only north and east steps. A dccp polyomino is a directed polyomino such that every column consists of contiguous cells [3]. Deutsch and Prodinger [8] give a bijection between the set of non-decreasing Dyck paths of length 2n and the set of dccp of area n, where the area of a polyomino is defined as its number of cells. Figure 3 shows a dccp of area 19. The numbers in the first (second) row represent the final (initial) altitude of each column. 4 0 2 0 4 1 4 1 5 1 4 2 3 2 Figure 3: A direct column-convex polyomino (dccp). The bijection from [8] can be described as follows. Given a dccp whose columns have initial altitudes A = (0, a2 , . . . , ak ) and final altitudes B = (b1 , b2 , . . . , bk ), from left to right, its corresponding non-decreasing Dyck path has valleys at heights (a2 , . . . , ak ), and peaks at heights (b1 , b2 , . . . , bk ), from left to right. For example, the dccp in Figure 3 is mapped to the path in Figure 1. We say that two consecutive columns in a dccp polyomino are at the same level if their initial altitudes are the same. For example, the polyomino in Figure 3 has 4 pairs of consecutive columns at the same level; columns 1 and 2, columns 3 and 4, columns 4 and 5, and columns 6 and 7. Thus, the sequence sn that we introduced in Section 2.2 also counts the total number of pairs of consecutive columns at the same level in all dccp polyominoes 14 Ars Math. Contemp. 21 (2021) #P2.04 of area n. Moreover, if we define the weight of a pair of consecutive columns at the same level as the number of cells in the first of these two columns, then the total weight over all dccp polyominoes of area n is given by wn . 4 Weakly symmetric peaks In this section we consider a variation of symmetric peaks. We recall from Section 1 that a peak is weakly symmetric if the maximal pyramid containing the peak is not preceded by an X. Figure 4 shows different possibilities for the steps preceding and following the maximal pyramid of a weakly symmetric peak. Note that the last configuration in Figure 4 can only occur in the last peak of a path. In Section 2, we gave generating functions to count symmetric and asymmetric peaks in non-decreasing Dyck paths, in this section we also give generating functions to count the number of weakly symmetric peaks. Surprisingly, the generating functions in this section have a simpler construction. We will find formulas, involving Fibonacci numbers, for the total number of weakly symmetric peaks, as well as the sum of their weights, using generating functions and recurrence relations. The results in this section are synthesized using Riordan arrays. Figure 4: Weakly symmetric peaks. 4.1 A generating function for the number of weakly symmetric peaks Let s̃n be the total number of weakly symmetric peaks in Dn . For example, we see from the paths in Figure 2 that s̃3 = 9. The first few values of s̃n for n ≥ 1 are 1, 3, 9, 27, 80, 234, 677, 1941, 5523, 15615, ..., which correspond to sequence A059502 in [23]. Given a non-decreasing Dyck path P , we denote by σ̃(P ) the number of weakly symmetric peaks of P , and recall that \|P \| denotes the semilength of P . We introduce the generating function X Dσ̃ (x, y) = x\|P \| y σ̃(P ) . P ∈D Theorem 4.1. The generating function Dσ̃ (x, y) is given by Dσ̃ (x, y) = (1 − x)xy . 1 − (2 + y)x + yx2 Proof. Recall the decomposition in (2.1). Non-empty paths in Bn can be written as XY or XT 0 Y , where T 0 is a non-decreasing Dyck paths. Paths in An are of the form X∆Y T 00 , where ∆ is a pyramid and T 00 is a non-decreasing Dyck paths. Figure 5 illustrates the three cases. S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 15 Figure 5: Decomposition of a non-decreasing Dyck path. Using the symbolic method, we obtain the relation xy x xy Dσ̃ (x, y) = xy + x(Dσ̃ (x, y) − Dσ̃ (x, y) + Dσ̃ (x, y) ) + Dσ̃ (x, y). 1−x 1−x 1−x \| {z } (a) The term (a) corresponds to the case where T 0 starts with a pyramid, which was symmetric in T 0 but is no longer weakly symmetric in the big path. This completes the proof. Corollary 4.2. The total number of weakly symmetric peaks in Dn satisfies these (i) The generating function for s̃n is given by ∞ X s̃n xn = n=1 (1 − x)(1 − 2x)x . (1 − 3x + x2 )2 (ii) The sequence s̃n satisfies the recurrence relation s̃n = 6s̃n−1 − 11s̃n−2 + 6s̃n−3 − s̃n−4 for n ≥ 5, with initial values s̃1 = 1, s̃2 = 3, s̃3 = 9 and s̃4 = 27. (iii) The sequence s̃n satisfies the recurrence relation s̃n = 3s̃n−1 − s̃n−2 + F2(n−2) for n ≥ 3, with initial values s̃1 = 1 and s̃2 = 3. (iv) For n ≥ 1, we have the convolution s̃n = n−1 X F2`−1 F2(n−`)−1 . `=0 (v) The sequence s̃n satisfies that s̃n = (3F2n + nL2n−2 ) /5. Proof. By Theorem 4.1, ∞ X n=0 s̃n xn = ∂Dσ̃ (x, y) ∂y = y=1 (1 − x)(1 − 2x)x . (1 − 3x + x2 )2 This proves part (i). The recurrence in part (ii) is obtained from this rational generating function. The proof of (iii) is similar to the proof of Theorem 2.3, but in this case we do not subtract the last pyramid at ground level of paths in Bn . 16 Ars Math. Contemp. 21 (2021) #P2.04 To prove part (iv), note that ∞ X s̃n xn = n=1 = (1 − x)x 1 − 3x + x2 1 − 2x 1 − 3x + x2 ∞ X ! ∞ X F2n−1 xn n=1 = ! F2n−1 xn n=0 ∞ X n−1 X n=1 `=0 ! F2`−1 F2(n−`)−1 xn . n Comparing coefficients of x yields the identity. Finally, it is easy to verify that the right side of part (v) satisfies the same recurrence relation as s̃n given in part (2), or alternatively in (3). From Part (v) of Corollary 4.2 and Equation (2.9), we conclude the following. Theorem 4.3. Among all peaks of all non-decreasing Dyck paths, the proportion of those that are weakly symmetric is asymptotically √ s̃n −1 + 5 lim ≈ 0.618034. = n→∞ tn 2 Notice that this coincides with the asymptotic proportion of symmetric peaks given in Theorem 2.6. 4.2 A connection with Riordan arrays In this section we use Riordan arrays to describe the distribution of the number of weakly symmetric peaks in non-decreasing Dyck paths. We start by giving some background on Riordan arrays [22]. We willP say that an infinite column vector (a0 , a1 , . . . )T has generating function f (x) if f (x) = n≥0 an xn , and we index rows and columns starting at 0. A Riordan array is an infinite lower triangular matrix whose kth column has generating function g(x)f (x)k for all k ≥ 0, for some formal power series g(x) and f (x) with g(0) 6= 0, f (0) = 0, and f 0 (0) 6= 0. Such a Riordan array is denoted by (g(x), f (x)). If we multiply this matrix by a column vector (c0 , c1 , . . . )T having generating function h(x), then the resulting column vector has generating function g(x)h(f (x)). This property is known as the fundamental theorem of Riordan arrays, or as the summation property. The product of two Riordan arrays (g(x), f (x)) and (h(x), l(x)) is defined by (g(x), f (x)) ∗ (h(x), l(x)) = (g(x)h(f (x)), l(f (x))) . (4.1) Under this operation, the set of all Riordan arrays is a group [22]. The identity element is I = (1, x), and the inverse of (g(x), f (x)) is (4.2) (g(x), f (x))−1 = 1/ g ◦ f <−1> (x), f <−1> (x) , where f <−1> (x) denotes the compositional inverse of f (x). Let rn,k be the number of paths in Dn with exactly k weakly symmetric peaks, that is, X Dσ̃ (x, y) = rn,k xn y k . n,k≥1 S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 17 Pn By definition, k=1 k rn,k = s̃n . Consider the matrix R = [rn,k ]n,k≥1 . The first few rows of R are  R = [rn,k ]n,k≥1        =       1 1 2 4 8 16 32 64 .. . 0 1 2 5 12 28 64 144 0 0 1 3 9 25 66 168 0 0 0 1 4 14 44 129 .. . 0 0 0 0 1 5 20 70 0 0 0 0 0 1 6 27 0 0 0 0 0 0 1 7 0 0 0 0 0 0 0 1 ···        ···  ,       .. . which correspond to array A105306 in [23]. Even though rows and columns of Riordan arrays are indexed starting at 0, the elements of R are shifted so that the entry in row 0 and column 0 is in fact r1,1 . The goal of this shift is to simplify some of our formulas. Theorem 4.4. The matrix R is a Riordan array given by 1 − x x(1 − x) R= , . 1 − 2x 1 − 2x Proof. Multiplying the right-hand side of the equality by the vector (1, y, y 2 , . . . )T , which 1 has generating function 1−xy , and using the summation property, the resulting vector has bivariate generating function 1 1−x 1 − x x(1 − x) 1 , = x(1 − x) 1 − 2x 1 − 2x 1 − xy 1 − 2x 1− y 1 − 2x 1−x Dσ̃ (x, y) = = , 1 − (2 + y)x + yx2 xy by Theorem 4.1. Theorem 4.5. For n, k ≥ 0, rn+1,k+1 = n X k+1 n−` `=0 ` k (−1)` 2n−k−` . Proof. From the definition of the Riordan array R, we have k 1 − x x(1 − x) 1 − 2x 1 − 2x n−k 1 − x k+1 = x 1 − 2x n XX k+1 k+n−` = xn−k (−1)` 2n−` xn . ` n−` rn+1,k+1 = [xn ] n≥0 `=0 18 Ars Math. Contemp. 21 (2021) #P2.04 Let P = defined by n k n,k≥0 , often called Pascal’s matrix, and let P = [pi,j ] be the matrix ( pi,j = (i+j)/2 j , 0, if i + j ≡ 0 otherwise. (mod 2); It is easy to show that P and P are Riordan arrays given by 1 x 1 x P= , and P = , . 1−x 1−x 1 − x2 1 − x2 Theorem 4.6. The matrix R factors as R = PP. Proof. By Equation (4.1), PP = x 1 , 1−x 1−x    = 1   1−x 1− 1 1 x , 1 − x2 1 − x2  x 1−x  2  , x 1−x 1− x 1−x   2  . Simplifying, PP = 1 − x x(1 − x) , 1 − 2x 1 − 2x = R. From above theorem and the product of matrices we obtain the following combinatorial identities. Theorem 4.7. For n, k ≥ 0, n rn+1,2k+1 = b 2 c X n `+k `=0 2` 2k , n rn+1,2k+2 = b2c X `=0 n 2` + 1 `+k+1 . 2k + 1 Rogers [21], observed that every element not belonging to row 0 or column 0 in a Riordan array can be expressed as a fixed linear combination of the elements in the preceding row. The A-sequence is defined to be the sequence coefficients of this linear combination. Similarly, Merlini et al. [19] introduced the Z-sequence, that characterizes the elements in column 0, except for the top one. Therefore, the A-sequence, the Z-sequence and the upper-left element completely characterize a Riordan array. We summarize this characterization in the following two theorems. Theorem 4.8 ([19]). An infinite lower triangular array F = [dn,k ]n,k≥0 is a Riordan array if and only if d0,0 6= 0 and there exist two sequences (a0 , a1 , a2 , . . . ), with a0 6= 0, and (z0 , z1 , z2 , . . . ) (called the A-sequence and the Z-sequence, respectively), such that dn+1,k+1 = a0 dn,k + a1 dn,k+1 + a2 dn,k+2 + · · · dn+1,0 = z0 dn,0 + z1 dn,1 + z2 dn,2 + · · · for n, k ≥ 0, for n ≥ 0. S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 19 Theorem 4.9 ([18, 19]). Let F = (g(x), f (x)) be a Riordan array with inverse F −1 = (d(x), h(x)). Then the A-sequence and the Z-sequence of F have generating functions A(x) = x , h(x) Z(x) = 1 (1 − d0,0 d(x)) , h(x) respectively. Next we describe the A-sequence and Z-sequence for the Riordan array R. Theorem 4.10. If Cn denotes the n-th Catalan number, then for n, k ≥ 2, rn,k = n X rn−1,k−1+` c` , `=0 where  if n = 0, 1;  1, n+2 cn = (−1) 2 C n−2 , if n ≥ 2 is even; 2   0, otherwise. Moreover, for n ≥ 2 rn,1 = n X rn−1,k−1+` c`+1 , `=0 with initial value r1,1 = 1. Proof. By Equation (4.2), the inverse of the matrix R is given by −1 R = ! √ √ 1 + 2x − 1 + 4x2 1 + 2x − 1 + 4x2 , . 2x 2 Therefore, by Theorem 4.9, the A-sequence and Z-sequence of the Riordan array R have generating functions given by √ √ 1 + 2x + 1 + 4x2 −1 + 2x + 1 + 4x2 A(x) = and Z(x) = . 2 2x We recall that the generating function of the Catalan numbers is given by √ X 1 − 1 − 4x n C(x) = Cn x = . 2x n≥0 P Therefore, A(x) = 1 + x + x2 C(−x2 ) = n≥0 cn xn , where cn is as in the statement of the theorem. Similarly, Z(x) = 1 + xC(−x2 ). The recurrences from Theorem 4.8 now give the desired result. The first few values of the sequence cn for n ≥ 0 are 1, 1, 1, 0, −1, 0, 2, 0, −5, 0, 14, 0, −42, 0, 132, .... 20 Ars Math. Contemp. 21 (2021) #P2.04 So, the recurrence for rn,k starts as rn−1,k−1 + rn−1,k + rn−1,k+1 − rn−1,k+3 + 2rn−1,k+5 − 5rn−1,k+7 + · · · . Next we analyze the central diagonal of the matrix R, that is, the sequence un = r2n+1,n+1 for n ≥ 0 (recall that the entry in row i and column j of R is ri+1,j+1 ). The first few values of un are 1, 2, 9, 44, 225, 1182, 6321, 34232, 187137, 1030490, 5707449, ..., which correspond to the sequence A176479 in [23]. Barry [4] proved that for any Riordan array (g(x), f (x)) = [dn,k ]n,k≥0 the generating function of its central diagonal is given by X v(x)g(v(x)) 0 d2n,n xn = v (x), f (v(x)) n≥0 where v(x) = x2 f (x) <−1> . Therefore, by Theorem 4.4, √ 3 − x + 1 − 6x + x2 √ un x = . 4 1 − 6x + x2 n≥0 X n Other combinatorial interpretations of the sequence un are given in [23]. For example, it counts the number of Dyck paths having exactly n peaks at height 1, n peaks at height 2, and no other peaks. It is also equal to n + 1 times the nth little Schröder number. The little Schröder numbers have several combinatorial interpretations in terms of leaves in plane trees, parenthesizations, and dissections of convex polygons [24]. 4.3 A generating function for total weight Let ω̃(P ) be the sum of the weights of the weakly symmetric peaks of a path P . Define the generating function X Dω̃ (x, y) = x\|P \| y ω̃(P ) . P ∈D Theorem 4.11. The generating function Dω̃ (x, y) is given by Dω̃ (x, y) = (1 − x)2 xy . 1 − 2(1 + y)x + 4yx2 − yx3 Proof. We again use the refinement of the decomposition (2.1) illustrated in Figure 5: every non-empty non-decreasing Dyck path can be written as either XY , XT 0 Y , or X∆Y T 00 , where T 0 and T 00 are non-decreasing Dyck paths and ∆ is a pyramid. It follows that xy x xy xy 2 Dω̃ (x, y) = xy + x(Dω̃ (x, y) − Dω̃ (x, y) + Dω̃ (x, y) − + )) 1 − xy 1−x 1 − xy 1 − xy \| {z } \| {z } (a) (b) xy + Dω̃ (x, y). 1 − xy S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 21 The correction term (a) corresponds to the case where T 0 consists of a pyramid followed by a non-empty path, whereas the term (b) corresponds to the case where T 0 is a pyramid. From Theorem 4.11 we obtain the following corollary, whose proof is similar to that of Corollary 4.2. Let w̃n be the sum of the weights of all weakly symmetric peaks of paths in Dn . Corollary 4.12. The sum of the weights of all weakly symmetric peaks in Dn satisfies the following: (i) The generating function for w̃n is given by ∞ X w̃n xn = n=1 (1 − 2x)x . (1 − 3x + x2 )2 (ii) The sequence w̃n satisfies the recurrence relation w̃n = 6w̃n−1 − 11w̃n−2 + 6w̃n−3 − w̃n−4 for n ≥ 5, with initial values w̃1 = 1, w̃2 = 4, w̃3 = 13 and w̃4 = 40. (iii) For n ≥ 1, we have the convolution w̃n = n X F2`−1 F2(n−`) = `=0 4F2n + nL2n−1 . 5 The first few values of w̃n for n ≥ 1 are 1, 4, 13, 40, 120, 354, 1031, 2972, 8495, 24110, ..., which correspond to the sequence A238846 in [23]. Let qn,k be the number of paths in Dn which have weakly symmetric weight k, that is, X Dω̃ (x, y) = qn,k xn y k . n,k≥1 Pn Notice that k=1 k qn,k = w̃n . Consider the matrix defined by Q = [qn,k ]n,k≥1 . The first few rows of Q are   1 0 0 0 0 0 0 0 ···  0  2 0 0 0 0 0 0    1  0 4 0 0 0 0 0    2  3 0 8 0 0 0 0    4 6 8 0 16 0 0 0 ···  Q = [qn,k ]n,k≥1 =  .  8 13 16 20 0 32 0  0    16 28 37 40 48 0 64 0     32 60 84 98 96 112 0 128    .. .. .. . . . Again, as in the matrix R, the elements of Q are shifted so that the entry in row 0 and column 0 is q1,1 . The proof of our last result is similar to that of Theorem 4.4. Theorem 4.13. The matrix Q is a Riordan array given by 1 − 2x + x2 2x − 4x2 + x3 , . Q= 1 − 2x 1 − 2x 22 Ars Math. Contemp. 21 (2021) #P2.04 ORCID iDs Sergi Elizalde https://orcid.org/0000-0003-4116-2455 Rigoberto Flórez https://orcid.org/0000-0002-3644-9358 José Luis Ramı́rez https://orcid.org/0000-0002-8028-9312 References [1] W. Asakly, Enumerating symmetric and non-symmetric peaks in words, Online J. Anal. Comb. (2018), 7, https://hosted.math.rochester.edu/ojac/articles.html. [2] E. Barcucci, A. Del Lungo, S. Fezzi and R. Pinzani, Nondecreasing Dyck paths and q-Fibonacci numbers, Discrete Math. 170 (1997), doi:10.1016/s0012-365x(97)82778-1. [3] E. Barcucci, R. Pinzani and R. Sprugnoli, Directed column-convex polyominoes by recurrence relations, in: TAPSOFT ’93: theory and practice of software development (Orsay, 1993), Springer, Berlin, volume 668 of Lecture Notes in Comput. Sci., pp. 282–298, 1993, doi:10.1007/3-540-56610-4 71. [4] P. Barry, On the central coefficients of Riordan matrices, J. Integer Seq. 16 (2013), Article 13.5.1, 12pp, https://cs.uwaterloo.ca/journals/JIS/VOL16/Barry1/ barry242.html. [5] E. Czabarka, R. Flórez and L. Junes, Some enumerations on non-decreasing Dyck paths, Electron. J. Combin. 22 (2015), Paper 1.3, 22, doi:10.37236/3941. [6] E. Czabarka, R. Flórez, L. Junes and J. L. Ramı́rez, Enumerations of peaks and valleys on non-decreasing Dyck paths, Discrete Math. 341 (2018), 2789–2807, doi:10.1016/j.disc.2018. 06.032. [7] A. Denise and R. Simion, Two combinatorial statistics on Dyck paths, Discrete Math. 137 (1995), 155–176, doi:10.1016/0012-365x(93)e0147-v. [8] E. Deutsch and H. Prodinger, A bijection between directed column-convex polyominoes and ordered trees of height at most three, Theoret. Comput. Sci. 307 (2003), 319–325, doi: 10.1016/s0304-3975(03)00222-6, random generation of combinatorial objects and bijective combinatorics. [9] S. Elizalde, Measuring symmetry in lattice paths and partitions, Sém. Lothar. Combin. 84B (2020), Art. 26, 12pp, https://www.mat.univie.ac.at/˜slc/. [10] S. Elizalde, The degree of symmetry of lattice paths, Ann. Comb. (2021), doi:10.1007/ s00026-021-00551-6. [11] S. Elizalde, Symmetric peaks and symmetric valleys in Dyck paths, Discrete Math. 344 (2021), 112364, doi:10.1016/j.disc.2021.112364. [12] P. Flajolet and R. Sedgewick, Analytic Combinatorics, Cambridge University Press, Cambridge, 2009. [13] R. Flórez, L. Junes and J. L. Ramı́rez, Enumerating several aspects of non-decreasing Dyck paths, Discrete Math. 342 (2019), 3079–3097, doi:10.1016/j.disc.2019.06.018. [14] R. Flórez, L. Junes and J. L. Ramı́rez, Counting asymmetric weighted pyramids in nondecreasing Dyck paths, Australas. J. Combin. 79 (2021), 123–140, https://ajc.maths. uq.edu.au/?page=get_volumes&volume=79. [15] R. Flórez and J. L. Ramı́rez, Some enumerations on non-decreasing Motzkin paths, Australas. J. Combin. 72 (2018), 138–154, https://ajc.maths.uq.edu.au/?page= get_volumes&volume=72. S. Elizalde et al.: Enumerating symmetric peaks in non-decreasing Dyck paths 23 [16] R. Flórez and J. L. Ramı́rez, Enumerating symmetric and asymmetric peaks in Dyck paths, Discrete Math. 343 (2020), 112118, doi:10.1016/j.disc.2020.112118. [17] R. Flórez and J. L. Ramı́rez, Enumerations of rational non-decreasing Dyck paths with integer slope, Graphs and Combinatorics (2021), doi:10.1007/s00373-021-02392-9. [18] T.-X. He and R. Sprugnoli, Sequence characterization of Riordan arrays, Discrete Math. 309 (2009), 3962–3974, doi:10.1016/j.disc.2008.11.021. [19] D. Merlini, D. G. Rogers, R. Sprugnoli and M. C. Verri, On some alternative characterizations of Riordan arrays, Canad. J. Math. 49 (1997), 301–320, doi:10.4153/cjm-1997-015-x. [20] H. Prodinger, Words, Dyck paths, trees, and bijections, in: Words, semigroups, & transductions, World Sci. Publ., River Edge, NJ, pp. 369–379, 2001, doi:10.1142/9789812810908 0028. [21] D. G. Rogers, Pascal triangles, Catalan numbers and renewal arrays, Discrete Math. 22 (1978), 301–310, doi:10.1016/0012-365x(78)90063-8. [22] L. W. Shapiro, S. Getu, W. Woan and L. Woodson, The Riordan group, Discrete Appl. Math. 34 (1991), 229–239, doi:10.1016/0166-218x(91)90088-e. [23] N. J. A. Sloane, The on-line encyclopedia of integer sequences, http://oeis.org/. [24] R. P. Stanley, Hipparchus, Plutarch, Schröder, and Hough, Amer. Math. Monthly 104 (1997), 344–350, doi:10.1080/00029890.1997.11990645. A Appendix. Notation tables number of such peaks in P total number over Dn vector of peak weights of P sum of peak weights of P total sum of weights over Dn total sum of heights over Dn symmetric σ(P ) sn ω(P ) = (ω1 (P ), . . . ) ω(P ) wn hn type of peaks asymmetric weakly symmetric σ(P ) σ̃(P ) sn s̃n all τ (P ) tn ω̃(P ) w̃n Table 1: Summary of notation for peak statistics. Notation Dn , D An , Bn , Cn,i Page 2 3 Notation ι(P ), ν(P ) S(x) Page 4 5 Notation rn,k qn,k Page 16 21 Table 2: Other notation, along with the page where it is first introduced.
https://openalex.org/W4282813257	https://hal.science/hal-03710230/file/aa43100-22.pdf	English	null	Bulge formation inside quiescent lopsided stellar disks: Connecting accretion, star formation, and morphological transformation in a <i>z</i> ∼ 3 galaxy group	Astronomy & astrophysics	2,022	cc-by	18,484	To cite this version: Boris S. Kalita, Emanuele Daddi, Frederic Bournaud, R. Michael Rich, Francesco Valentino, et al.. Bulge formation inside quiescent lopsided stellar disks: connecting accretion, star formation and mor- phological transformation in a z 3 galaxy group. Astronomy and Astrophysics - A&A, 2022, 666, pp.A44. 10.1051/0004-6361/202243100. hal-03710230 Bulge formation inside quiescent lopsided stellar disks: connecting accretion, star formation and morphological transformation in a z 3 galaxy group Boris S. Kalita, Emanuele Daddi, Frederic Bournaud, R. Michael Rich, Francesco Valentino, Carlos Gómez-Guijarro, Sandrine Codis, Ivan Delvecchio, David Elbaz, Veronica Strazzullo, et al. Distributed under a Creative Commons Attribution 4.0 International License Bulge formation inside quiescent lopsided stellar disks: Connecting accretion, star formation, and morphological transformation in a z ∼3 galaxy group Boris S. Kalita1 , Emanuele Daddi1 , Frederic Bournaud1, Robert Michael Rich2, Francesco Valentino3,4 , Carlos Gómez-Guijarro1 , Sandrine Codis1, Ivan Delvecchio5 , David Elbaz1, Veronica Strazzullo6,7,8 , Victor de Souza Magalhaes9 , Jérôme Pety9, and Qinghua Tan10,1 Boris S. Kalita1 , Emanuele Daddi1 , Frederic Bournaud1, Robert Michael Rich2, Francesco Valentino3,4 , Carlos Gómez-Guijarro1 , Sandrine Codis1, Ivan Delvecchio5 , David Elbaz1, Veronica Strazzullo6,7,8 , Victor de Souza Magalhaes9 , Jérôme Pety9, and Qinghua Tan10,1 1 CEA, Irfu, DAp, AIM, Université Paris-Saclay, Université de Paris, CNRS, 91191 Gif-sur-Yvette, France e-mail: boris.kalita@cea.fr 2 Department of Physics & Astronomy, University of California Los Angeles, 430 Portola Plaza, Los Angeles, CA 90 Cosmic Dawn Center (DAWN), Denmark 4 2 Department of Physics & Astronomy, University of California Los Angeles, 430 Portola Plaza, Los Angeles, CA 90095, USA 3 Cosmic Dawn Center (DAWN), Denmark 4 i l h i i i f h j h k 4 Niels Bohr Institute, University of Copenhagen, Jagtvej 128, 2200 Copenhagen N, Denmark 5 els Bohr Institute, University of Copenhagen, Jagtvej 128, 2200 Copenhagen N, Denmark 6 University of Trieste, Piazzale Europa, 1, 34127 Trieste, TS, Italy 6 University of Trieste, Piazzale Europa, 1, 34127 Trieste, TS, Italy INAF – Osservatorio Astronomico di Brera, Via Brera 28, 20121 Milano, Italy 7 INAF – Osservatorio Astronomico di Brera, Via Brera 28, 20121 Milano, Italy 8 8 INAF – Osservatorio Astronomico di Trieste, Via Tiepolo 11, 34131 Trieste, Italy INAF – Osservatorio Astronomico di Trieste, Via Tiepolo 11, 34131 Trieste, Italy 9 Institut de Radioastronomie Millimétrique, 300 Rue de la Piscine, 38406 Saint-Martin d’Hères, France 10 9 Institut de Radioastronomie Millimétrique, 300 Rue de la Piscine, 38406 Saint-Martin d’Hères, France 10 10 Purple Mountain Observatory & Key Laboratory for Radio Astronomy, Chinese Academy of Sciences, 10 Yuanh Nanjing 210023, PR China Received 12 January 2022 / Accepted 10 June 2022 Received 12 January 2022 / Accepted 10 June 2022 Received 12 January 2022 / Accepted 10 June 2022 Received 12 January 2022 / Accepted 10 June 2022 ABSTRACT We present well-resolved near-IR and submillimeter analysis of the three highly star-forming massive (>1011 M⊙) galaxies within the core of the RO-1001 galaxy group at z = 2.91. Each of them displays kpc scale compact starbursting cores with properties consistent with forming galaxy bulges, embedded at the center of extended, massive stellar disks. Surprisingly, the stellar disks are unambiguously both quiescent and severely lopsided. Therefore, “outside-in” quenching is ongoing in the three group galaxies. We propose an overall scenario in which the strong mass lopsidedness in the disks (ranging from factors of 1.6 to >3) likely generated under the eﬀects of accreted gas and clumps, is responsible for their star-formation suppression, while funnelling gas into the nuclei and thus creating the central starbursts. The lopsided side of the disks marks the location of impact of accretion streams, with additional matter components (dust and stars) detected in their close proximity directly tracing the inﬂow direction. The interaction with the accreted clumps, which can be regarded as minor mergers, leads the major axes of the three galaxies to be closely aligned with the outer Lyman-α-emitting feeding ﬁlaments. These results provide the ﬁrst piece of observational evidence of the impact of cold accretion streams on the formation and evolution of the galaxies they feed. In the current phase, this is taking the form of the rapid buildup of bulges under the eﬀects of accretion, while still preserving massive quiescent and lopsided stellar disks at least until encountering a violent major merger. Key words. galaxies: high-redshift – galaxies: evolution – submillimeter: galaxies – galaxies: groups: individual: RO-1001 – galaxies: star formation – galaxies: structure HAL Id: hal-03710230 https://hal.science/hal-03710230v1 Submitted on 12 Apr 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 4.0 International License Astronomy & Astrophysics Astronomy & Astrophysics A&A 666, A44 (2022) https://doi.org/10.1051/0004-6361/202243100 c⃝B. S. Kalita et al. 2022 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is published in open access under the Subscribe-to-Open model. Subscribe to A&A to support open access publication. A44, page 1 of 16 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is published in open access under the Subscribe-to-Open model. Subscribe to A&A to support open access publication. A4 1. Introduction On one hand, simulations predict that smooth-gas-accretion driven vio- lent disk instabilities result in the gas being driven to the core of secularly evolving disk-like galaxies, once the galaxy mass reaches ∼109.5 M⊙and thereby creating most of the stellar mass in a compact spheroidal structure (wet compaction; Dekel et al. 2013; Zolotov et al. 2015; Tacchella et al. 2016). Whereas obser- vational studies of large statistical samples advocate the rapid eﬀects of major mergers between galaxies leading to the for- mation of such objects (Cimatti et al. 2008; Ricciardelli et al. 2010; Fu et al. 2013; Ivison et al. 2013; Toft et al. 2014, 2017; Elbaz et al. 2018; Gómez-Guijarro et al. 2018). Both scenarios have its issues however. Wet compaction predicts the presence of high-gas fractions in cSFGs that is simply not observed (Puglisi et al. 2021; Gómez-Guijarro et al. 2022a,b). Whereas, a dominance of the merger-driven starbursts (pushing them a factor of 3−4 above the main sequence) would suggest that the galaxy simply transitions through the star-forming main- sequence on its way to becoming quiescent, making the tight correlation between the SFR and stellar masses diﬃcult to repro- duce. Although, Elbaz et al. (2018) does provide an tentative solution through a subpopulation of star-bursts hidden within the main-sequence. It needs to be noted that whenever we refer to accretion in this work, we are including both smooth gas as well as clumpy material. The latter is expected to make up ∼one-third of the accreted matter (Dekel et al. 2009a) and its interaction with the galaxies could be observationally characterized as minor merg- ers of varying mass ratios. In this paper, we discuss the obser- vational data in Sect. 2, followed by the analysis and results (Sect. 3). We then discuss galaxy morphology, star formation and their link to accretion in Sect. 4. Finally, we provide the conclusions in Sect. 8. Throughout, we adopt a concordance ΛCDM cosmology, characterized by Ωm = 0.3, ΩΛ = 0.7, and H0 = 70 km s−1 Mpc−1. We use a Chabrier initial mass function. Magnitudes and colors are on the AB scale. All uncertainties on measured parameters that have been quoted indicate the 90% conﬁdence interval determined from the limits of ∆χ2 = 2.71. All images are oriented such that north is up and east is left. q The morphology of cSFGs could hold clues to disentan- gling the processes. 1. Introduction Besides their characteristic submillime- ter bright highly star-forming cores, the surrounding stellar regions can also provide valuable information, which is only possible through near-IR follow-ups of ALMA (submillime- ter) detected cSFGs. In the wet compaction scenario, once the galaxy enters its peak compaction phase at ∼109.5 M⊙, the stel- lar disk (or a proto-disk) stays constant or shrinks while the core rapidly builds up mass (Tacchella et al. 2016). Whereas in case of major mergers between massive galaxies, clear signs of disturbed morphology, especially in the form of clumpy stel- lar structures (at least during the initial phases) is anticipated (Lotz et al. 2006; Bournaud et al. 2011; Rujopakarn et al. 2019; Calabrò et al. 2019) and extended tidal-tails (e.g., Bridge et al. 2010; Wen & Zheng 2016; Guo et al. 2016). Attempts at char- acterizing the stellar regions are already underway (e.g., Puglisi et al. 2021; Gómez-Guijarro et al. 2022a), with the narrative swaying in favour of mergers (Elbaz et al. 2018; Rujopakarn et al. 2019). Nevertheless, it is imperative to deter- mine whether this is a universal trend, especially in regions where galaxies are expected to grow under the inﬂuence of high levels of accretion, which might be more conducive for the wet compaction scenario. 1 https://github.com/gbrammer/grizli 2 http://www.iram.fr/IRAMFR/GILDAS 1. Introduction On one hand, simulations predict that smooth-gas-accretion driven vio- lent disk instabilities result in the gas being driven to the core of secularly evolving disk-like galaxies, once the galaxy mass reaches ∼109.5 M⊙and thereby creating most of the stellar mass in a compact spheroidal structure (wet compaction; Dekel et al. 2013; Zolotov et al. 2015; Tacchella et al. 2016). Whereas obser- vational studies of large statistical samples advocate the rapid eﬀects of major mergers between galaxies leading to the for- mation of such objects (Cimatti et al. 2008; Ricciardelli et al. 2010; Fu et al. 2013; Ivison et al. 2013; Toft et al. 2014, 2017; Elbaz et al. 2018; Gómez-Guijarro et al. 2018). Both scenarios have its issues however. Wet compaction predicts the presence of high-gas fractions in cSFGs that is simply not observed (Puglisi et al. 2021; Gómez-Guijarro et al. 2022a,b). Whereas, a dominance of the merger-driven starbursts (pushing them a factor of 3−4 above the main sequence) would suggest that the galaxy simply transitions through the star-forming main- sequence on its way to becoming quiescent, making the tight correlation between the SFR and stellar masses diﬃcult to repro- duce. Although, Elbaz et al. (2018) does provide an tentative solution through a subpopulation of star-bursts hidden within the main-sequence. on one of these cases, we investigate the galaxy-group RO-1001 at z = 2.91 presented in Daddi et al. (2021). The structure hosts three massive (>1011 M⊙) star-forming galaxies in its inner core (along with an additional quiescent galaxy; Kalita et al. 2021a), at the center of its ∼4 × 1013 M⊙dark-matter halo potential well. They are all <70 kpc (9′′) from the peak of the giant 300 kpc- wide Lyman-α halo centered on the group, and spectroscopically coincident with the intensity averaged Lyman-α emission within ±500 km s−1 (Fig. 1). Finally, the Lyman-α contours provide tentative identiﬁcation of accretion-streams that are expected to feed the central halo and hence the three star-forming galaxies within. the compact star-forming core that is expected to form the spheroidal bulge characteristic of the compact QGs. When gas is driven into the central compact region, the increase in the gas surface density results in a rise in star-formation that rapidly builds up the stellar mass of the bulge. However, the mode of formation of these cSFGs has been a matter of debate. 1. Introduction pliﬁed by the tight correlation between their star formation rates (SFRs) and the stellar mass at least up to z ∼4−6 (the star-forming main-sequence; Daddi et al. 2007; Elbaz et al. 2007; Whitaker et al. 2012; Speagle et al. 2014; Schreiber et al. 2015). Morphologically, this phase is expected to feature sec- ularly growing disks (Daddi et al. 2010a; Tacconi et al. 2010; Dekel & Krumholz 2013; Feldmann & Mayer 2015). The formation of massive galaxies (>1011 M⊙) that happened within the ﬁrst ∼3 Gyr after the Big Bang and was sustained by cold streams of gas (Birnboim & Dekel 2003; Kereš et al. 2005) is still not fully understood (Somerville & Davé 2015; Vogelsberger et al. 2020). Cold gas accretion, which includes smooth gas as well as clumpy material, is expected to be ubiquitous in high redshift dense environments (Dekel et al. 2009a). Encouraging observational evidence for cold streams was recently obtained for a sample of galaxy groups and clus- ters at 2.0 < z < 3.3 and for statistical samples of mas- sive galaxies (Daddi et al. 2022a,b). Yet there remains a paucity of direct observations of how this critical process aﬀects indi- vidual galaxies. In general, the need of gas accretion fueling the star formation in galaxies is abundantly clear and exem- Meanwhile, to accomodate for the existence of compact (∼1 kpc), spheroidal, quiescent galaxies (QGs) observed at z ∼ 2−4 (e.g., Valentino et al. 2020a; Lustig et al. 2021; D’Eugenio et al. 2021) in contrast to the relatively more extended star-forming disk galaxies (van der Wel et al. 2014), an intermediate population of compact star-forming galaxies (cSFGs) has been proposed and widely observed (Barro et al. 2013, 2014; van Dokkum et al. 2015; Puglisi et al. 2019, 2021). The feature at the heart of this hypothesis, quite literally, is A&A 666, A44 (2022) the compact star-forming core that is expected to form the spheroidal bulge characteristic of the compact QGs. When gas is driven into the central compact region, the increase in the gas surface density results in a rise in star-formation that rapidly builds up the stellar mass of the bulge. However, the mode of formation of these cSFGs has been a matter of debate. 2. Observational data To study the three star-forming galaxies (A, B and C) in RO- 1001 (Fig. 1), we make use of HST-WFC3 imaging in 3 bands (F160W, F125W and F606W) over a total of 11 orbits during Cycle 27 (Proposal ID: 15190, PI: E. Daddi). The data reduction was executed using the pipeline grizli1. The 5σ point-source sen- sitivities reached are 26.25 (F160W), 26.47 (F125W) and 26.39 (F606W) with a pixel scale of 0.06′′ and a half-power beam- width of 0.24′′ for F160W. Public COSMOS F814W imaging (Scoville et al. 2007) was also incorporated into the analysis. Furthermore, we used the Ks-band image from data release 4 of Ultra-VISTA (McCracken et al. 2012). Finally, IRAC 3.6 µm and 4.5 µm images were taken from the Spitzer Matching Survey of the Ultra-VISTA Deep Stripes (SMUVS; Ashby et al. 2018), while those at 5.7 µm and 7.9 µm are obtained from the public COSMOS database (Laigle et al. 2016). New Atacama Large Millimetre Array (ALMA) band 7 observations, taken in Cycle 7 (Project ID: 2019.1.00399.S, PI: R.M. Rich) provide the submillimeter information of our sources. The data reduction was carried out using the Common Astronomy Software Application (CASA) and a maximum sen- sitivity of 28 µJy beam−1 was reached, with a synthesized beam size of 0.49′′×0.46′′. Finally, we utilize NOrthern Extended Mil- limter Array (NOEMA) spectroscopy data to map the CO[3−2] transition in each of the three galaxies. The synthesized beam size at 88.3 GHz is 4.0′′ × 1.8′′. The spectra creation and analy- sis is carried out using a GILDAS-based pipeline2, and has been already discussed in a previous work (Daddi et al. 2021). Therefore, to attempt a resolution, one will have to speciﬁ- cally investigate high-redshift massive dark-matter halos which are expected to allow the penetration and survival of cold-gas streams into a hot halo (Kereš et al. 2005; Dekel et al. 2009a). This is primarily driven by the expected evolution with halo mass (∝MDM) and redshift (∝(1 + z)α with α ≈2.25−2.50). Studying cSFG counterparts within such accretion-rich environ- ments is our best bet at disentangling the eﬀects of accretion and mergers. However, observationally establishing the pres- ence of infalling cold-gas streams in halos has proved to be extremely challenging, with only a handful of convincing cases yet (using Ly-α emission as a tracer; Martin et al. 2015, 2019; Umehata et al. 2019; Daddi et al. 2021, 2022a,b). 2. Observational data Following up 1 https://github.com/gbrammer/grizli 2 http://www.iram.fr/IRAMFR/GILDAS A44, page 2 of 16 B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks Fig. 1. Location of the massive star-forming galaxies in the network of Lyman-α ﬁlaments. Left: color image composite of RO-1001, using observations in F160W, F125W and F606W. The ALMA data has been coadded to that in F606W since both trace star-formation, diﬀering based on dust obscuration. The scaling of the ALMA data was done based on its ﬂux to SFR ratio in comparison to the same for that in F606W. The white dashed contours trace the Lyman-α halo whereas the cyan arrows show the tentative directions of the three gas-accretion ﬁlaments converging onto the center of the group potential well (the correspondence to the galaxies is discussed in detail in Sect. 5.2). Right: zoom on the three star-forming galaxies (A, B and C) as seen in ALMA submillimeter dust-emission while the white contours trace the F160W ﬂux tracing stellar light. The contours begin at 4σ with increments of 4σ. The arrows indicate the direction of the streams, which are shown in green if they are aligned with the major axis of the galactic disks (in dashed white lines), as discussed in Sect. 5.2. Those which are not, are in red. In case of Galaxy-A, we rather show the major axis of the ALMA contour with the white dashed line. Fig. 1. Location of the massive star-forming galaxies in the network of Lyman-α ﬁlaments. Left: color image composite of RO-1001, using observations in F160W, F125W and F606W. The ALMA data has been coadded to that in F606W since both trace star-formation, diﬀering based on dust obscuration. The scaling of the ALMA data was done based on its ﬂux to SFR ratio in comparison to the same for that in F606W. The white dashed contours trace the Lyman-α halo whereas the cyan arrows show the tentative directions of the three gas-accretion ﬁlaments converging onto the center of the group potential well (the correspondence to the galaxies is discussed in detail in Sect. 5.2). Right: zoom on the three star-forming galaxies (A, B and C) as seen in ALMA submillimeter dust-emission while the white contours trace the F160W ﬂux tracing stellar light. The contours begin at 4σ with increments of 4σ. 3. Analysis the images in all other observed wavelength windows to ascer- tain consistency. We ﬁnd no >1σ ﬂuctuations in the respective residual images. We ﬁnd that the two Sérsic proﬁles for Galaxy- B are clearly not spatially coincident with the submillimeter bright compact core (discussed below in Sect. 3.2), but rather fall ∼0.8′′ on either side of it (and hence with a spatial sep- aration of ∼0.16′′ between the two Sérsic proﬁle centers). We also ﬁnd remarkable consistency of the axes ratio and position angles of the two Sérsic proﬁles, hence likely part of a continu- ous stellar structure. We repeat this procedure for Galaxies A and C. Galaxy-A is found to have a complex morphology which is ﬁt with a double Sérsic in diﬀering (almost orthogonal) orienta- tions. Galaxy-C on the other hand is found to resemble Galaxy- B in terms of an accordance with two coaligned Sérsic proﬁles with low indices (<1) on either side of submillimeter core. But in this case the separation is ∼0.2′′, making it more compact than Galaxy-B. We use the combination of the data listed above (Sect. 2) to derive the properties of the three star-forming galaxies in RO-1001. Each of them show extremely compact submillime- ter bright rotating cores primarily centered on the much more extended (4−9×) near-IR detected stellar components (Fig. 1, right panel), with both being coplanar in at least two-thirds of the cases (Galaxies B and C). The stellar regions also show marked lopsidedness in their emission. We investigate in detail each of these aspects of the galaxies in this section. Given that Galaxy-B is the most extended and hence best resolved, we always begin with the description of its analysis for simplicity. We then pro- ceed to Galaxies A and C, where the same prescription as that for Galaxy-B is used. 2. Observational data The arrows indicate the direction of the streams, which are shown in green if they are aligned with the major axis of the galactic disks (in dashed white lines), as discussed in Sect. 5.2. Those which are not, are in red. In case of Galaxy-A, we rather show the major axis of the ALMA contour with the white dashed line. 3.1. Near-IR surface brightness ﬁtting To measure integrated eﬀective radii (re) of each of the galax- ies, we did an additional round of ﬁtting using single Sérsic pro- ﬁles. Besides using the generic version of the Sérsic proﬁle, we also invoke the m = 1 Fourier component to account for the lopsidedness of the stellar components. This is due to quantify the emission asymmetry we observe in each of the cases which is later discussed in Sect. 5.1. We also measure growth curves using circular apertures with their centers ﬁxed at the ALMA core of the galaxies to independently determine the eﬀective radius. For Galaxy-A, we only use the northern component of its complex near-IR double emission, which we determine as the primary We investigate the distribution of rest-frame optical emission in Galaxy-B by model-ﬁtting (Fig. 2) the intensity proﬁles using the software GALFIT (Peng et al. 2002, 2010). The H-band (F160W) image is used since it has the highest signal-to-noise among the available bands. We ﬁnd that ﬁtting and subtracting a double Sérsic proﬁle (with indices ∼0.5) gives the best result, assessed by the maximum ﬂuctuations in the residual image (found to be <1σ). Care is taken to simultaneously subtract all nearby sources within ∼8′′. We also ﬁt the same proﬁle, con- volved with the appropriate point-source-functions (PSFs), to A44, page 3 of 16 A44, page 3 of 16 A&A 666, A44 (2022) Fig. 2. Morphological proﬁle ﬁtting of stellar emission. Left panel: HST F160W cutouts of Galaxies-A and B (top) and Galaxy-C (bottom) used for the morphological model ﬁtting. Middle and right panels: GALFIT returned models and residuals of the same cutouts. In the latter, only the RO-1001 galaxy models have been subtracted. The rest of the sources, although used during the ﬁt, have been left in the image. Fig. 2. Morphological proﬁle ﬁtting of stellar emission. Left panel: HST F160W cutouts of Galaxies-A and B (top) and Galaxy-C (bottom) used for the morphological model ﬁtting. Middle and right panels: GALFIT returned models and residuals of the same cutouts. In the latter, only the RO-1001 galaxy models have been subtracted. The rest of the sources, although used during the ﬁt, have been left in the image. phase center. Finally, the pointings are combined into a single measurement set which provides the maximum sensitivity avail- able with our data. stellar region due to its proximity to the submillimeter emission. 3.1. Near-IR surface brightness ﬁtting We use the average of all three methods that are consistent within 15−20%, to obtain the ﬁnal measurements reported in Table 1: 3±1 kpc, 9±2 kpc and 4±1 kpc for Galaxies-A, B and C respec- tively. It is noteworthy that for each galaxy, the single proﬁles are characterized by Sérsic indices <1, indicating disk-like morphol- ogy (and hence we refer to them as disks throughout this work, a conclusion which will be reinforced in Sect. 4.2). The elliptici- ties measured for the three galaxies are 0.5±0.1, 0.15±0.05 and 0.5 ± 0.2 (A, B and C), indicating an almost edge-on orientation for Galaxy-B. stellar region due to its proximity to the submillimeter emission. We use the average of all three methods that are consistent within 15−20%, to obtain the ﬁnal measurements reported in Table 1: 3±1 kpc, 9±2 kpc and 4±1 kpc for Galaxies-A, B and C respec- tively. It is noteworthy that for each galaxy, the single proﬁles are characterized by Sérsic indices <1, indicating disk-like morphol- ogy (and hence we refer to them as disks throughout this work, a conclusion which will be reinforced in Sect. 4.2). The elliptici- ties measured for the three galaxies are 0.5±0.1, 0.15±0.05 and 0.5 ± 0.2 (A, B and C), indicating an almost edge-on orientation for Galaxy-B. Since the Sérsic proﬁle, commonly used to study galaxy mor- phologies, cannot be easily extended to the UV space due to its Fouriertransformnotbeinganalyticallyexpressible,weuseasub- stitute. We exploit the Spergel proﬁle (Spergel 2010), recently incorporated within the MAPPING procedure of GILDAS. This hasbeenfoundtocorrelatewellwiththeSérsicproﬁleallowingus to extract Sérsic parameters corresponding to the Spergel param- eters we determine. To also determine the robustness of the ﬁt- ting procedure (Fig. 3; Table 2), we perturb the initial parameters within a factor of 5 over a total of ∼200 times and repeat the ﬁt- ting. We also artiﬁcially inject 1000 times the best ﬁtting model of eachgalaxyatdiﬀerentemptylocations(oneatatime)andﬁtthem individually to estimate reliable error for our measurements based on the a-posteriori dispersion of resulting parameters. This proce- dure also shows that there are no detectable systematic biases in the measured quantities. A44, page 4 of 16 3.2. Submillimeter emission analysis (2) Derived from the 870 µm ﬂux of individual galaxies assuming the same SED shape as for their coaddition (Daddi et al. 2021). (3) This high upper-limit from the composite-τ model is in agreement with a constant star formation model being within 90% conﬁdence interval. (4) Full Width at Zero Velocity as previously reported (Daddi et al. 2021). (5) Dynamical mass primarily associated with the core, rather than the whole galaxy as described in the main text. (∗∗)3σ upper-limits. Table 1. Properties of the star-forming massive galaxies in RO-1001. Notes. (1) Estimated from the composite-τ model ﬁtting to optical and near-IR photometry and therefore primarily associated with the disks. (2) Derived from the 870 µm ﬂux of individual galaxies assuming the same SED shape as for their coaddition (Daddi et al. 2021). (3) This high upper-limit from the composite-τ model is in agreement with a constant star formation model being within 90% conﬁdence interval. (4) Full Width at Zero Velocity as previously reported (Daddi et al. 2021). (5) Dynamical mass primarily associated with the core, rather than the whole galaxy as described in the main text. (∗∗)3σ upper-limits. Notes. (1) Estimated from the composite-τ model ﬁtting to optical and near-IR photometry and therefore primarily associated with the disks. (2) Derived from the 870 µm ﬂux of individual galaxies assuming the same SED shape as for their coaddition (Daddi et al. 2021). (3) This high upper-limit from the composite-τ model is in agreement with a constant star formation model being within 90% conﬁdence interval. (4) Full Width at Zero Velocity as previously reported (Daddi et al. 2021). (5) Dynamical mass primarily associated with the core, rather than the whole galaxy as described in the main text. (∗∗)3σ upper-limits. Table 2. Derived morphological Spergel parameters in submillimeter. Table 2. Derived morphological Spergel parameters in submillimeter. Table 2. Derived morphological Spergel parameters in submillimeter. Table 2. Derived morphological Spergel parameters in submillimeter. ID A B C Eﬀective radiusmajor (∗∗) arcsec 0.092 ± 0.004 0.148 ± 0.002 0.122 ± 0.054 kpc 0.72 ± 0.02 1.15 ± 0.01 0.95 ± 0.42 Ellipticity 0.62 ± 0.03 0.40 ± 0.01 0.47 ± 0.22 Position angle degrees −90.6 ± 0.6 −27.4 ± 0.3 −46.4 ± 0.6 ∆angledisk,bulge (1) degrees – 3.4 ± 1.5 2.9 ± 1.5 Spergel index (2) −0.6 ± 0.2 0.5 ± 0.3 −0.5 ± 0.3 Notes. 3.2. Submillimeter emission analysis The compact highly star-forming regions of the galaxies are detected thanks to their dust emission using ALMA at 870 µm. It is noteworthy that due to a well characterized PSF and high signal-to-noise, we have the ability to map structures much smaller than the integrated ALMA PSF (Rujopakarn et al. 2019). We begin with a detailed morphological analysis of the emission region in Galaxy-B. Due to the data being distributed over mul- tiple partially overlapping pointings, we combine the ones with Galaxy-B within their primary beam. To do so, we follow the procedure outlined in Gómez-Guijarro et al. (2022a), for combi- nation and stacking of individual pointings in ALMA. We phase shift each of them to set the source coordinates at their respective For Galaxy-B, we obtain an almost-perfect exponential pro- ﬁle characteristic of a disk of eﬀective radius ∼1.1 kpc, with a Spergel index of 0.55 ± 0.12 (Sérsic index ∼1) and an ellipticity of 0.40 ± 0.01. From the model hence returned, we measure the 870 µm observed-frame ﬂux. This is converted to a total-infrared luminosity (LIR; 8−1000 µm) using the integrated SED from the coadded photometry of RO-1001 (Daddi et al. 2021). This can then be converted to a SFR of 674±106 M⊙yr−1 using the widely A44, page 4 of 16 B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks Table 1. Properties of the star-forming massive galaxies in RO-1001. ID A B C RA 10:01:23.174 10:01:22.964 10:01:22.369 Dec 02:20:05.57 02:20:05.87 02:20:02.63 zspec 2.9214 2.9156 2.9064 log M⋆(1) M⊙ 11.50+0.15 −0.19 11.20+0.07 −0.14 11.26+0.14 −0.10 S ν(870 µm) mJy 4.44 ± 0.05 8.69 ± 0.03 4.04 ± 0.11 SFRcore (2) M⊙yr−1 345 ± 55 674 ± 106 313 ± 50 SFRdisk_ALMA (2) M⊙yr−1 <60 (∗∗) <19 (∗∗) <43 (∗∗) SFRdisk_SED (1) M⊙yr−1 66+446 −49 (3) 42+51 −37 94+66 −94 t50 (1) Gyr 1.7+0.3 −0.7 0.5+0.7 −0.2 0.2+1.3 −0.1 re_disk kpc 3 ± 1 9 ± 2 4 ± 1 rdisk/rcore 4 ± 1 9 ± 2 5 ± 1 log Mmol M⊙ 9.8 10.7 10.6 FWZVCO[3−2] (4) km s−1 381 1114 1098 log Mdyn,tot (5) M⊙ 10.4 11.1 11.2 AV 1.82.1 1.3 1.01.2 0.4 1.61.8 0.4 Notes. (1) Estimated from the composite-τ model ﬁtting to optical and near-IR photometry and therefore primarily associated with the disks. adoptedconversionrelation(Kennicutt1998;Daddi et al.2010b): 3.3. Determining the quiescence of the stellar regions adoptedconversionrelation(Kennicutt1998;Daddi et al.2010b): 3.3. Determining the quiescence of the stellar regions Besides the measurements for the submillimeter bright compact cores, the high resolution 870 µm map (Sect. 2) also provides us with constraints on the levels of star formation in the surround- ing regions. We are mainly interested in the estimates for the extended stellar regions detected in near-IR, that lack any sub- millimeter emission. Using the H-band models as ﬁxed priors, we measure 3σ upper-limits for each of the galaxies by simulta- neously ﬁtting them along with the primary proﬁles for the cores in the ALMA data (uv plane). For Galaxy-B, we estimate a 3σ ﬂux upper-limit of 0.25 mJy (corresponding to an SFR upper- limit of 19 M⊙yr−1) for the stellar disk, while we obtain 0.77 mJy and 0.55 mJy (60 M⊙yr−1 and 43 M⊙yr−1) for Galaxies A and C respectively. This provides clear evidence of almost all star formation being concentrated at the core leaving the massive stellar disks devoid of it and well below the star-forming main- sequence (Fig. 4). The error for this measurement is determined from the 1σ dis- persion in the dust-temperature dependent parameter ⟨U⟩, that is usually invoked during the conversion of the 870 µm ﬂux to LIR. We use this to account for variations in dust temperature of individual galaxies with respect to the average value. This approach of evaluating SFR uncertainties is over-conservative, as it does not take into account that the integrated bolometric IR luminosity (hence SFR) of the 3 galaxies that is known to better than 10% accuracy, given the well sampled IR SED including Herschel (Daddi et al. 2021). In Galaxy-A, we obtain a much diﬀerent Spergel index (−0.61±0.04) that translates to a Sérsic index ∼4. The situation is similarinGalaxy-CwithaSpergelindexof−0.48±0.12,although it has an additional point source adjacent to it (at 0.14′′). The sizes of these proﬁles are very compact with eﬀective radii ∼0.7 kpc and 0.9 kpc. We measure the SFR using the same prescription as that for Galaxy-B, and estimate values of 345 ± 55 M⊙yr−1 and 313 ± 50 M⊙yr−1 for Galaxies A and C respectively. We note that the estimation of the star formation upper lim- its in the stellar disks should be considered as overly conserva- tive. 3.2. Submillimeter emission analysis (1) The diﬀerence between position angles of the stellar disk in HST F160W and that of the submillimeter core. The lack of a value in case of Galaxy-A is due to its stellar emission having a complex morphology without a clear position angle for the whole galaxy. (2) Spergel index values 0.5 and −0.6 correspond to Sérsic indices 1 and 4 respectively. (∗∗) ∼eﬀective radius of the corresponding Sérsic proﬁle, as has been determined through modeling Sérsic and Spergel proﬁles (Tan et al., in prep.). Notes. (1) The diﬀerence between position angles of the stellar disk in HST F160W and that of the submillimeter core. The lack of a value in case of Galaxy-A is due to its stellar emission having a complex morphology without a clear position angle for the whole galaxy. (2) Spergel index values 0.5 and −0.6 correspond to Sérsic indices 1 and 4 respectively. (∗∗) ∼eﬀective radius of the corresponding Sérsic proﬁle, as has been determined through modeling Sérsic and Spergel proﬁles (Tan et al., in prep.). adoptedconversionrelation(Kennicutt1998;Daddi et al.2010b): 3.3. Determining the quiescence of the stellar regions adoptedconversionrelation(Kennicutt1998;Daddi et al.2010b): 3.3. Determining the quiescence of the stellar regions Components of the three galaxies on the star-forming main- sequence: star-bursting bulges and quiescent disks. The SFR vs. stellar- mass values of the star-bursting core, the quiescent stellar disk and the combination of the two placed with respect to the star-forming main- sequence (Schreiber et al. 2015) at z = 3, shown as the black solid line. The shaded region demarcates the 0.3 dex uncertainty in the relation. We use the 3σ ALMA upper-limits as the value for SFR with the asso- ciated gray error-bars showing the range allowed by our SED model ﬁtting.The latter have been artiﬁcially oﬀset in the x-axis to emphasize the diﬀerence in the method of measurement. g y We use the 3σ ALMA upper-limits as the value for SFR with the asso- ciated gray error-bars showing the range allowed by our SED model ﬁtting.The latter have been artiﬁcially oﬀset in the x-axis to emphasize the diﬀerence in the method of measurement. Fig. 3. Top panel: ALMA 870 µm image of the core of RO-1001 with the three star-forming galaxies clearly visible. Each of them were ﬁt with intensity proﬁles which were then subtracted to obtain the residual image displayed in the bottom panel. The ALMA PSF at 870 µm is shown in the inset on the top right. suggest progressively higher ﬂuxes in the IRAC bands, leading to oﬀsets from SED models. To limit the inﬂuence of the rapidly star-forming core, primarily in the IRAC bands with insuﬃcient resolution to disentangle the core from the disk, we execute the SED ﬁtting in three stages. The ﬁrst is done using photometry up to the bands where the galaxies are well-resolved (2.1 µm). We then include the remaining 4 bands in pairs of two. We ﬁnd that the results from the ﬁrst two stages are almost identical within their errors and hence use the results of the second stage (up to the 4.5 µm band; due to better constraints). We show in Fig. 6 that the net photometry at the third stage is reproducible using the SED derived at stage-two and an additional highly obscured component for the core. of the emission from all three submillimeter bright galactic cores in RO-1001 that have a much higher SFR surface den- sity (ΣSFR; for example see Fig. 5). adoptedconversionrelation(Kennicutt1998;Daddi et al.2010b): 3.3. Determining the quiescence of the stellar regions This is because the conversion to SFR is done based on the average IR SED in the group which is driven by the coaddition A44, page 5 of 16 A&A 666, A44 (2022) Fig. 3. Top panel: ALMA 870 µm image of the core of RO-1001 with the three star-forming galaxies clearly visible. Each of them were ﬁt with intensity proﬁles which were then subtracted to obtain the residual image displayed in the bottom panel. The ALMA PSF at 870 µm is shown in the inset on the top right. Fig. 4. Components of the three galaxies on the star-forming main- sequence: star-bursting bulges and quiescent disks. The SFR vs. stellar- mass values of the star-bursting core, the quiescent stellar disk and the combination of the two placed with respect to the star-forming main- sequence (Schreiber et al. 2015) at z = 3, shown as the black solid line. The shaded region demarcates the 0.3 dex uncertainty in the relation. We use the 3σ ALMA upper-limits as the value for SFR with the asso- ciated gray error-bars showing the range allowed by our SED model ﬁtting.The latter have been artiﬁcially oﬀset in the x-axis to emphasize the diﬀerence in the method of measurement. suggest progressively higher ﬂuxes in the IRAC bands, leading to oﬀsets from SED models To limit the inﬂuence of the rapidly Fig. 3. Top panel: ALMA 870 µm image of the core of RO-1001 with the three star-forming galaxies clearly visible. Each of them were ﬁt with intensity proﬁles which were then subtracted to obtain the residual image displayed in the bottom panel. The ALMA PSF at 870 µm is shown in the inset on the top right. Fig. 4. Components of the three galaxies on the star-forming main- sequence: star-bursting bulges and quiescent disks. The SFR vs. stellar- mass values of the star-bursting core, the quiescent stellar disk and the combination of the two placed with respect to the star-forming main- sequence (Schreiber et al. 2015) at z = 3, shown as the black solid line. The shaded region demarcates the 0.3 dex uncertainty in the relation. We use the 3σ ALMA upper-limits as the value for SFR with the asso- ciated gray error-bars showing the range allowed by our SED model ﬁtting.The latter have been artiﬁcially oﬀset in the x-axis to emphasize the diﬀerence in the method of measurement. Fig. 4. adoptedconversionrelation(Kennicutt1998;Daddi et al.2010b): 3.3. Determining the quiescence of the stellar regions Hence, the dust tempera- ture in the disks would likely be lower due to a softer radiation ﬁeld (based on the star-formation surface density vs. dust tem- perature relation, Valentino et al. 2020b, see also Magdis et al. 2012; Magnelli et al. 2014; Daddi et al. 2015), which would in turn drive our 3σ upper-limit even lower, probably at least by factors of 2−3. p For a description of the SED model ﬁtting, we refer the readers to Appendix A. The procedure for Galaxy-B returns the lowest reduced χ2 of 2.0 for the two declining SFH mod- els used (composite-τ and delayed-τ). In case of a constant star- formation model this value is found to be 6.8 that translates to a ∆χ2 = 38, making it highly unlikely. Hence, this indicates that the disk in Galaxy-B is undergoing a decline in star-formation, as already conclusively demonstrated by the ALMA upper lim- its. We also determine this decline to be signiﬁcant by measur- ing the age vs. τ ratio from the delayed-τ models and it is found to be 7 ± 1. Given that the composite-τ models are relatively more robust, we use them to measure the look-back time of half- mass formation (t50 = 0.5+0.7 −0.2 Gyr), that can be considered to be the approximate epoch at which the last major star formation episode had occurred. However, the results from delayed-τ mod- els are also in agreement. Furthermore, we obtain a SFR estimate of 42+51 −37 M⊙yr−1, consistent with the ALMA 3σ upper-limit. 3.4. Star formation history modeling We make use of the photometry obtained in observed frame opti- cal and near-IR, to estimate a star formation history (SFH) and thereby the age of the stellar population as well as stellar masses. The measurements can primarily be attributed to the stellar disks since the prodigious submillimeter ﬂux from the cores is by design at the expense of UV rest-frame ﬂux. However, during our ﬁtting procedure, we do ﬁx an additional core component in each case using positional priors obtained in Sect. 3.2. Except in the IRAC bands, all others return ﬂuxes <5% of the total galaxy ﬂux. However for IRAC, the PSF is a factor ∼10 larger than that of HST and a factor ∼5 compared to Ultra-VISTA. Hence in this case, the sources are unresolved and this leads to the pho- tometry corresponding to the integration over the whole galaxy. Albeit, in each of the cases the stellar mass within the core is expected to be <10% (based on estimates discussed in Sect. 3.5) that ensures a minimal contribution. Nevertheless, Fig. 6 does 37 The picture is slightly less strict in Galaxy-C followed by Galaxy-A. In the former, the declining models still give the best A44, page 6 of 16 B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks Fig. 5. Spatial distribution of Galaxy-B stellar mass and star formation rates. Left: upper panel shows SFR distribution model of Galaxy-B, with the morphology determined by the F160W stellar emission in case of the disk, and the ALMA dust emission for the core. The amplitudes are determined by the SFR of the two components. Lower panel: is a 1D representation of the plot above, after a rotation the make the major axis horizontal. Middle: stellar mass distribution following the same procedure. Here the mass in the core is determined from the diﬀerence between the dynamical mass and gas-mass determined from the CO[3−2] emission line. Right: speciﬁc SFR provided by the ratio of the SFR and the stellar mass. Fig. 5. Spatial distribution of Galaxy-B stellar mass and star formation rates. Left: upper panel shows SFR distribution model of Galaxy-B, with the morphology determined by the F160W stellar emission in case of the disk, and the ALMA dust emission for the core. The amplitudes are determined by the SFR of the two components. 3.4. Star formation history modeling 7) not so much to ascer- tain quiescence that is already unambiguously demonstrated by ALMA, but rather to compare them to more classical examples A44, page 7 of 16 A&A 666, A44 (2022) mass is ﬁnally computed as Mmol = αCO × L′ CO(1−0) by adopting a conversion factor αCO = 0.8 M⊙(K km s−1 pc2)−1 as expected in starbursts (given location of the cores, with respect to the star-forming main-sequence in Fig. 4, and their very high SFR surface densities). This provides values of 4.6 × 1010 M⊙, 0.7 × 1010 M⊙, 4.2 × 1010 M⊙for Galaxy-B, A and C respectively. Although, we do acknowledge that this esti- mate is dependent on our choice of conversion factors. Neverthe- less, we can place a strict upper-limit on the gas mass using the dynamical mass estimates calculated below (that is, in the lim- iting case in which there was no stellar mass nor dark matter in the cores), which is less than 2× above the current values in each case. A Milky-Way-like αCO would have resulted in a fac- tor of 5 higher gas-mass. Hence, these are in direct conﬂict with the dynamical mass values. Moreover, any increase in gas-mass would also indicate almost a negligible stellar mass, which in turn would place the galaxy-cores at a much higher oﬀset from the star-forming main-sequence. This would suggest even more star-burst-like characteristics. by Fig. 7. UVJ colors of high-z QGs (z > 2.5) adapted from D’Eugenio et al. (2020). The post-star burst region is demarcated with the red box as speciﬁed in Belli et al. (2019). The marker sizes are proportional to the stellar mass. The red and blue stars are the dust uncorrected and corrected measurements of the quiescent stellar disks in the three star-forming galaxies within our sample. The ﬁnal star in orange marks the location of Galaxy-D, a quiescent galaxy photometrically conﬁrmed to be part of RO-1001 (Kalita et al. 2021a). Fig. 7. UVJ colors of high-z QGs (z > 2.5) adapted from D’Eugenio et al. (2020). The post-star burst region is demarcated with the red box as speciﬁed in Belli et al. (2019). The marker sizes are proportional to the stellar mass. The red and blue stars are the dust uncorrected and corrected measurements of the quiescent stellar disks in the three star-forming galaxies within our sample. 3.4. Star formation history modeling Lower panel: is a 1D representation of the plot above, after a rotation the make the major axis horizontal. Middle: stellar mass distribution following the same procedure. Here the mass in the core is determined from the diﬀerence between the dynamical mass and gas-mass determined from the CO[3−2] emission line. Right: speciﬁc SFR provided by the ratio of the SFR and the stellar mass. Fig. 6. Best-ﬁt SED models in optical and near-IR for each of the three star-forming galaxies in RO-1001. The photometry with their respective errorbars are shown in red. The cyan contours show a modiﬁed SED with an addition of an example 0.1 Gyr old highly obscured (AV = 6.5−7.5) constant star-formation history SED in each case. In each case, the resulting stellar-masses are in agreement with our CO[3−2] estimates within 0.1 dex and are hence a representation of contributions of the star-bursting cores. Fig. 6. Best-ﬁt SED models in optical and near-IR for each of the three star-forming galaxies in RO-1001. The photometry with their respective errorbars are shown in red. The cyan contours show a modiﬁed SED with an addition of an example 0.1 Gyr old highly obscured (AV = 6.5−7.5) constant star-formation history SED in each case. In each case, the resulting stellar-masses are in agreement with our CO[3−2] estimates within 0.1 dex and are hence a representation of contributions of the star-bursting cores. these cases (94+66 −94 M⊙yr−1 for Galaxy-A and 66+446 −49 M⊙yr−1 for Galaxy-C) are still in general agreement with the much more stringent ALMA 3σ upper-limit. ﬁt (reduced-χ2 = 1.9) and a t50 = 0.2+1.3 −0.1 Gyr. But the con- stant star formation model is relatively less unlikely than for Galaxy-B, with a reduced-χ2 of 3.7. Galaxy-A on the other hand has values of 0.8 and 1.1 for almost equally plausible declin- ing and constant star formation models, allowing for possible ongoing star formation in the disk. Nevertheless, the SFR in both g pp We also derive UVJ colors (Fig. 3.4. Star formation history modeling However, given their cores have morphology closer to a Sér- sic index of 4, we re-estimate the Mdyn,tot using the following formula applicable to an elliptical galaxy dominated by random motion. p q q by the parameter FWZVCO[3−2] is already reported in Table 1, while the inclination angle (i) is determined based on the ellip- ticity of the submillimeter proﬁle. Subsequently, we measure the total dynamical mass, Mdyn,tot = 2×Mdyn,re = 1.3±0.1×1011 M⊙. The factor of 2 in invoked since only half of the mass is con- tained within re. We use the same procedure to obtain Mdyn,tot = 2.6 ± 0.3 × 1010 M⊙and 1.7 ± 0.2 × 1011 M⊙for Galaxies A and C. However, given their cores have morphology closer to a Sér- sic index of 4, we re-estimate the Mdyn,tot using the following formula applicable to an elliptical galaxy dominated by random motion. Finally for Galaxy-B, we pay special attention to the spatial decomposition of the stellar mass estimates (listed in Table 1) derived from the SED ﬁtting procedure – by obtaining separate photometry of the northern and southern sections of its elongated disk. This is to quantify the lopsidedness in the stellar structure. We hence ﬁnd the total stellar mass of 1.6 ± 0.4 × 1011 M⊙for the complete stellar region is divided into 1.1 × 1011 M⊙and 0.5 × 1011 M⊙for the northern and southern segments. It is note- worthy that for this analysis, we only use observations up to the 2.1 µm band beyond which the resolution is too low to spatially disentangle the two parts. Mdyn,re ≈5.0 re σ2 e G · (3) Mdyn,re ≈5.0 re σ2 e G · (3) We measure the total dynamical masses of ∼2.2 × 1010 M⊙ and 2.4 × 1011 M⊙for Galaxies A and C respectively, reason- ably consistent with our earlier estimates. Although the rela- tively high discrepancy in case of Galaxy-C likely points to the rotational-dominance expected in a gas-rich structure. Neverthe- less, we do not expect the structures to fully conform to such a dynamical arrangement due to the inherent dissipative nature of gas within them. 3.4. Star formation history modeling The ﬁnal star in orange marks the location of Galaxy-D, a quiescent galaxy photometrically conﬁrmed to be part of RO-1001 (Kalita et al. 2021a). The width of the emission line in Galaxy-B is used to deter- mine the dynamical mass within the dusty core where the gas is expected to be conﬁned. Given the submillimeter emission proﬁle of the core is found to resemble a disk, we measure the Mdyn,re within the eﬀective radius re using the following relation (Daddi et al. 2010a): of high redshift quiescent galaxies. Of special interest are the widely studied post-starburst (PSB) galaxies that have expe- rienced quenching of star-formation over the last <0.8 Gyr and generally show low dust attenuation (Gobat et al. 2012; Schreiber et al. 2018; Forrest et al. 2020; D’Eugenio et al. 2020; Valentino et al. 2020a). The PSB ages are potentially similar to the mass-weighted ages derived for our sample, while attenua- tion in our case is larger (Table 1). We ﬁnd that the observed UVJ colors of our quiescent disks are much redder than those of known population of quiescent systems at z ∼3. Also, at least in two cases they are scattered just outside of the formal UVJ- passive boundary. However, when correcting for dust reddening the colors of the three galaxies in RO-1001 more closely resem- ble those of PSBs as well as of the massive quiescent galaxy with a mass-weighted age >1 Gyr (Kalita et al. 2021a) within the same group (Fig. 7). Mdyn,re = 1.3 × re × FWZVCO[3−2]/22 G sin2i ± 12.5%. (2) (2) The eﬀective radius returned from the submillimeter emission proﬁle is used in this equation. The width of the line, quantiﬁed The eﬀective radius returned from the submillimeter emission proﬁle is used in this equation. The width of the line, quantiﬁed by the parameter FWZVCO[3−2] is already reported in Table 1, while the inclination angle (i) is determined based on the ellip- ticity of the submillimeter proﬁle. Subsequently, we measure the total dynamical mass, Mdyn,tot = 2×Mdyn,re = 1.3±0.1×1011 M⊙. The factor of 2 in invoked since only half of the mass is con- tained within re. We use the same procedure to obtain Mdyn,tot = 2.6 ± 0.3 × 1010 M⊙and 1.7 ± 0.2 × 1011 M⊙for Galaxies A and C. 4. Results ing cores and extended near-IR detected coplanar stellar disks showing varying levels of lopsidedness (Fig. 1, right panel). The size contrast is evident from the high near-IR to submillimeter radius ratios: ∼5−8 for Galaxy-B, while in case of Galaxy-A and C it is 4 ± 1 and 5 ± 1. Majority of the star formation is concen- trated in the cores, with the IR-based SFR in the cores being a 3.5. Rotating gas traced by CO[3−2] transition nd C with three individual subimages: (top-left) the NOEMA CO[3−2] he average galaxy proﬁle shown as contours superimposed on the F160W line in blue and the local Lyman-α proﬁle in red; (right) The contours of pact core (white; beginning at 100σ with increments of 50σ for Galaxy-B of the blue and red-shifted CO[3−2] emission locations with the size of the en also been provided for each component – disk in red, core in blue and tainties are large enough to make the diﬀerence with the other component Fig. 8. Two panels showing the rotational properties in Galaxies B and C with three individual subimages: (top-left) the NOEMA CO[3−2] emission proﬁles for the blue and red-shifted sections with respect to the average galaxy proﬁle shown as contours superimposed on the F160W image; (bottom-left) The NOEMA spectra with the CO[3−2] emission line in blue and the local Lyman-α proﬁle in red; (right) The contours of the stellar disk (black; beginning at 8σ with increments of 4σ), the compact core (white; beginning at 100σ with increments of 50σ for Galaxy-B and at 50σ with increments of 25σ for Galaxy-C) and a representation of the blue and red-shifted CO[3−2] emission locations with the size of the compact core as seen in ALMA. The expected rotational axes have been also been provided for each component – disk in red, core in blue and CO[3−2] emission as black dashed lines. The NOEMA spin axes uncertainties are large enough to make the diﬀerence with the other component not statistically signiﬁcant. 3.5. Rotating gas traced by CO[3−2] transition Finally, two separate continuum images are created for Galaxies B and C, by dividing the whole spectral range into two segments that are separated by the average location of the CO[3−2] emission of the whole galaxy. The oﬀset between the peaks trace the plane of rotation, which are found to be in agree- ment with the major axes of the respective galaxies in both sub- millimeter and near-IR (Fig. 8). A similar analysis is not possible for Galaxy-A due to a low signal-to-noise of the CO[3−2] detec- tion. A direct spectroscopic tracer of the gas content of the galax- ies is available in the form of NOEMA CO[3−2] transition observations. For Galaxy-B, we use its integrated luminosity (log(L′ CO(3−2)/(K km−1 pc2)) = 10.46) to estimate a gas mass. This is calculated from the line ﬂux (Daddi et al. 2021) using the relation prescribed in a recent work (Silverman et al. 2018). Using a standard R31 = 0.5 (Daddi et al. 2015; Valentino et al. 2020a,b), we hence derive log(L′ CO(1−0)) = 10.76. The gas A44, page 8 of 16 B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks 8. Two panels showing the rotational properties in Galaxies B and C with three individual subimages: (top-left) the NOEM ssion proﬁles for the blue and red-shifted sections with respect to the average galaxy proﬁle shown as contours superimposed o ge; (bottom-left) The NOEMA spectra with the CO[3−2] emission line in blue and the local Lyman-α proﬁle in red; (right) Th tellar disk (black; beginning at 8σ with increments of 4σ), the compact core (white; beginning at 100σ with increments of 50σ at 50σ with increments of 25σ for Galaxy-C) and a representation of the blue and red-shifted CO[3−2] emission locations with pact core as seen in ALMA. The expected rotational axes have been also been provided for each component – disk in red, cor 3−2] emission as black dashed lines. The NOEMA spin axes uncertainties are large enough to make the diﬀerence with the oth statistically signiﬁcant. 4.1. Compact star-forming cores in extended quiescent stellar disks The spatial decomposition of surface-brightness proﬁles of the galaxies reveals extremely compact submillimeter bright rotat- A44, page 9 of 16 A&A 666, A44 (2022) Galaxies B and C. We therefore identify it as the primary stel- lar disk of Galaxy-A. The additional component is likely an in- falling equal-mass galaxy. factor of 6−36× higher than the 3σ upper-limits in the disks. The mass concentrations also displays this dichotomy between the cores and the stellar components. Using the diﬀerence between the CO[3−2] derived dynamical masses and the gas masses, we obtain approximate stellar masses of the cores for each galaxy (≈2 × 1010 M⊙, 8 × 1010 M⊙and 12 × 1010 M⊙). These are con- centrated within the areas >60× compact than the extended stel- lar regions that feature stellar masses of 3.2 ± 1.3 × 1011 M⊙, 1.6 ± 0.4 × 1011 M⊙and 1.8 ± 0.5 × 1011 M⊙. factor of 6−36× higher than the 3σ upper-limits in the disks. The mass concentrations also displays this dichotomy between the cores and the stellar components. Using the diﬀerence between the CO[3−2] derived dynamical masses and the gas masses, we obtain approximate stellar masses of the cores for each galaxy (≈2 × 1010 M⊙, 8 × 1010 M⊙and 12 × 1010 M⊙). These are con- centrated within the areas >60× compact than the extended stel- lar regions that feature stellar masses of 3.2 ± 1.3 × 1011 M⊙, 1.6 ± 0.4 × 1011 M⊙and 1.8 ± 0.5 × 1011 M⊙. In at least two of the galaxies (B and C), the major axes of both the core and the disk are aligned within ∼3◦, as well as with the major axis implied by the rotational patterns of the gas in the respective cores, determined from the CO[3−2] emis- sion (Fig. 8). This is dependent on a reasonable assumption that the gas is conﬁned within the same area as the dust. Hence, we conclude that these two galaxies have coplanar (and possibly corotating) stellar-disks and compact cores. The current data does not allow us to measure the major axis direction of the rota- tional pattern, or the severely lopsided stellar disk (by a factor >3, toward the south) of Galaxy-A. For the latter, we are likely observing only the “heavier” part of the disk and hence the disk size and shape parameters cannot be satisfactorily determined. 4.3. Young cores within older disks For the three cores, we ﬁnd short gas-consumption timescales of 20 Myr, 70 Myr and 130 Myr and time taken to build the stel- lar masses, assuming constant rates of star-formation, ∼60 Myr, 120 Myr and 400 Myr. However, the short timescales ∼100 Myr for three such neighboring objects makes them statistically unlikely to be simultaneously observed. Hence we propose that the extreme star formation levels is rather the culmination of an increase over a larger period of time. The rate and the beginning of this increase cannot be ascertained. Nevertheless, these are still similar or smaller compared to the time scales characteriz- ing the decline in star-formation of the stellar components, based on the goodness-of-ﬁt from the best-ﬁt SED composite τ-models (Schreiber et al. 2018): the look-back times to the epoch of half- mass formation (t50) are 1.7+0.3 −0.7 Gyr, 0.5+0.7 −0.2 Gyr and 0.2+1.3 −0.1 Gyr for Galaxies A, B and C. Hence, the star-bursting cores only started getting built after the disks had already formed about half of their stellar mass and were past their last major episode of star-formation. For Galaxies B and C, we do not expect their stellar disks to have been perturbed by major mergers involving equal-mass galaxies (with ratios 1:1 to ∼1:3), which have been studied in multiple simulations over the years (e.g., Bendo & Barnes 2000; Bournaud et al. 2005a; Nevin et al. 2019). Firstly, we conclude a lack of any visible disturbance in the disks from our well resolved and high signal-to-noise HST imaging (Fig. 1, right). Had these undergone major mergers, they would have had to consist of two perfectly aligned disks at a very early stage of collision (precoalescence), the probability of which is 0.1% and 0.4% for Galaxies B and C respectively based on the proba- bility of the speciﬁc geometrical orientation (having uncertain- ties equal to the thickness of the disks). Coupled with the con- currence of both these galaxies are detected in the same exact scenario, the probability reduces to a negligible 0.04%. More- over, the temporal aspect also makes such a scenario even more unlikely – given that both Galaxy-B and C are detected in the same situation within the observation window. 4.3. Young cores within older disks Furthermore, had a major merger been the case, one would have detected a bimodality in the mass distribution associated with each partic- ipating galaxy rather than a centrally peaked proﬁle, especially seen in Galaxy-B (Fig. 5). 4.2. Morphological characterization The single-peaked proﬁles point to these galaxies consisting of singular (albeit, lopsided) disks with central cores. This is corroborated by the single Sérsic proﬁle ﬁts (with indices ≲1; Sect. 3.1) to their near-IR surface-brightness proﬁles. About 10−40% of the stellar mass is concentrated in the cores occu- pying <1% of the surface area of the whole galaxy. This is illus- trated in Fig. 5 for Galaxy-B, the most extended of the three galaxies. We ﬁnd comparable contrasts in the other two galaxies (based on their near-IR to submillimeter ratio, Table 1). Hence, the cores of these galaxies are likely forming the future stellar bulges. This is characterized by their nuclear location, masses, the extreme compactness similar to z ∼2−3 passive galaxies (Daddi et al. 2005), and their submillimeter surface brightness proﬁles with Sérsic index ∼4 for Galaxies-A and C, which is relatively unusual (Barro et al. 2016; Martig & Bournaud 2008, indicate that the indices should be closer to that for a disk). Galaxy-B although has an index ∼1, but the higher ellipticity of the core (0.40 ± 0.01) in comparison to the disk (0.15 ± 0.05) still supports the idea of bulge formation. 4.1. Compact star-forming cores in extended quiescent stellar disks For Galaxy C, the ellipticities of the core and the stellar compo- nent are found to be in agreement (within 0.1). But in Galaxy-B, where the stellar component is observed edge-on (with an ellip- ticity of 0.15±0.05), the core has a larger ellipticity (0.40±0.01). However, it should be noted that all the three cores have values within 0.4−0.6. 5. Discussion In this section, we revisit the key observational results and dis- cuss how their interplay leads to a coherent picture of the evolu- tion of the three star-forming galaxies in RO-1001. Each of them feature extended stellar disks that are lopsided and quiescent, along with highly star-forming compact cores within them. An alternate scenario would have been the near-IR detected stellar disk and the submillimeter core are actually two sepa- rate galaxies. However, we also reject such a scenario due to the submillimeter bright segment being perfectly at the geo- metric center of the stellar emission region in not one but two separate cases within the same group. In Galaxy-A, with its disturbed stellar morphology however, the situation is diﬀerent from its neighbors. As discussed earlier, the stellar emission can be decomposed into two almost perpendicular orientations. We observe that one of the two (the northern component) is bet- ter coinciding with the compact ALMA core, similar to that in A44, page 10 of 16 5.1. Lopsidedness and quiescence of the disks Since lopsidedness is usually contributed to tidal interactions and in-situ star-formation asymmetry (Sancisi et al. 2008), we attribute the strong lopsidedness in our sample to the accre- tion of material onto the galaxies, that is asymmetric gas- accretion (Bournaud et al. 2005b) (that is reﬂected by stellar- mass distribution hence created) or the tidal interaction (or minor mergers) with in-falling satellite subhalos (Zaritsky & Rix 1997; Kazantzidis et al. 2008, 2009), that may also include their assim- ilation into the primary galaxy halo. These less-severe inter- actions (with mass-ratios from 1:4 or lower) are expected to preserve the stellar disks (Bournaud et al. 2005b; Hopkins et al. 2009). Only in Galaxy-A, can we also associate the imminent major merger to also be playing a role. p p y g j The only possible scenarios capable of self-suﬃciently explaining the observed suppression of star-formation are found to be connected to the aforementioned heavy lopsidedness in the disks. Introduction of such asymmetry, in a diﬀerentially rotating disk-like structure induces loss of angular momentum, causing mass to rapidly fall into the central regions (Combes & Gerin 1985). This process can be characterized as the triggering of the m = 2 Fourier component, denoting a bar, by the m = 1 component that refers to the asymmetry or lopsidedness (Bournaud et al. 2005b), observed in our sample. Bars have already been found to eﬃciently suppress star-formation up to factors of ∼10 in the disk (Khoperskov et al. 2018), while driv- ing gas into the core (Carles et al. 2016), albeit on less spectacu- lar scales than what observed here. This could be a possible case of an early-phase bar-formation, which are usually scarce at high redshifts (e.g., Sheth et al. 2008). However, the resolution and sensitivity is not suﬃcient to conﬁrm it. The accreted clumpy material driving the lopsidedness is also expected to feed the star-forming core, and in the process leading to a stabilization of the disk (Dekel et al. 2009b). Therefore the same outside-in quenching can be expected in all scenarios involving lopsided- ness and compact star formation. j g p y g Meanwhile, the quiescence of the stellar disks (albeit pos- sibly not entirely passive), is the ﬁrst indication of the pres- ence of an apparent “outside-in” quenching mode in high- redshift massive galaxy populations, in contradiction to the clas- sic “inside-out” conﬁguration (Lang et al. 2014; Tacchella et al. 5.1. Lopsidedness and quiescence of the disks We ﬁrst begin with the severe lopsidedness of the stellar disks (Fig. 1, right panel). For Galaxy-B, the asymmetry in the stellar emission (of a factor of 2.0 in F160W-HST) manifests as a mass lopsidedness of 2.2 (Sect. 3.4) along the major-axis (Fig. 9). In Galaxy-C, the emission asymmetry, also along the major axis, is 1.6. The current data does not allow us to measure the major axis direction of the stellar disk of Galaxy-A, but only the lopsided- ness upper limit (>3) and direction (South), that is nearly perpen- dicular to the East-West ALMA core major axis (Fig. 9). Such B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks Fig. 9. Additional stellar and dust components tracing the direction of accretion. The three panels showing the three residual F160W images of the galaxies after the subtraction of the primary disks (in green contours; starting at 4σ with increments of 4σ). The red solid contours show the ALMA 870 µm emission with the lines starting at 50σ with increments of 50σ. The red solid lines indicate the respective ALMA major axes. In case of Galaxies A and B, the dashed red contours starting at 3σ with steps of 0.5σ displays the ALMA residual emission after the subtraction of the primary emission regions. In case of Galaxy-C, the same contours show the additional point source adjacent to the core that was found during the ﬁtting procedure. In this case, the contours are at 15, 25σ. Finally, the arrows indicate the likely directions of gas-accretion onto the galaxies based on the asymmetric distribution of all the aforementioned components, which also coincide with the direction in which the disks are lopsided. Fig. 9. Additional stellar and dust components tracing the direction of accretion. The three panels showing the three residual F160W images of the galaxies after the subtraction of the primary disks (in green contours; starting at 4σ with increments of 4σ). The red solid contours show the ALMA 870 µm emission with the lines starting at 50σ with increments of 50σ. The red solid lines indicate the respective ALMA major axes. In case of Galaxies A and B, the dashed red contours starting at 3σ with steps of 0.5σ displays the ALMA residual emission after the subtraction of the primary emission regions. 5.1. Lopsidedness and quiescence of the disks In case of Galaxy-C, the same contours show the additional point source adjacent to the core that was found during the ﬁtting procedure. In this case, the contours are at 15, 25σ. Finally, the arrows indicate the likely directions of gas-accretion onto the galaxies based on the asymmetric distribution of all the aforementioned components, which also coincide with the direction in which the disks are lopsided. gas from external regions of galaxies (Bravo-Alfaro et al. 2000; Vollmer et al. 2001; Fumagalli et al. 2009; Boselli et al. 2014; Loni et al. 2021), therefore resulting in an apparent outside-in quenching (for a review, Boselli et al. 2022). It could also lead to compression of the gas in the galaxy which could result in the lopsidedness (although the stellar morphology would not be aﬀected). However, as shown in Appendix B, a conservative lower-limit of the radius up to which RPS can remove gas from the sample in RO-1001 would be 10−15 kpc. This is more than an order of magnitude higher than the kpc-scale cores beyond which the galaxies have suppressed star-formation. Hence we do not expect RPS to be playing a major role. asymmetries, not yet recognized at high redshifts, are in fact a common characteristic among spiral galaxies in the local Uni- verse (Sancisi et al. 2008; Rix & Zaritsky 1995; Zaritsky & Rix 1997; Reichard et al. 2008), although relatively less pronounced. Since lopsidedness is usually contributed to tidal interactions and in-situ star-formation asymmetry (Sancisi et al. 2008), we attribute the strong lopsidedness in our sample to the accre- tion of material onto the galaxies, that is asymmetric gas- accretion (Bournaud et al. 2005b) (that is reﬂected by stellar- mass distribution hence created) or the tidal interaction (or minor mergers) with in-falling satellite subhalos (Zaritsky & Rix 1997; Kazantzidis et al. 2008, 2009), that may also include their assim- ilation into the primary galaxy halo. These less-severe inter- actions (with mass-ratios from 1:4 or lower) are expected to preserve the stellar disks (Bournaud et al. 2005b; Hopkins et al. 2009). Only in Galaxy-A, can we also associate the imminent major merger to also be playing a role. asymmetries, not yet recognized at high redshifts, are in fact a common characteristic among spiral galaxies in the local Uni- verse (Sancisi et al. 2008; Rix & Zaritsky 1995; Zaritsky & Rix 1997; Reichard et al. 2008), although relatively less pronounced. 5.1. Lopsidedness and quiescence of the disks 2015; Breda & Papaderos 2018). We consider various possi- ble modes of quenching. Since we observe the galaxies to be undergoing an outside-in quenching, this could not have pri- marily occurred due to feedback from active galactic nuclei (AGN; Alatalo et al. 2015), known to quench galaxies inside- out (Tacchella et al. 2018). We also ﬁnd no evidence of AGN activity from X-ray observations (Daddi et al. 2021), although a radio excess in Galaxy-C is indicative of weak past AGN activity in that galaxy. The process of morphological quench- ing (Martig et al. 2009), where the formation of a stellar bulge stabilizes the disk against further star-formation, is also improb- able. The galaxies are far from being bulge-dominated, essen- tial for this mode of quenching to be applicable, based on their stellar mass distributions. Also unlikely is cosmologi- cal starvation (Feldmann & Mayer 2015) since the availabil- ity of gas has already been established in RO-1001. Finally, ram-pressure stripping (RPS; Gunn & Gott 1972) could be regarded as a possible contributor as it is known to remove Moreover, the expected damping time of the lopsidedness is comparable to the Hubble time (Jog & Combes 2009), explain- ing their detection throughout our sample and allowing them to act as catalysts of bulge formation under the high redshift accre- tion conditions. This timescale is not however associated with the gas being funneled into the core, but rather the survival of A44, page 11 of 16 A44, page 11 of 16 A44, page 11 of 16 A&A 666, A44 (2022) Another characteristic of cSFGs seemingly in conﬂict with our results is the expectation of major merger dominance within such samples (Elbaz et al. 2018). Both in galaxies B and C, the stellar disks appear to be well-preserved (Sect. 4.2). the asymmetry that drives this process. We propose that accreted material is resulting in the compact star-forming cores within outside-in quenching disks, either directly (mergers) or indi- rectly (lopsidedness funneling gas to the core). The accretion may vary from smooth dust (hence presumably gas) compo- nents, to subhalo stellar clumps as in Galaxies B and C (while preserving the disks in the process), to additional massive stellar structures as observed in Galaxy-A. pp p ( ) We can however provide possible reasons behind these apparent discrepancies. 5.2. Determining the direction of accretion Due to the conﬂuence of evidence for inﬂuence of accretion on the galaxies, we try to discuss possible tracers of this process. In an accreting galaxy, the lopsided part of the disks would be expected to mark the location of impact for the accreted mate- rial. Corroborative evidence can be found in our sample when the models of the primary disks in the F160W image and the submillimeter bright cores in the ALMA image are subtracted. Residual emission (stars in near-IR emission and dust in submil- limeter) is clearly detected in each case (Fig. 9). In Galaxy-A, its F160W and the associated ALMA residuals demonstrate the presence of the similarly-massive companion merging into the disk. For Galaxies-B and C, their respective F160W residuals are much fainter (by ∼3 orders of magnitude) and are likely in- ﬂowing small-mass clumps within or adjacent to the disks. These are found to be preferentially located in the direction of the lopsidedness of the stellar-disks. We hence suggest that these indicators could be tracing the mass inﬂow into the core. 5.1. Lopsidedness and quiescence of the disks Our high-resolution imaging capabilities in both near-IR and submillimeter for RO-1001 is usually not reached in larger statistical studies of cSFGs. Hence we might have simply been able to resolve the galaxies better to deter- mine their properties. This allowed us to study the lopsidedness, the lack of dynamical perturbations (in Galaxies B and C) and the apparent outside-in quenching, based on which we draw the conclusions of our work. Although, we can also hypothesize a physical reasoning. Large statistical samples usually comprise of galaxies in the ﬁeld which do not experience the high levels of accretion of gas and satellites expected within dense gas-rich environments like RO-1001 (accretion rate increases with halo mass; Dekel et al. 2009a). Hence the cSFGs in the regions with less accretion would be more likely to be results of major merg- ers driving gas into compact cores while destroying extended stellar disks. This is further exempliﬁed by our direct visual investigation ﬁnding a complete lack of similarly massive galax- ies with extended and continuous stellar morphology (like in Galaxy B) in the redshift window of 2.6 < z < 3.2 in both COSMOS and GOODS-South ﬁelds. If this is true, cSFGs with compact star-forming cores and very extended quiescent stellar disks (similar to our sample) might be more easily found in dense accretion-rich regions. 6. A generalized picture of accretion-driven galaxy evolution Extending on our hypothesis of accretion onto the three star- forming galaxies in RO-1001, it is imperative to associate the discussed properties with the large-scale narrative of accretion for the galaxy-group (Daddi et al. 2021). Any ongoing accretion in RO-1001 would likely be coupled with the established Lyman- α-emitting ﬁlaments in RO-1001 (Daddi et al. 2021). Hydro- dynamical simulations predict that massive galaxies evolving under the inﬂuence of nearby accretion-streams have their major axes aligned along the stream direction (Dubois et al. 2014; Codis et al. 2018). We observe a consistency with this predic- tion in our observations (Fig. 1, right panel). We quantify this by ﬁnding the probability for the chance alignment of the major axis of each galaxy with one of the three ﬁlaments. The range of allowed angles is approximated using the center of the galaxies and the edges of the −18.0 contour (corresponding to a surface brightness of 10−18 s−1 erg cm−2 arcsec−2) in Fig. 1, since it is the highest brightness level at which the Ly-α traced ﬁlaments are detected. We also ensure that the tentative direction of the ﬁl- aments, qualitatively reported in Daddi et al. (2021), are within this subtended angle (<±10◦). We ﬁnd total chance probabili- ties of 13%, 17% and 12%. Additionally, the probability of all three galaxies being aligned with one of the ﬁlaments (which is what we observe) is hence <1%. This alignment, especially in case of disk-like galaxies, is attributable to the process of tidal torquing (Codis et al. 2015) by the massive accretion ﬁlaments. However, at the core of this lies the tidal interactions the galaxies experience due to infalling satellites (or minor mergers). Hence, the apparent alignments should be regarded as an indication of the galaxies accreting clumpy material, which has already been suggested in Sect. 5.1. In this section, we put forward a way to ﬁt our work within the general framework of high-z massive galaxy evolution. Through- out, we suggest how our results indicating accretion onto galax- ies is well suited to provide a general picture of galaxy mass- buildup at these redshifts. 6.3. A uniﬁed picture of accretion-driven evolution Based on the results of the three star-forming galaxies in RO- 1001, we ﬁnd evidence of massive disk-like galaxies expected to have secularly evolved featuring compact star-forming regions at their cores. We propose that the massive disks build up lop- sidedness under the combined eﬀects of gas and satellite accre- tion which in turn results in loss of angular momentum of the material. This drives the prodigous star-formation in the central regions. Such a scenario is likely being observed in Galaxies B and C. Although, gas can also be driven to the core by much more violent major mergers (Toft et al. 2017; Elbaz et al. 2018), but this leads to a destruction of the stel- lar disks which is only anticipated in Galaxy-C. However, we conclude that the “wet compaction” scenario proposed by sim- ulations (Dekel et al. 2013; Zolotov et al. 2015; Tacchella et al. 2016), where compact stellar bulges get rapidly built up as a result of accretion-driven violent disk instabilities, is too extreme to reproduce the observations in RO-1001. Such a compaction simply does not allow for creation of extended and massive stel- lar disks (≳109.5 M⊙) while in our sample we are observing them with masses >1011 M⊙. g p p Furthermore, this will also allow the tracing of the metal- licity of the stellar disks. It is hypthesized that accreted gas being metal-poor leads to a fall in the metallicity in the accreting galaxy (Lehner et al. 2013; Sánchez Almeida et al. 2013, 2014; Wotta et al. 2019). Hence, RO-1001 could be a test-bed for such theories. Finally, spectroscopy will also provide the kinematics of the stellar disk and determine the level of rotational domi- nance within the structure. This would be necessary to model the evolution of the galaxies as well as similar galaxies in the future featuring signs of accretion. This is especially of inter- est since we have suggested the possible prevalence of clumps migrating to the core of the galaxies, driving the high levels of star-formation. The timescales of in-ﬂowing material is coupled with the rotation timescales of the respective disks (Dekel et al. 2009b). Finally, the presence of massive stellar disks points to the need for a mode of destruction, since they are not usually observed in dense environments at lower redshifts (Bundy et al. 2010). 7.1. Further investigations In our study, we are still lacking information about certain facets of the three galaxies in RO-1001. A spatially resolved spectroscopic study of the quiescent stellar regions would be extremely useful to constrain their star-formation histories. A lot of work in this direction has been done for massive QGs (e.g., Schreiber et al. 2018; Forrest et al. 2020; Valentino et al. 2020a), with signatures of rapid quenching of star-formation observed in their samples. Although we do determine rapid quenching of the stellar disks in our sample (Sect. 3.4), conclusive proof will only be available after obtaining spectroscopic data. 6.3. A uniﬁed picture of accretion-driven evolution Dynamical disturbances caused by mergers or galaxy harassment can easily be invoked for this (Toth & Ostriker 1992; Kazantzidis et al. 2006; Bullock et al. 2009; Purcell et al. 2009; Bournaud et al. 2011). We might already be observing such a scenario in action in Galaxy-A, with its signs of a forthcom- ing major merger. Finally, a possible end product may be the compact quiescent spheroidal galaxy (Kalita et al. 2021a) that has been photometrically conﬁrmed to also be within the group, although it never grew beyond 1011 M⊙. It is curious however, that we have stumbled upon a galaxy-group with three massive galaxies close together with recent gas-rich star-bursting cores 6.1. Comparison to high-z compact star-forming galaxies We start of by making a comparison with the widely studied cSFG population at similar redshifts (Cimatti et al. 2008; Ricciardelli et al. 2010; Fu et al. 2013; Ivison et al. 2013; Toft et al. 2014, 2017; Elbaz et al. 2018; Gómez-Guijarro et al. 2018, 2019; Puglisi et al. 2019, 2021). These cSFGs also feature compact kpc-scale submillimeter bright regions, as is observed in our sample. In fact, Puglisi et al. (2019) already suggested these regions (or cores) to be above the star-forming main sequence (as seen in our sample, Fig. 4). However, the surround- ing disks being well below the star-forming main sequence as in our sample (Fig. 4), making galaxies appear to be quench- ing outside-in, have not yet been recognized. A similar sce- nario might hold for post-starbust galaxies (e.g., Baron et al. 2022). Another apparent diﬀerence is the average ratio of the near-infrared and submillimeter radii for cSFGs, that is ∼3 (Puglisi et al. 2021; Smercina et al. 2018; Lang et al. 2019). Although this is likely a lower limit due to many cSFGs remain- ing unresolved in large sample studies. It remains thus unclear if examples like the three galaxies in RO-1001 with ratios 4−9 could also present among cSFGs. Moreover, simply the size of Galaxy-B being a factor of 2.0−2.5 above the mass-size relation (van der Wel et al. 2014) for star-forming galaxies at z = 3, adds to the rarity of our sample, especially with regards to cSFGs. In each case however, we would still expect a level of bend- ing of the accretion streams before the ﬁnal impact on to the galaxies, revealed by oﬀsets between the direction of accretion A44, page 12 of 16 B. S. Kalita et al.: Bulge formation inside quiescent lopsided stellar disks (Fig. 9) and the preferred ﬁlament (Fig. 1, right panel). We propose a scenario where the Galaxy-A, B and C are being fed by the southwestern, northern and southeastern ﬁlaments. The swap of the preferred ﬁlament for Galaxies A and C from those in their proximity has been made due to better alignment with the major-axis of the galaxies traced by near-IR and submil- limeter emissions as well as the direction of accretion traced by the lopsidedness and residual emission (Fig. 9). Otherwise, for Galaxy-C the accretion direction would have been oppo- site to direction of approach of the ﬁlament. 6.1. Comparison to high-z compact star-forming galaxies Moreover, there is an additional inconsistency of the line-of-sight velocities of the Lyman-α in the southeastern ﬁlament at the galaxy position (Daddi et al. 2021). For Galaxy-A, it has a large spatial displace- ment from the direction of the nearest southwestern ﬁlament. In each case hence, the actual streams of in-falling material would have needed to drastically bend before the ﬁnal impact onto the galaxies. Nevertheless, even in our preferred scenario there is still a level of bending of the streams necessary as they approach the inner halos of the galaxies. For Galaxy-A, the major axis (measured here from ALMA) versus the lopsidedness and in- falling material direction are nearly orthogonal. Hence the bend- ing is likely occurring in the line-of-sight. The same could also be true in Galaxy-B, where the northern ﬁlament with its reduced length likely has a major component along the line-of-sight. Hence, the in-falling stream would need to bend if it has to agree with the expected direction of accretion along the major-axis, on the plane of the sky (Fig. 9). created within the last few 100 Myr. From each of their observed properties, they seem to have undergone a recent shift in evolu- tion with a preference for the central region. A possible reason has recently been discussed in Noguchi (2022), where bulges get rapidly built-up in >1011 M⊙galaxies within dark-matter halo cores that are accessible to cold-gas ﬁlaments (Dekel et al. 2009a). This limit is related to the dependence of migration timescales of clumpy accreted material (already suggested in our sample by their lopsidedness, as discussed in Sect. 5.1) that feed the central star-forming region on the stellar mass of the galaxy. Hence, the galaxies in RO-1001, all of which are >1011 M⊙, could simply be experiencing ideal conditions for a rapid buildup of bulges. It should be noted however that we refrain from esti- mating the timescale of migration of accreted clumps since such a calculation depends on the relative angular momentum of the inﬂowing material with respect to the disk. This can result in a range between a few tens to a few hundred Myrs, which is highly uncertain and heavily dependent on assumptions that we are not in a position make with the current data. 7.2. Extending the search to other structures Asymmetries in the stellar distribution as well as relative posi- tion of the submillimeter bright cores of cSFGs are rather com- mon. However, they are mostly associated with galaxy-galaxy interactions (Elbaz et al. 2018; Cibinel et al. 2019; Puglisi et al. 2019). However, we demonstrate that if studied in higher res- olution, they might reveal eﬀects of accretion of gas and clumps especially in dark matter halos where cold-gas ﬁla- ments are expected to survive in spite of the surrounding hot medium (Dekel et al. 2009a). Hence, deep observations of star- forming galaxies within dense-environments would be instru- mental in establishing a generalized picture of massive galaxy evolution in dense environments. A few examples are in fact A44, page 13 of 16 A44, page 13 of 16 A&A 666, A44 (2022) already available. CLJ1449+0856 (Gobat et al. 2011, 2013; Strazzullo et al. 2016; Coogan et al. 2018) has three highly star- forming galaxies that showcase clear oﬀsets between their near- IR and submillimeter (or radio) contours, with one of them also featuring AGN activity (S7-N7 system; Kalita et al. 2021b). Using unresolved submillimeter data, they were regarded as mergers. However, reconsideration after obtaining high resolu- tion data might reveal the asymmetries to be due to compara- ble features as those seen in RO-1001. Another such case is the highly star-forming galaxy, GN20, which is part of a proto- cluster at z = 4.05 (Daddi et al. 2009; Hodge et al. 2015). This too features a similar oﬀset and is a conﬁrmed member of a high-z dense environment. Additional sources of interest include galaxies within seven other high-z structures with Ly-α halos suggesting abundance of gas (Daddi et al. 2022a,b). Future well- resolved studies of these and additional structures hold much promise. accretion-stream with the galaxies. We detect these signatures in each of the cases. Hence we make the ﬁrst direct connection between accretion streams and the morphological transformation of galaxies driving compact star-formation in massive galaxies at high redshifts. Finally, we show that in the presence of accretion, galaxies can build-up compact highly star-forming cores without the need of major mergers and therefore preserving their stellar disks. A key feature however is an asymmetry in the stellar distribution, which needs to be studied in high resolution to determine if it is driven by accretion of gas and clumps or by violent interactions like major mergers. 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However, the ubiquity of such a scenario can only be revealed with more well-resolved observations of massive galaxies at the heart of well-studied high-redshift dense environments, which will likely become more accessible in the upcoming James Webb Telescope era. 8. Summary and conclusions Within a z = 2.91 galaxy-group of dark matter halo mass of ∼4 × 1013 M⊙, we observe three massive (>1011 M⊙galax- ies (Fig. 1). They have a combined star-formation rate of ∼1250 M⊙yr−1, and are studied here with high resolution near- IR (HST) and submillimeter (ALMA) data. Each of the galaxies have extremely compact (eﬀective radius ∼1 kpc) submillimeter bright cores where almost all of the star-formation and 10−40% of the stellar mass is conﬁned. They will likely form the future bulges of their respective galaxies. These are embedded in copla- nar stellar disk-like structures which are 4−8× more extended in radius. Acknowledgements. First and foremost, we would like to express our gratitude to the anonymous referee for the valuable suggestions. We would also like to thank Gabriel Brammer for assistance with the use of grizli for the HST data reduction. R.M.R. acknowledges support from GO15910. V.S. acknowledges the support from the ERC-StG ClustersXCosmo grant agreement 716762. F.V. acknowledges support from the Carlsberg Foundation Research Grant CF18- 0388 “Galaxies: Rise and Death” and from the Cosmic Dawn Center of Excel- lence funded by the Danish National Research Foundation under then Grant No. 140. This paper makes use of the following ALMA data: 2019.1.00399. ALMA is a partnership of ESO (representing its member states), NSF (USA) and NINS (Japan), together with NRC (Canada), MOST and ASIAA (Taiwan), and KASI (Republic of Korea), in cooperation with the Republic of Chile. The Joint ALMA Observatory is operated by ESO, AUI/NRAO and NAOJ. The ﬁrst key new result that emerge from our work is that all three galaxies show marked stellar disk lopsidedness of varying degrees (from 1.6 to >3.0). The stellar morphology of two-thirds of galaxies (B and C) do not show signs of extreme dynamical disturbances however, hence limiting the role of major mergers. Although, the third (Galaxy A) is likely at an initial phase of collision with an equally massive counterpart. These are ﬁrst, unique cases of high redshift galaxies in which lopsidedness has been recognized to play an important role. A44, page 14 of 16 Acknowledgements. First and foremost, we would like to express our gratitude to the anonymous referee for the valuable suggestions. We would also like to thank Gabriel Brammer for assistance with the use of grizli for the HST data reduction. R.M.R. acknowledges support from GO15910. V.S. acknowledges the support from the ERC-StG ClustersXCosmo grant agreement 716762. 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(2006) for the minimum radius up to which gas may be stripped from a galaxy (rstrip): In order to ﬁt SFH models to the galaxies, we use the results of the surface brightness distributions in F160W (which also pro- vides the ﬂux in the band) and then obtain the rest of the pho- tometry using the best-ﬁt models as priors in all other bands. We ﬁt this photometry with BC03 stellar population models (Bruzual & Charlot 2003) to derive the properties of the three massive galaxies (Fig. 8). The redshift is ﬁxed to the spectro- scopic values previously published for the sources (Daddi et al. 2021). We use FAST++3 to determine the best ﬁtting SED tem- plates through a χ2 minimization procedure. We ﬁx the metal- licity to the solar value as is expected in massive galaxies and implement the Calzetti dust attenuation law (Calzetti et al. 2000), with a range of extinction values, AV = 0−5 in steps of 0.02. Finally, we use three diﬀerent sets of SFH models. rstrip = 0.5 ro × ln  G Mstar Mgas v2 gal ρICM 2π r2o . (B.1) (B.1) Here, the ro is the scale radius of the galaxies, while Mstar, Mgas corresponds to the gas and stellar mass of the galaxies. We note that vgal is the relative velocity between the galaxy and the ICM, for which we use the approximate virial velocity of the galaxies in RO-1001 (500 km s−1; Daddi et al. 2021). Finally, the density of the ICM (ρICM) is determined using (Boselli et al. 2022): Here, the ro is the scale radius of the galaxies, while Mstar, Mgas corresponds to the gas and stellar mass of the galaxies. We note that vgal is the relative velocity between the galaxy and the ICM, for which we use the approximate virial velocity of the galaxies in RO-1001 (500 km s−1; Daddi et al. 2021). Finally, the density of the ICM (ρICM) is determined using (Boselli et al. 2022): (B.2) ρICM = 1.15 ne mp. (B.2) ρICM = 1.15 ne mp. Firstly, we use constant star formation models, characterized by no decline in star formation (it remains constant throughout) to check if a high attenuation star-forming scenario is formally consistent with the available photometry. 3 https://github.com/cschreib/fastpp L29 Lotz, J. M., Madau, P., Giavalisco, M., Primack, J., & Ferguson, H. C. 2006, ApJ, 636, 592 Wotta, C. B., Lehner, N., Howk, J. C., et al. 2019, ApJ, 872, 81 Zaritsky, D., & Rix, H.-W. 1997, ApJ, 477, 118 Lustig, P., Strazzullo, V., D’Eugenio, C., et al. 2021, MNRAS, 501, 2659 Magdis G E Daddi E Béthermin M et al 2012 ApJ 760 6 Lustig, P., Strazzullo, V., D’Eugenio, C., et al. 2021, MNRAS, 501, 2659 Magdis, G. E., Daddi, E., Béthermin, M., et al. 2012, ApJ, 760, 6 y p Zolotov, A., Dekel, A., Mandelker, N., et al. 2015, MNRAS, 450, 2327 Magdis, G. E., Daddi, E., Béthermin, M., et al. 2012, ApJ, 760, 6 A44, page 15 of 16 A&A 666, A44 (2022) Appendix B: Prevalence of Ram Pressure Stripping We also use delayed τ-models. These feature an exponentially declining SFH that is characterized as ∝(t/τ2) e−t/τ with a peak of star formation at t = τ. The τ varies within [100 Myr, tobs] with steps of 0.1 dex, where tobs is the age of the universe at the redshift of the galaxy. Finally, we include the composite τ-models(Schreiber et al. 2018) which are similar to the τ-models, however with diﬀerent timescales (τrise and τdecl) for the rising and declining phases separated by the epoch tburst. We use the value of 10−3 cm−3 for the electron density (ne) as is expected in the core of clusters and groups (Vikhlinin et al. 2009; Sun et al. 2009; Pratt et al. 2010). We hence get an rstrip = 10 −15 kpc for the galaxies in RO- 1001. However, this should be regarded as a conservative lower- limit since RPS eﬃciency is expected the steeply decrease at high-redshifts. This is primarily due to the contribution of non- gravitational heating which eﬀectively reduces the ICM con- tent (Fujita 2001). Moreover, the decreasing eﬃciency of RPS at high redshifts (z ≳2) is also suggested through simulations in (Pfeﬀer et al. 2022). Hence, in each of our sample galaxies, the rstrip is found to be a factor of ≳2 larger than the stellar disks. SFRbase(t) ∝ ( e(tburst−t)/τrise fort > tburst, e(t−tburst)/τdecl fort ≤tburst. (A.1) SFRbase(t) ∝ ( e(tburst−t)/τrise fort > tburst, e(t−tburst)/τdecl fort ≤tburst. (A.1) (A.1) Both τrise and τdecl are varied within the range of [10 Myr, 3 Gyr], while for tburst a grid of [10 Myr, tobs] is used. Both τrise and τdecl are varied within the range of [10 Myr, 3 Gyr], while for tburst a grid of [10 Myr, tobs] is used. A44, page 16 of 16
https://openalex.org/W2767022532	https://repositorio.ul.pt/bitstream/10451/29351/1/Feola%20-%20II%20bozze.pdf	Italian	null	Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele	Pisa University Press eBooks	2,018	cc-by	9,691	* Il presente articolo è pubblicato nell’ambito del progetto di ricerca PRIN 2012: L’universalità e i suoi limiti: meccanismi di inclusione ed esclusione nella storia della filosofia e nei dibattiti filosofici contemporanei (Unità locale di Pisa, Scuola Normale Superiore), finanziato dal Ministero dell’Università e della Ricerca. Desidero ringraziare Tiziano Dorandi per i suggerimenti bibliografici circa le vicende della biblioteca di Aristotele, e Maria Michela Sassi per la supervisione durante il lavoro di ideazione, stesura e correzione. Ove non altrimenti indicato, tutte le traduzioni dal greco, dal latino e da altre lingue straniere sono mie. Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele Giuseppe Feola, Centro de Filosofia da Universidade de Lisboa Giuseppe Feola, Centro de Filosofia da Universidade de Lisboa 3 Moraux, ibid., p. 20. Nelle pagine seguenti Moraux nota che, a causa di questa diaspora, già 50 anni dopo la morte di Aristotele ben quattro città avrebbero potuto vantare copie delle opere del Nostro provenienti dalla sua stessa biblioteca, o comunque copiate direttamente dagli originali: Atene (sede del Liceo), Scepsi (dove forse si trovavano quelle copie che Neleo, avendole ricevute per testamento da Teofrasto, non aveva venduto a Tolomeo d’Egitto), Alessandria (dove si trovavano quelle vendute a Tolomeo), Rodi (dove aveva fatto ritorno Eudemo). 1 P. Moraux, Der Aristotelismus bei den Griechen von Andronikos bis Alexander von Aphrodisia, Berlin-New York, Walter de Gruyter, 1973-1984; trad. it. di G. Girgenti, L’Aristotelismo presso i Greci, Milano, Vita e Pensiero, 2000. 1. Proposito In questo contributo tratterò di alcune classiche questioni circa l’ordinamento e la coerenza dottrinaria del Corpus Aristotelicum, limitando il problema al corpus psicobiologico. Le questioni relative all’ordinamento sono le seguenti: qual è il rapporto tra le varie opere che lo compongono? Qual è il rapporto tra le singole parti di ciascuna opera? In che misura l’indice di ciascuna singola opera e del corpus nel suo insieme risale alla volontà ordinatrice di Aristotele, e in che misura invece l’eventuale lavoro di redattori posteriori ha sovraimposto all’opera di Aristotele criteri sistematici estranei alle sue intenzioni? Le questioni relative alla coerenza dottrinaria sono queste altre: in che misura le dottrine esposte nelle varie opere (e nelle varie parti di ciascuna opera) si armonizzano tra loro? Come vanno spiegate eventuali incongruenze? Si tratta di questioni che ho definito “classiche” perché appartengono al repertorio di domande che il XX secolo ha imparato a porre al corpus, e per Giuseppe Feola 36 rispondere alle quali si è addirittura originata una branca della bibliografia dotata di un suo specifico metodo e di sue particolari caratteristiche: una branca che si è sviluppata enormemente tra gli anni ’20 e gli anni ’80 del secolo scorso. p 2 Moraux, L’Aristotelismo, cit., vol. I, p. 20 n. 21. 4 E qui emerge il primo nodo problematico: Stratone, il successore di Teofrasto, avrebbe mai permesso a Neleo di portar via le copie delle opere di Aristotele, se il Liceo non ne avesse avute di altre? La difficoltà è sollevata da J. Barnes, Roman Aristotle, in Philosophia togata II. Plato and Aristotle at Rome, a cura di J. Barnes, M. Griffin, Oxford, Clarendon Press, 1997, p. 14. Anche secondo H.J. Drossaart-Lulofs (Neleus of Scepsis and the Fate of the Library of the Peripatos, in Les textes philosophiques et scientifiques grecs au Moyen Âge latin. Hommage à Fernand Bossier, a cura di R. Beyers, J. Brams, D. Sacré, K. Verrycken, Leuven, Leuven University Press, 1999, pp. 9-24) è lecito supporre che, se pure il testamento di Teofrasto avesse previsto il lascito di tutta la biblioteca a Neleo, è a dir poco implausibile che Stratone lasciasse partire la biblioteca per Scepsi senza opporsi: avrebbe ad esempio potuto ricorrere all’aiuto di uno dei moltissimi avvocati che in Atene erano specialisti nell’impugnare testamenti. 5 A questo elemento della storia Barnes è propenso a credere, proprio perché inverosimile. Barnes argomenta che, in generale, chi inventa storie allo scopo di essere creduto tende a inventare storie verisimili, e la nostra storia non lo è: perché mai i successori di Neleo avrebbero dovuto seppellire i libri, piuttosto che venderli a caro prezzo al re di Pergamo? (Barnes, Roman Aristotle, cit., p. 8). Ciò che Barnes nega, dunque, non è la veridicità della storia qui narrata; bensì che le copie di cui è questione in questa storia, e che furono sotterrate e rovinate dalle tarme, fossero tutte le copie disponibili (ibid., p. 14). 6 Cfr. Moraux, L’Aristotelismo, cit., vol. I, pp. 53-56. Barnes, Roman Aristotle, cit., p. 24, data la pubblicazione del lavoro di Andronico a dopo la morte di Cicerone (43 a.C.), 4 E qui emerge il primo nodo problematico: Stratone, il successore di Teofrasto, avrebbe mai permesso a Neleo di portar via le copie delle opere di Aristotele, se il Liceo non ne avesse avute di altre? La difficoltà è sollevata da J. Barnes, Roman Aristotle, in Philosophia togata II. Plato and Aristotle at Rome, a cura di J. Barnes, M. Griffin, Oxford, Clarendon Press, 1997, p. 14. Anche secondo H.J. Drossaart-Lulofs (Neleus of Scepsis and the Fate of the Library of the Peripatos, in Les textes philosophiques et scientifiques grecs au Moyen Âge latin. Hommage à Fernand Bossier, a cura di R. Beyers, J. Brams, D. Sacré, K. Verrycken, Leuven, Leuven University Press, 1999, pp. 9-24) è lecito supporre che, se pure il testamento di Teofrasto avesse previsto il lascito di tutta la biblioteca a Neleo, è a dir poco implausibile che Stratone lasciasse partire la biblioteca per Scepsi senza opporsi: avrebbe ad esempio potuto ricorrere all’aiuto di uno dei moltissimi avvocati che in Atene erano specialisti nell’impugnare testamenti. 5 A questo elemento della storia Barnes è propenso a credere, proprio perché inverosimile. Barnes argomenta che, in generale, chi inventa storie allo scopo di essere creduto tende a inventare storie verisimili, e la nostra storia non lo è: perché mai i successori di Neleo avrebbero dovuto seppellire i libri, piuttosto che venderli a caro prezzo al re di Pergamo? (Barnes, Roman Aristotle, cit., p. 8). Ciò che Barnes nega, dunque, non è la veridicità della storia qui narrata; bensì che le copie di cui è questione in questa storia, e che furono sotterrate e rovinate dalle tarme, fossero tutte le copie disponibili (ibid., p. 14). 6 Cfr. Moraux, L’Aristotelismo, cit., vol. I, pp. 53-56. Barnes, Roman Aristotle, cit., p. 24, data la pubblicazione del lavoro di Andronico a dopo la morte di Cicerone (43 a.C.), – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. in base al fatto che non ne troviamo menzione nelle opere e nell’epistolario dell’Arpinate, sempre attento alle novità bibliografiche. La datazione proposta da Barnes è chiaramente volta a ridimensionare l’importanza del lavoro di Andronico nel processo di diffusione delle opere esoteriche, visto che, se da un lato Cicerone non menziona Andronico, dall’altro però conosce e menziona una distinzione tra opere esoteriche ed essoteriche (De finibus bonorum et malorum, V, 5, 12). Secondo J. Dillon, The Reception of Aristotle in Antiochus and Cicero, in Brill’s Companion to the Reception of Aristotle in Antiquity, a cura di A. Falcon, Leiden-Boston, Brill, 2016, pp. 183-201, le opere esoteriche tornarono nel circuito librario proprio durante la vita di Cicerone, il quale, avendo accesso alla biblioteca di Silla, avrebbe potuto conoscerle e sfogliarle, ma, avendo in precedenza ammirato l’Aristotele dei dialoghi come maestro di stile, sarebbe in qualche modo rimasto deluso dalla sciatteria dell’esposizione di queste nuove opere, al punto da trascurarle completamente (ibid., p. 186): ciò spiegherebbe come mai le menzioni ciceroniane delle opinioni aristoteliche mostrino così poche somiglianze con le dottrine a noi familiari. Dillon esamina svariate menzioni di Aristotele nel corpus Ciceronianum, mostrando che in nessun caso abbiamo la certezza di avere a che fare con le opere a noi note, mentre viceversa in molti casi la discrepanza tra le dottrine delle opere esoteriche e quelle riportate da Cicerone è palese. La datazione proposta da Dillon per il ritorno in circolazione delle opere esoteriche è coerente coi racconti di Strabone e Plutarco. 7 Una voce fuori dal coro è Barnes, Roman Aristotle, cit., il quale attribuisce scarsissima importanza al lavoro di Andronico, a suo avviso molto maldestro: «I suspect that Andronicus’ textual activity aroused little interest among Aristotelian scholars and left little mark on Peripatetic scholarship. […] Andronicus merely published copies of corrupt manuscripts» (pp. 30-31; cfr. anche p. 33). E ancora: «There is no reason to think that the Peripatetic renascence was any more dependent on books; and there is no reason to think that Andronicus played midwife at the rebirth» (ibid., p. 66). Mi sembra però che Barnes avrebbe dovuto fornire una spiegazione alternativa di questa coincidenza cronologica tra l’operazione andronicea e il rifiorire del Peripato, piuttosto che limitarsi a negarne la correlazione. Una posizione più equilibrata è, forse, quella di M. 8 Cfr. Moraux, L’Aristotelismo, cit., vol. I, p. 57. Interessante l’osservazione di D. Lefebvre, Aristotle and the Hellenistic Peripatos: From Theophrastus to Critolaus, in Falcon (a cura di), Brill’s Companion, cit., pp. 13-34, secondo cui la lettura dei cataloghi delle opere degli immediati successori di Stratone alla guida del Peripato dà l’impressione che questi filosofi non conoscessero le opere di Aristotele a noi note (p. 29). 2. Posizione del problema A legittimare il tipo di domande che ho appena ricordato è in primo luogo la trasmissione del corpus. Pochissimi corpora della grecità (lasciando da parte il caso particolarissimo di Omero) vedono la loro autorialità gravata da così tante ipoteche come quello di Aristotele. Lo studio ritenuto più autorevole sulla sua trasmissione nelle generazioni immediatamente successive alla morte dell’autore, quello di Paul Moraux1, evidenzia la totale incertezza in cui ci troviamo circa la continuità della tradizione dopo la morte di Aristotele. Moraux afferma ad esempio che già la seconda generazione di intellettuali successiva ad Aristotele (quella di Posidonio e di Panezio) potrebbe non aver avuto accesso agli originali delle opere di scuola2. Tuttavia, lo stesso Moraux, avvalendosi per lo più di procedimenti congetturali fondati sulla qualità, quantità ed esattezza delle menzioni di Aristotele da parte di autori di quest’epoca, ipotizza che esistessero copie variamente circolanti di singole opere, di varia qualità filologica: da quelle che potremmo considerare fedelissime, provenienti dalla biblioteca di Aristotele e appartenenti a esponenti della ‘diaspora’ del Peripato3, alle copie non autorizzate prodotte dal mercato librario per venire incontro alla curiosità intellettuale di collezionisti e pubblico colto. Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 37 37 La fonte principale sulla sorte della biblioteca di Aristotele è Strabone, sulla veridicità della cui narrazione sono stati però avanzati forti dubbi. Strabone afferma che il figlio di Corisco, Neleo, dopo essere stato uditore di Aristotele e Teofrasto, ne avrebbe ereditato i libri e li avrebbe portati con sé a Scepsi4. Alla morte di Neleo, i suoi eredi, non interessati al sapere, tennero i libri in pessime condizioni; quando poi seppero che i re Attalidi cercavano libri per la costituzione della biblioteca di Pergamo, addirittura li nascosero in un fosso, dove furono maltrattati dall’umidità e dalle tarme5. Passò altro tempo, e i libri furono venduti al bibliofilo Apellicone di Teo, il quale avrebbe cercato di emendare maldestramente i testi corrotti dalle pessime condizioni di conservazione, mettendo così in circolazione libri pieni di errori. Quando Silla conquistò Atene, confiscò la biblioteca di Apellicone; di essa si curò il grammatico Tirannione; anche lui, però, non si sarebbe dimostrato (a giudizio di Strabone) all’altezza del compito (Geografia, XIII, 1, 54, 608-609). 2. Posizione del problema E molte vicissitudini ancora interverranno, prima di giungere all’intervento di Andronico di Rodi, che per primo (secondo la ricostruzione di Plutarco, Vita di Silla, 26)6 stabilisce un corpus riconosciuto, grazie al quale 38 Giuseppe Feola – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. Hatzamichali (Andronicus of Rhodes and the Construction of the Aristotelian Corpus, in Falcon (a cura di), Brill’s Companion, cit., pp. 81- 100): sarà pur vero che varie copie di singole opere dell’Aristotele esoterico circolavano già in età ellenistica; ma Andronico avrebbe reso disponibili al pubblico, in una veste unitaria e in un ordinamento sistematico, lavori che prima circolavano poco e ‘in ordine sparso’, e che perciò erano di difficile reperimento e di ancor più difficile consultazione. Anche se è esagerato dire che Andronico fece riemergere l’Aristotele esoterico dall’oblio, l’immissione sul mercato di copie nuove in un ordine sistematico sarebbe appunto – secondo Hatzamichali – quel fattore che può spiegare l’improvvisa crescita dell’interesse per Aristotele a partire dal I sec. a.C. e soprattutto la grande differenza tra il catalogo laerziano e quello di Tolomeo (cfr. infra nota 11). 39 Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 39 Quali che siano state queste vicissitudini, quali che siano stati i caratteri dell’intervento di Andronico, ciò che è importante per i nostri scopi in questa sede non è stabilire la portata del suo intervento, bensì la netta differenza che si può notare tra il catalogo pre-androniceo che troviamo in Diogene Laerzio e quelli post-andronicei9: nel primo gli scritti di scuola compaiono in scarso numero10, nel secondo compaiono massicciamente, e già con titoli tali da restituire la facies a noi familiare del corpus11. È importante a tal proposito una notizia che ci dà Porfirio, paragonando il proprio lavoro di editore di Plotino a quello di Andronico: Ἐπεὶ δὲ αὐτὸς [sc. Πλωτῖνος] τὴν διάταξιν καὶ τὴν διόρθωσιν τῶν βιβλίων ποιεῖσθαι ἡμῖν ἐπέτρεψεν, ἐγὼ δὲ κἀκείνῳ ζῶντι ὑπεσχόμην καὶ τοῖς ἄλλοις ἑταίροις ἐπεγγειλάμην ποιῆσαι τοῦτο, 9 Cfr. Moraux, L’Aristotelismo, cit., vol. I, pp. 68-69. I cataloghi delle opere di Aristotele sono riportati in V. Rose, Aristotelis qui ferebantur librorum fragmenta, editio stereotypa editionis primæ (MDCCCLXXXVI), Stuttgart, Teubner, 1966, pp. 1-22. Essi sono tre: quello di Diogene Laerzio (Vite dei filosofi, V, 21-27), quello di Esichio, e quello di Tolomeo. 10 Se dunque è vero che la lista di Diogene riflette i pìnakes delle biblioteche di Pergamo e Alessandria (come suppone T. Dorandi, Diogene Laerzio e la tradizione catalogica. Liste di libri nelle Vite e opinioni dei filosofi, in «Antiquorum philosophia», VII (2013), pp. 107-126, e in particolare p. 9 Cfr. Moraux, L’Aristotelismo, cit., vol. I, pp. 68-69. I cataloghi delle opere di Aristotele sono riportati in V. Rose, Aristotelis qui ferebantur librorum fragmenta, editio stereotypa editionis primæ (MDCCCLXXXVI), Stuttgart, Teubner, 1966, pp. 1-22. Essi sono tre: quello di Diogene Laerzio (Vite dei filosofi, V, 21-27), quello di Esichio, e quello di Tolomeo. 12 Barnes, Roman Aristotle, cit., passim, affronta anche la questione se quest’opera di riordinamento diede luogo a opere la cui costituzione interna era sensibilmente diversa da quella precedente, o se invece Andronico si limitò a indicare un ordine preferenziale di lettura per opere i cui indici erano già quelli che troviamo nella tradizione posteriore. Il paragone con ciò che Porfirio fece con le opere di Plotino spinge Barnes verso la seconda soluzione: «Porphyry says nothing at all about the creation of our Aristotelian treatises – he does not say that Andronicus invented Top. or put together EN or cobbled up Met. Nor he does imply such creative activity. His own work on Plotinus’ manuscripts did not involve the creation of ‘new’ works. Porphyry did not take an essay here and a squib there and unite them into a tract or treatise. On the contrary, he found fifty- four essays and he left fifty-four essays. He found the fifty-four essays in no order than the order of their writing, and he left the same fifty-four essays in what he took to be a more satisfactory and a more philosophical order» (ibid., pp. 39-40). Questo punto dell’argomentazione di Barnes non mi sembra convincente: il parallelo che Porfirio sta istituendo potrebbe essere benissimo tra i singoli trattati di Plotino (da lui ordinati in Enneadi) e i singoli libri di Aristotele che, in base a questo parallelismo, Andronico avrebbe ordinato in trattazioni più ampie; e, in tal caso, la testimonianza di Porfirio starebbe implicando proprio ciò che Barnes nega: che fu Andronico a ‘cucire insieme’ quelle trattazioni più ampie quali p.es. i Topici, la Fisica o la Metafisica, a partire dai singoli libri. – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. 123), allora dovremmo inferirne che nelle principali biblioteche del mondo ellenistico circolasse un corpus la cui facies era molto diversa da quella a noi familiare. Barnes, Roman Aristotle, cit., pp. 43-44 e p. 54, suggerisce che la differenza tra il corpus noto al pubblico ellenistico e quello post-androniceo rifletta solo il fatto che fino ad Andronico i medesimi libri che poi sarebbero comparsi nel nostro corpus circolassero già, ma come trattazioni separate, e dunque con titoli diversi da quelli con i quali noi li conosciamo; nel seguito dell’articolo Barnes ridimensiona ulteriormente la portata dell’intervento di Andronico. 11 Se avessimo solo il catalogo di Diogene Laerzio sarebbe per noi impossibile congetturare che Aristotele abbia scritto opere in qualche modo afferenti al corpus quale noi lo conosciamo, laddove, almeno in alcune sezioni del catalogo di Esichio, il Corpus Aristotelicum è già in qualche modo riconoscibile: in particolare è riconoscibile (cfr. la sez. 155-159) il blocco HA-MA-PA-GA. Sulla difficile questione delle liste delle opere di Aristotele, e sui problemi posti dal loro reciproco confronto, cfr. Dorandi, Diogene Laerzio, cit., p. 116: «Qualunque sia la fonte del catalogo delle opere di Aristotele che Diogene trasmette – se pure mai potrà essere determinata – quello che resta evidente e indiscutibile è il fatto che, all’epoca in cui questa lista venne redatta, alcuni libri di differenti trattati circolavano separati come unità indipendenti, che molte opere avevano titoli diversi da quelli con i quali le conosciamo oggi e che mancava inoltre una divisione delle opere in gruppi tematici». Giuseppe Feola 40 πρῶτον μὲν τὰ βιβλία οὐ κατὰ χρόνους ἐᾶσαι φύρδην ἐκδεδομένα ἐδικαίωσα, μιμησάμενος δὲ Ἀπολλόδωρον τὸν Ἀθηναῖον καὶ Ἀνδρόνικον τὸν Περιπατητικὸν, ὧν ὁ μὲν Ἐπίχαρμον τὸν κωμῳδιογράφον εἰς δέκα τόμους φέρων συνήγαγεν, ὁ δὲ τὰ Ἀριστοτέλους καὶ Θεοφράστου εἰς πραγματείας διεῖλε τὰς οἰκείας ὑποθέσεις εἰς ταὐτὸν συναγαγών κτλ. πρῶτον μὲν τὰ βιβλία οὐ κατὰ χρόνους ἐᾶσαι φύρδην ἐκδεδομένα ἐδικαίωσα, μιμησάμενος δὲ Ἀπολλόδωρον τὸν Ἀθηναῖον καὶ Ἀνδρόνικον τὸν Περιπατητικὸν, ὧν ὁ μὲν Ἐπίχαρμον τὸν κωμῳδιογράφον εἰς δέκα τόμους φέρων συνήγαγεν, ὁ δὲ τὰ Ἀριστοτέλους καὶ Θεοφράστου εἰς πραγματείας διεῖλε τὰς οἰκείας ὑποθέσεις εἰς ταὐτὸν συναγαγών κτλ. Poiché egli stesso [sc. – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. Plotino] mi aveva incaricato di effettuare la disposizione e la correzione dei [suoi] libri, ed io stesso a lui ancora vivo l’avevo promesso, e avevo annunciato agli altri compagni che l’avrei fatto, anzitutto ritenni giusto licenziare i libri (che erano stati prodotti [da Plotino] alla rinfusa) non in base ai tempi [di composizione], bensì imitando Apollodoro di Atene e Andronico il Peripatetico, di cui il primo raccolse le opere del commediografo Epicarmo riportandole in dieci libri, mentre il secondo divise in trattati le opere di Aristotele e Teofrasto riunendo in un’identica rubrica gli argomenti affini etc. (Porfirio, Vita di Plotino, 24, 2-12, trad. mia). Porfirio afferma di aver fatto, per le opere di Plotino, quel che Andronico avrebbe fatto per quelle di Aristotele: distribuire le opere in pragmatìe, mettendo insieme le trattazioni di argomento affine12. Il passo Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 41 di Porfirio sembra infatti implicare che fu Andronico a creare per esempio i Topici o la Fisica, mettendo assieme libri che prima circolavano come opere indipendenti. Questa ricostruzione ha il vantaggio di esser coerente con la differenza – che già abbiamo riscontrato13 – tra i cataloghi pre- e quelli post-andronicei. Se dunque questa informazione dataci da Porfirio è affidabile, ad Andronico dovrebbe risalire l’ordinamento delle opere aristoteliche. Se ora proviamo a estrarre un sunto da questa messe confusa di dati e di opinioni, vediamo che di certo vi è molto poco: un’eclissi dell’interesse per Aristotele nei 250 anni successivi alla sua morte e una tendenza ad attribuirgli dottrine diverse da quelle a noi note; un’improvvisa crescita dell’interesse per lui nel I sec. a.C., in concomitanza con le vicende narrate da Strabone e Plutarco e con l’opera di Andronico, di cui però sappiamo poco o nulla. Alla luce di ciò, il parere di Hatzamichali, secondo cui Andronico, pur senza produrre una vera e propria edizione critica, sarebbe il responsabile dell’ordinamento e della reimmissione nel circuito librario delle opere esoteriche, che prima circolavano in scarso numero e in ordine sparso, mi sembra condivisibile14. Ecco perché è legittimo porsi la questione se (e in che misura) l’ordinamento androniceo del corpus rispecchi le intenzioni di Aristotele (ammesso che Aristotele avesse delle precise intenzioni riguardo all’ordinamento delle sue opere). Cfr. supra, nota 12. 14 Hatzamichali, Andronicus of Rhodes, cit., passim. 13 Cfr. supra, nota 12. 14 h l d 15 W. Jæger, Aristoteles. Grundlegung einer Geschichte seiner Entwicklung, Berlin, Weidmannsche Buchhandlung, 1923; trad. it. di G. Calogero, Aristotele. Prime linee di una storia della sua evoluzione spirituale, con aggiunte e appendice dell’Autore, Firenze, La Nuova Italia, 1935, rist. 1968. 16 F. Nuyens, Ontwikkelingsmomenten in de zielkunde van Aristoteles, Njimegen-Utrecht, Dekker & Van de Vegt, 1939; trad. fr. di T. Schillings, L’évolution de la psychologie d’Aristote, Louvain, Insitut Supérieur de Philosophie, 1948. 17 D. Lanza, M. Vegetti, Aristotele. Opere biologiche, Torino, UTET, 1971, rist. 1996; cfr. in particolare il breve saggio Origini e metodi della zoologia aristotelica nella Historia Animalium (ibid., pp. 77-128): secondo Vegetti e Lanza, la Historia animalium, concepita in origine come summa generale del sapere di Aristotele in campo biologico, sarebbe stata poi degradata dall’autore a mera raccolta e collezione di dati, quando l’articolarsi sempre maggiore dell’approccio ilomorfico e della tematica della potenza-atto avrebbe imposto l’elaborazione di una nuova biologia: quella del De partibus e del De generatione (cfr. ibid., p. 82). g q p g ( p ) 18 D.M. Balme, The Place of Biology in Aristotle’s Philosophy, in Philosophical Issues in Aristotle’s Biology, a cura di A. Gotthelf, J.G. Lennox, Cambridge-New York, Cambridge University Press, 1987, pp. 9-20. – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 43 Viceversa, Lennox insiste alquanto sul ruolo di ciascun trattato nell’insieme organico del corpus, giungendo, in nome di tale organicità, a sposare un’ipotesi a suo tempo proposta da Balme18, e a rovesciare l’ipotesi abbastanza diffusa della priorità temporale della Historia rispetto al De partibus: la Historia contiene un maggior numero di osservazioni naturalistiche rispetto al De partibus, e costituirebbe l’implementazione empirica del programma schizzato nel De partibus19. Nell’affrontare la questione, nelle prossime pagine mi volgerò ai due seguenti problemi: (1) qual è il rapporto che intrattengono le singole opere biologiche tra loro? (2) qual è il rapporto del corpus biologico col De anima? – almeno secondo l’opinione più diffusa7 – il Peripato, prima in declino, riconquista un posto di primo piano tra le scuole filosofiche8. Si noti infatti che Andronico vive in un’epoca nella quale, proprio dopo la svolta nell’organizzazione e nell’esposizione del sapere segnata da Aristotele, la forma del trattato sistematico è ormai divenuta la forma paradigmatica della comunicazione del sapere scientifico e filosofico, cosa che certo non può ancora dirsi della prima metà del IV sec. a.C., quando l’esposizione della scienza e della filosofia avveniva per lo più tramite la forma dello scritto breve oppure del dialogo. 42 Giuseppe Feola Se, da un lato, non è più molto frequente vedere proposte ermeneutiche simili a quelle di Jæger15 e Nuyens16, che si sentivano in grado di stabilire quali capitoli e capoversi della tale o tal altra opera andassero assegnati a quale periodo, di discutere della successione cronologica di singole frasi che differiscono tra loro solo per diverse sfumature nella presentazione lessicale dello stesso concetto, e di ricostruire la biografia intellettuale di Aristotele sulla base di questi indizi, non è però più nemmeno possibile (ed è questo il guadagno sostanziale della prospettiva jægeriana) assumere ingenuamente che il corpus sia espressione di una dottrina unitaria e coerente dal punto di vista dei contenuti: è certo lecito sostenere questa opinione, ma bisogna argomentarla; meno ancora è possibile assumere che il corpus sia la manifestazione di un insegnamento monolitico dal punto di vista dell’esposizione, simile alle trattazioni sistematiche, accuratamente pianificate anche dal punto di vista dell’architettonica generale, proposte da autori moderni come Spinoza o Cartesio: ancora una volta, è possibile sostenerlo, ma bisogna argomentarlo. In questo quadro è normale che appaiano proposte radicalmente opposte tra loro, come vedremo subito affrontando finalmente i problemi specifici posti dalle opere biologiche. Ad esempio, Vegetti e Lanza affermano che la Historia animalium apparterrebbe a una fase del pensiero scientifico di Aristotele i cui principii sarebbero stati sconfessati dagli sviluppi successivi della sua metafisica, e che il corpus biologico sarebbe nato dalla saldatura artificiale tra i due blocchi, di diversa impostazione epistemologica, costituiti l’uno dalla Historia, l’altro dal De partibus e dal De generatione animalium17. g q p g 18 D.M. Balme, The Place of Biology in Aristotle’s Philosophy, in Philosophical Issues in Aristotle’s Biology, a cura di A. Gotthelf, J.G. Lennox, Cambridge-New York, Cambridge University Press, 1987, pp. 9-20. 19 J.G. Lennox, Aristotle. On the Parts of Animals, Oxford, Clarendon Press, 2001, rist. 2004, Introduction, p. xiv. 20 Cfr. Jæger, Aristotele, cit., pp. 417-418 e 448. 19 J.G. Lennox, Aristotle. On the Parts of Animals, Oxford, Clarendon Press, 2001, rist. 2004, Introduction, p. xiv. 20 Cfr. Jæger, Aristotele, cit., pp. 417-418 e 448. y pp 19 J.G. Lennox, Aristotle. On the Parts of Animals, Oxford, Clarendon Press, 2001, rist. 19 J.G. Lennox, Aristotle. On the Parts of Animals, Oxford, Clarendon Press, 2001, rist. 2004, Introduction, p. xiv. 20 f l 4 4 44 University Press, 1987, pp. 9-20. 19 J.G. Lennox, Aristotle. On the Parts of Animals, Oxford, Clarendon Press, 2001, rist. 2004, Introduction, p. xiv. 20 Cfr. Jæger, Aristotele, cit., pp. 417-418 e 448. p 22 Ibid., p. 448: «È ormai difficile stabilire esattamente quale parte abbia avuta di persona Aristotele nella redazione della Storia degli animali». 21 Ibid., p. 330. , pp 24 Ibid., p. 398: «Quel che si ottiene in tal modo è, nel migliore dei casi, solo la successione prevista dallo stesso Aristotele, al termine della sua attività letteraria, dal punto di vista pedagogico e contenutistico». 23 Ibid., pp. 451-452. 21 Ibid., p. 330. 25 Ibid., pp. 510-511: «Ciò accade per la Storia degli animali non altrimenti che per la Metafisica e la Politica. Abbozzi di ordinamento sistematico, spesso inseriti solo durante il g 23 Ibid., pp. 451-452. 3. Opinioni autorevoli circa il rapporto tra le varie opere del corpus biologico Per quanto riguarda i rapporti interni al corpus biologico, circa i quali abbiamo già menzionato le opinioni di Vegetti e Lanza e di Lennox, sarà ora il caso di vedere cosa ne dice il fondatore dell’approccio ‘evolutivo’ al pensiero di Aristotele, cioè Jæger. Jæger non dedica al corpus psicobiologico un capitolo specifico nel suo trattato: menziona le opere biologiche solo per asserire, come se si trattasse di un corollario ovvio della sua argomentazione principale, che opere così piene di dettagli empirici devono necessariamente essersi originate nel momento in cui Aristotele era massimamente lontano dallo spirito dell’Accademia platonica, e cioè alla fine della sua vita20. Jæger apporta anche argomentazioni più circostanziate per una datazione tarda 44 Giuseppe Feola delle opere biologiche: per esempio il fatto che le notizie sugli animali esotici contenute nella Historia animalium sarebbero potute giungere in Grecia solo tramite la spedizione di Alessandro21. Per quanto riguarda le relazioni interne al corpus biologico, Jæger ipotizza che la Historia stesse alle opere più speculative nel medesimo rapporto in cui la raccolta delle Costituzioni stava alla Politica: si tratterebbe di raccolte di dati, che Aristotele avrebbe delegato a più persone; in tal modo si spiegherebbero le incongruenze che di tanto in tanto è possibile rinvenire nell’esposizione22. È poi notorio lo iato che Jæger crede di individuare tra il III libro del De anima e tutto il resto della psicologia di Aristotele: il III libro sarebbe un relitto della fase platonica del pensiero di Aristotele23. Si noti che Jæger non nega che sia ammissibile anche un ordine sistematico di lettura del corpus; egli però afferma che tale ordine restituirebbe solo l’ordine che Aristotele, alla fine della sua carriera, assegnò al corpus, e non quello – a suo avviso più significativo – in cui l’autore compose le sue opere24. posteriore lavoro di fusione, restano senz’alcuna applicazione o vengono applicati solo a metà. Quella della sistemazione esteriore non è stata un’idea originaria di questo architetto, e quindi pp t l i t i p ò ‘ i t i ’ pp p 26 La tesi è esposta diffusamente in Nuyens, L’évolution, cit., passim. 27 Balme, The Place, cit. 28 Ibid., p. 12. 27 Balme, The Place, cit. poster ore avoro d fus o e, resta o se z a cu a app caz o e o ve go o app cat so o a età. Quella della sistemazione esteriore non è stata un’idea originaria di questo architetto, e quindi neppure tale sistema si può ‘ricostruire’». Q g q neppure tale sistema si può ‘ricostruire’». 3. Opinioni autorevoli circa il rapporto tra le varie opere del corpus biologico Se da un lato Jæger ha di certo ragione nell’affermare che è arbitrario (e anche ingenuo) presumere che l’ordine di composizione debba rispecchiare quello indicato come preferibile per la lettura sistematica, dall’altro la giustificazione del fatto che l’ordine sistematico (quale che sia) dovrebbe essere meno significativo di quello cronologico di composizione sta tutta in una intuizione che Jæger ritiene di avere circa la natura dello spirito di Aristotele: Jæger considera del tutto estraneo allo spirito di Aristotele qualsiasi tentativo di cercare o costruire un’architettura generale del sapere; la precedenza, almeno nel tardo Aristotele, sarebbe data sempre al particolare25, e l’importanza Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 45 delle ricerche del tardo Aristotele starebbe nell’aver dato l’avvio a una nuova stagione dello spirito greco, quello della scienza ellenistica; quindi: nessuna architettonica generale del sapere (se non a un livello molto estrinseco), e dunque nessun ordine sistematico delle opere. L’unico ordine che conta è quello genealogico, che Jæger crede di aver scoperto. Com’è noto, Nuyens tentò di estendere al corpus psicobiologico il metodo jægeriano, giungendo a una diversa cronologia relativa delle opere del Nostro: le opere biologiche, nella misura in cui manifestano la tendenza a descrivere la relazione anima-corpo mediante la metafora ‘artigiano- strumento’ (concezione da Nuyens chiamata “strumentalismo vitalistico”), apparterrebbero non alla fase più tarda dell’attività di Aristotele, ma ad una intermedia tra il (presunto) dualismo giovanile e l’ilomorfismo ‘compiuto’ di De anima II26. In un contributo della metà degli anni ’80 D.M. Balme ha riproposto (ma in modo più minimalista di quanto fosse consueto nell’approccio jægeriano, e senza pretese di disegnare l’intero sviluppo intellettuale dell’autore), l’idea che vi sia stato uno sviluppo del pensiero di Aristotele, mettendone al centro l’impatto che sulle sue concezioni metafisiche avrebbero avuto gli studi biologici27. Balme osserva che il De partibus animalium dispiega una descrizione degli animali per classi, cioè per specie, mentre il De generatione animalium si concentra sulla trasmissione del patrimonio genetico individuale28. 29 Da qui in poi parlerò, a questo proposito, di forme “sub-specifiche” e non “individuali”, perché (a) ai fini della presente discussione basta ipotizzare che Aristotele sia giunto a porre delle forme sub-specifiche; (b) credo che le forme individuali pongano dei problemi ulteriori, che è meglio non sollevare qui. g 30 Ibid., pp. 18-20. p 34 Ibid., p. 13. HA costituirebbe il punto di riferimento databile, visto che, com’è noto, moltissimi dati lì esposti si riferiscono ad animali tipici della zona di Lesbo, in particolare della famosa laguna di Pyrrha, oggi Kalloni, e a Lesbo Aristotele visse tra il 344 e il 342 a.C. 35 Ibid., pp. 17-18. 33 Ibid., p. 16. 31 Ibid., p. 11. 32 Ibid., p. 17. 3. Opinioni autorevoli circa il rapporto tra le varie opere del corpus biologico Il cambiamento di approccio sarebbe stato dovuto alla scoperta, avvenuta al momento di lavorare alla Historia animalium, di differenze che stanno, nell’albero classificatorio, al di sotto del livello della specie, e così fini (secondo Balme) da poter giungere a definire l’individuo: Balme osserva che la teoria della trasmissione del patrimonio genetico fornita nel De generatione Giuseppe Feola 46 animalium, di fatto, suppone che vi sia una forma individuale, ulteriormente specificata rispetto a quella della specie: che si tratti di forme individuali o di forme sub-specifiche29, resta il fatto che GA propone, sulla base di ricerche empiriche, una soluzione a una questione sulla quale, invece, Metafisica VII si dibatte in discussioni dialettiche che non portano a risultati conclusivi30. Quindi la Historia e il De generatione sarebbero posteriori a Metaph. VII31. Viceversa, De partibus II-IV, con le sue considerazioni assiologiche che ricorderebbero quelle del Timeo, dovrebbe essere anteriore, addirittura del periodo accademico32. Balme può sostenere questa ricostruzione perché afferma, en passant, che le informazioni empiriche contenute nel De partibus non sarebbero più accurate di quelle a disposizione di qualunque persona colta, e che il vero salto nella raccolta dei materiali Aristotele l’avrebbe compiuto solo con la Historia33. L’ordine di scrittura proposto da Balme è dunque: (1) PA II-IV e IA al tempo dell’Accademia; (2) PA I al tempo di Physica II e Metaph. VII, pure nel periodo accademico ma dopo i trattati prima menzionati; (3) PN alla fine del periodo accademico; (4) HA iniziato nel periodo accademico ma continuato durante i viaggi34; (5) GA35. Come si vede, la ricostruzione di Balme si fonda su due assunti non proprio pacifici: (I) che tanta parte dell’opera di Aristotele (inclusa la grande elaborazione della metafisica della sostanza, fondamentale per l’impostazione del De partibus) risalga al periodo accademico, (II) che le informazioni zoologiche, anatomiche, embriologiche, del De partibus e del De generatione non richiedessero una ricerca Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 47 specifica, sul campo, che superasse in dettaglio e accuratezza le informazioni già disponibili mediante la così detta ‘cultura generale’. Questo secondo assunto, in particolare, mi sembra insostenibile. Secondo Balme, al momento di redigere la Historia, Aristotele vi avrebbe inserito un riassunto schematico delle informazioni già raccolte nelle altre due opere, e vi avrebbe aggiunto quelle raccolte di persona, alcune delle quali sarebbero anche in conflitto con le teorie espresse nei trattati preesistenti36. 36 Ibid., pp. 13-15. Esempi assortiti di luoghi in cui le osservazioni raccolte nella Historia confliggerebbero con le teorie del De partibus e del De generatione sono esposti alle pp. 14-15. 37 Ibid., p. 17. 38 Ibid., pp. 12-13. gg 37 Ibid., p. 17. p 38 Ibid., pp. 12-13. 39 M. Furth, Aristotle’s Biological Universe: an Overview, in Philosophical Issues in Aristotle’s Biology, a cura di A. Gotthelf, J.G. Lennox, Cambridge-New York, Cambridge University Press, 1987, pp. 21-52. 3. Opinioni autorevoli circa il rapporto tra le varie opere del corpus biologico La Historia sarebbe, nella ricostruzione di Balme, un work-in-progress, un laboratorio di appunti iniziato a Lesbo, e che andò crescendo per tutta la durata della vita di Aristotele, e alla quale non fu mai data l’ultima mano37. Perciò l’ordinamento enunciato in HA 491a7-14, che pone HA prima di PA e GA, si riferirebbe invece a un ordine di lettura stabilito a scopo didattico in una fase successiva, nella quale sarebbero stati anche aggiunti i riferimenti incrociati, i quali, sia dal punto di vista del contenuto sia da quello della sintassi, possono esser rimossi senza alterare l’ordine dell’esposizione. Ad avviso di Balme, infine, informazioni più dettagliate sull’ordine di lettura consigliato da Aristotele sono per noi inaccessibili, a causa dell’opera di riedizione di Andronico, che avrebbe stabilito l’archetipo dei manoscritti che abbiamo, dopo tutte le vicissitudini narrate in Strabone (al cui racconto Balme ritiene si possa dar credito)38. Ci limiteremo qui a rilevare che, visto che la correlazione tra la strutturazione del vivente delineata nel De partibus animalium e l’ontologia sviluppata in Metaph. VII-IX sembra evidente al di là di qualunque dubbio, è chiaro che le proposte di Balme non potranno trovare accoglienza in chi non sia disposto ad ammettere una datazione così alta di lavori (i libri centrali della Metafisica) normalmente considerati rappresentativi del pieno sviluppo filosofico dello Stagirita. Ricordiamo fra l’altro che Furth ha addirittura Giuseppe Feola 48 proposto un’analisi dell’ontologia di Aristotele interamente incentrata sull’idea che Metaph. VII-IX sia una discussione formale sui principii della biologia e su come si debba concepire il che cos’è di un organismo, in particolare di un animale39, idea che trovo tutt’altro che stravagante, e anzi notevolmente centrata. y pp 40 C. Natali, Aristotele, Roma, Carocci, 2014. La biologia è trattata sotto la rubrica generale de Lo studio del mondo fisico (pp. 85-188), e in particolare alle pp. 139-166 (La fisica della natura vivente). 41 Cfr. Natali, Aristotele, cit., p. 141. Di tanto in tanto, però, anche Natali avanza qualche dubbio circa il fatto che la ripulitura dell’opera sia stata perfettamente compiuta, come quando osserva che il GA fa talora sorgere il sospetto di una doppia redazione (cfr. p. 159). 42 Cfr. Natali, Aristotele, cit., p. 149 per il rapporto tra PA e HA, e pp. 160 e 162 per quello tra HA e IA; quanto a GA, si tratterebbe del naturale completamento di PA (cfr. p. 142, dove si cita GA 715a1-18, e p. 154), e di conseguenza starebbe, con HA, in un rapporto analogo a quello in cui si trova PA (lo studio delle cause, 782a22, cfr. p. 159). 4. Stato dell’arte Mentre la questione della cronologia relativa della composizione delle diverse opere sembra rimanere irresolubile, circa la questione della corrispondenza tra le intenzioni di Aristotele e l’indice del corpus (quale è possibile ricostruirlo dai richiami incrociati tra le diverse opere, e in particolare dalle dichiarazioni programmatiche poste negli incipit e negli explicit) si segnala, negli ultimi anni, un maggiore ottimismo rispetto alle posizioni di Jæger, Nuyens, o anche di Balme. Senza voler discutere decine di autori e contributi, che spesso prendono posizione su questo o quel dettaglio dell’indice del corpus solo nella misura in cui la questione è rilevante per la loro esegesi delle dottrine dello Stagirita, si può osservare, come tendenza generale, una certa fiducia a ‘prendere per buono’ l’ordinamento del corpus a noi tramandato. Un buon esempio è il recente volume di Carlo Natali40, il quale – proprio perché informato da finalità quasi divulgative – non entra nelle minuzie dell’argomentazione e ci offre un ‘precipitato’ di quel che oggi quasi tutti assumono nel momento in cui si apprestano a interpretare il corpus psicobiologico: l’ordine di lettura suggerito dalla rete dei richiami incrociati non rispecchierebbe l’ordine cronologico della stesura, ma comunque sarebbe stato escogitato dall’autore, a scopo didattico41. Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 49 E il perno fondamentale di tale ordinamento è la distinzione tra esposizione dei ‘fatti’ che troviamo nella Historia animalium ed esposizione delle cause, che troviamo nel De partibus e nel De generatione. Quanto Aristotele scrive in HA I 6, 491a7-14, sembra volerci suggerire questo orientamento: Ταῦτα μὲν οὖν τοῦτον τὸν τρόπον εἴρηται νῦν ὡς ἐν τύπῳ, γεύματος χάριν περὶ ὅσων καὶ ὅσα θεωρητέον· δι’ ἀκριβείας δ’ ὕστερον ἐροῦμεν, ἵνα πρῶτον τὰς ὑπαρχούσας διαφορὰς καὶ τὰ συμβεβηκότα πᾶσι λαμβάνωμεν. Μετὰ δὲ τοῦτο τὰς αἰτίας τούτων πειρατέον εὐρεῖν. Queste cose [sc. l’introduzione generale alla Historia fornita nei capp. precedenti] sono state ora dette in questo modo come in abbozzo, per dare un assaggio circa le varie cose che bisogna indagare: in seguito ne parleremo con precisione, affinché anzitutto afferriamo le differenze esistenti [tra i vari animali] e gli accidenti di tutti [gli animali]. Dopo di ciò, bisognerà tentare di trovare le cause di queste cose. (Historia animalium, I 6, 491a7- 14, trad. mia). Queste cose [sc. l’introduzione generale alla Historia fornita nei capp. 43 Questa la ricostruzione dell’indice della Historia animalium fornita da G.E.R. Lloyd, Aspects of the Relationship between Aristotle’s Psychology and his Zoology, in Essays on Aristotle’s De Anima, a cura di M.C. Nussbaum, A.O. Rorty, Oxford, Clarendon Press, 1992, pp. 147- 167 (cfr. in particolare p. 155); ristampato con qualche adattamento come The Relationship of psychology to zoology, in G.E.R. Lloyd Aristotelian Explorations, Cambridge-New York, Cambridge University Press, 1996, pp. 38-66. 4. Stato dell’arte precedenti] sono state ora dette in questo modo come in abbozzo, per dare un assaggio circa le varie cose che bisogna indagare: in seguito ne parleremo con precisione, affinché anzitutto afferriamo le differenze esistenti [tra i vari animali] e gli accidenti di tutti [gli animali]. Dopo di ciò, bisognerà tentare di trovare le cause di queste cose. (Historia animalium, I 6, 491a7- 14, trad. mia). Dunque, stando al proposito qui esplicitato, la Historia esporrebbe i fatti, il “che”, mentre PA e GA (e IA) esporrebbero le cause dei fatti, il “perché”42. Come però è stato osservato da numerosi interpreti, i “fatti” esposti in HA non vanno concepiti come un mero centone di osservazioni ‘pure’: oltre al fatto che le osservazioni sono sempre (in Aristotele come in ogni altro scienziato) guidate da un’intenzione teorica, vi è da notare, nel caso particolare di HA, che tale intenzione teorica è esplicita; per giunta, le osservazioni vi sono catalogate in base a quattro grandi rubriche, stabilite secondo un criterio sulla cui scelta Aristotele si interroga consapevolmente: 50 Giuseppe Feola dopo aver discusso, appunto, tale questione (HA I 1-6), da HA I 7 fino a tutto l’intero libro IV si estende la descrizione ragionata delle differenze tra le parti organiche (μόρια), nei libri V-VIII sono descritte le differenze tra le “operazioni vitali” (πράξεις, βίοι), mentre il libro IX discute le “abitudini” (ἤθη) degli animali43. Vediamo adesso quali sono le ricadute di queste considerazioni sullo status di quel (particolarissimo) testo che è il corpus biologico. 5. Che tipo di testo è il corpus aristotelico? Nell’iniziare a lavorare su questo problema, non avevo alcuna opinione consolidata circa tutte le questioni che finora abbiamo affrontato. Ma mi sono convinto via via che gli indizi relativi alle discrepanze di contenuto tra le diverse opere, che gli interpreti usano per valutare l’anteriorità o la posteriorità di un’opera rispetto a un’altra, sono spesso (anzi, direi quasi sempre) interpretabili sia nel modo voluto dall’interprete, sia in modo specularmente opposto. Un ottimo esempio sono i casi in cui la Historia presenta una forma ‘compatta’, o addirittura dà solo un accenno, di descrizioni che nel De partibus si trovano in forma estesa: Ἐν μὲν οὖν ἐνίοις τὰ αἰσθητήρια φανερώτατά ἐστι, τὰ μὲν τῶν ὀμμάτων καὶ μᾶλλον. In alcuni [animali] gli organi di senso sono evidentissimi, e soprattutto quelli degli occhi. (Historia animalium IV, 8, 533a19-20, trad. mia). 51 Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 51 E qui finisce quel che Aristotele, nella Historia, ha da dire sugli occhi. La corrispondente trattazione, nel De partibus, comprende l’intero capitolo II 13, per un totale di una colonna e mezza circa dell’edizione Bekker44. Cos’è avvenuto, qui? che Aristotele ha espanso, in PA, materiali osservativi precedentemente stoccati in forma più schematica in HA (ipotesi standard, rappresentata p.es. da Natali)? oppure che HA riassume le informazioni proprio perché PA le aveva già fornite in maniera più dettagliata (ipotesi Balme)? La questione è chiaramente indecidibile. L’idea che HA esponga il ‘che’, mentre PA, IA e GA ricerchino il ‘perché’, è evidentemente da prendere con la massima serietà, visto che è quella ufficiale di Aristotele; ma, come già detto qui e come più volte osservano Vegetti e Lanza, la Historia non può certo considerarsi come una mera collezione di osservazioni, e non è certo priva di indagini eziologiche: quindi anche qui c’è qualcosa di non chiaro. Presenterò adesso qualche mia osservazione, sulla base dell’idea di Furth, già menzionata, che i libri centrali della Metafisica siano una discussione sul che cos’è di un organismo, e dell’osservazione di Balme, secondo cui il De generatione animalium prende partito a favore dell’esistenza delle forme sub-specifiche, idea che né in De partibus animalium né in Metaph. VII- IX è ancora articolata (nel migliore dei casi, è presentata come un qualcosa di problematico). 44 In compenso, subito dopo il brevissimo accenno alla vista, la Historia si addentra, senza in alcun modo avvertire il lettore che verrà introdotta una digressione, in una minuta discussione sulle esperienze dei pescatori che dimostrano che i pesci hanno il senso dell’olfatto. Sono giunture come queste che conferiscono alla Historia il suo tipico aspetto da work-in-progress. 45 Che Aristotele avesse in mente un’opera di etologia, analoga a ciò che PA è per l’anatomia e GA per l’embriologia? È lecito supporre che il De motu animalium fosse il I libro di quest’opera, che ne esponeva l’architettura eziologica, più o meno come fa PA I per l’insieme del De partibus? 5. Che tipo di testo è il corpus aristotelico? Si assuma che GA sia l’ultima (per i motivi detti) tra le opere biologiche; si può poi ipotizzare (come è ampiamente accettato dagli studiosi) che Metaph. VII-IX e PA appartengano ad un unico periodo, anteriore a quello di GA. In questo periodo Aristotele non ha ancora accettato come pacifica l’esistenza di forme a livello sub-specifico: questo periodo però non può essere (come vorrebbe Balme) quello tardo-accademico, perché PA presuppone un corpus di conoscenze che non è (a differenza di ciò che Balme 52 Giuseppe Feola pensa) quello di una normale persona colta, ma che richiede (almeno una parte sostanziale del)le osservazioni raccolte in HA. Si dovrà dunque tornare al vecchio schema di successione HA-PA-GA, con HA a funger da punto di riferimento databile grazie al suo ancoramento al periodo dei viaggi? Non necessariamente, se diamo il debito peso ai fatti evidenziati da Balme: che solo una parte delle osservazioni contenute in HA sono sfruttate in PA, e che proprio alcune delle osservazioni contenute in HA potrebbero aver contribuito a portare dal paradigma di PA a quello di GA e alla scoperta delle varietà sub-specifiche. In altri termini, io difendo (con la communis opinio) l’idea che la Historia animalium sia la grande raccolta di materiali dalla quale Aristotele ha tratto la maggior parte dei dati contenuti nelle altre opere, ma con i seguenti caveat: Aristotele (a differenza di quanto pensano Vegetti e Lanza) non ha ‘degradato’ HA dal livello di trattato a quello di raccolta di materiali quando la sua ontologia è cambiata, ma ha da subito concepito HA come tronco principale di cui tutti gli altri trattati biologici sono i rami; e (soprattutto) non ha mai smesso di arricchire HA, la cui stesura si deve dunque concepire come una concrezione progressiva fermatasi solo con la morte dell’autore. Non difendo, quindi, l’idea che HA fosse una raccolta di materiali strettamente ancillare alla stesura dei due trattati teorici (e di quelli soli): basti pensare a tutti gli spunti sull’etologia degli animali, che non trovano sviluppo nei trattati teorici45; ma non difendo nemmeno l’idea che HA (come vogliono Vegetti e Lanza) rappresenti uno stadio precoce della scienza aristotelica, anteriore e addirittura alternativo a quello di PA e GA. Il testo della Historia animalium corrisponde, a mio avviso, al risultato Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 53 dell’ultimo stadio di riordinamento degli appunti personali di Aristotele biologo. 5. Che tipo di testo è il corpus aristotelico? Stadio necessariamente provvisorio, come provvisorio è ogni work- in-progress, e dunque approssimativo: così si spiegherebbero le incongruenze di contenuto, le descrizioni e le ‘schede’ a volte incompiute, le intromissioni di determinate osservazioni in contesti inappropriati se valutati dal punto di vista sistematico; così si potrebbero (forse) spiegare persino i contenuti dei due ultimi libri, i cui tratti non ortodossi sono stati notati da più studiosi: potrebbe trattarsi di raccolte di informazioni che Aristotele non avrebbe avuto il tempo di vagliare ed emendare. Che poi questa raccolta di materiali possa anche essere stata ampliata da successori di Aristotele, o che Aristotele stesso possa aver delegato la raccolta dei materiali a vari collaboratori, che cioè HA possa avvicinarsi allo statuto di opera ad autorialità collettiva (e che ciò possa valere in particolare per i due ultimi libri), è un fatto aggiuntivo, che può integrarsi con l’ipotesi che ora ho delineato. 46 Il principio è enunciato chiaramente in de An. II 1, 412a27-b1. Una nota conseguenza di questo postulato (noto come “principio di ominimia”) viene esplicitata in Mete. IV 12, 390a10-20: ciò che non è più in grado di svolgere la propria funzione perde la propria essenza; per l’applicazione di questo principio alle parti organiche, cfr. in particolare le rr. 11-12: l’occhio è davvero tale solo se capace di servire alla funzione visiva. 6. Rapporto tra il De anima e il corpus biologico Potremmo dire, in estrema sintesi, che nelle opere biologiche Aristotele tratta della natura animale da due punti di vista, il punto di vista delle parti e quello delle funzioni; i due punti di vista sono quasi sempre integrati tra loro; e ciò, in conformità con l’idea fondamentale dell’anatomia funzionale tipica dello Stagirita, in base alla quale la parte è “in vista” della funzione che svolge ed è da essa definita46: Ἐπεὶ δὲ τὸ μὲν ὄργανον πᾶν ἕνεκά του, τῶν δὲ τοῦ σώματος μορίων ἕκαστον ἕνεκά του, τὸ δ’ οὗ ἕνεκα πρᾶξίς τις, φανερὸν ὅτι καὶ τὸ Ἐπεὶ δὲ τὸ μὲν ὄργανον πᾶν ἕνεκά του, τῶν δὲ τοῦ σώματος μορίων μ ργ μ μ ρ ἕκαστον ἕνεκά του, τὸ δ’ οὗ ἕνεκα πρᾶξίς τις, φανερὸν ὅτι καὶ τὸ 46 Il principio è enunciato chiaramente in de An. II 1, 412a27-b1. Una nota conseguenza di questo postulato (noto come “principio di ominimia”) viene esplicitata in Mete. IV 12, 390a10-20: ciò che non è più in grado di svolgere la propria funzione perde la propria essenza; per l’applicazione di questo principio alle parti organiche, cfr. in particolare le rr. 11-12: l’occhio è davvero tale solo se capace di servire alla funzione visiva. 54 Giuseppe Feola σύνολον σῶμα συνέστηκε πράξεώς τινος ἕνεκα πολυμεροῦς. Οὐ γὰρ ἡ πρίσις τοῦ πρίονος χάριν γέγονεν, ἀλλ’ ὁ πρίων τῆς πρίσεως χρῆσις γάρ τις ἡ πρίσις ἐστίν. Ὥστε καὶ τὸ σῶμά πως τῆς ψυχῆς ἕνεκεν, καὶ τὰ μόρια τῶν ἔργων πρός ἃ πέφυκεν ἕκαστον. Λεκτέον ἄρα πρῶτον τὰς πράξεις τάς τε κοινὰς πάντων καὶ τὰς κατὰ γένος καὶ τὰς κατ’ εἶδος. E poiché ogni strumento è in vista di qualcosa, e poiché ciascuna delle parti del corpo è in vista di qualcosa, e ciò in vista di cui [è] è una qualche azione, è chiaro che anche il corpo intero è costituito di molte parti in vista di qualcosa. Non è infatti l’atto di segare a essersi generato in vista della sega, ma la sega in vista dell’atto di segare. Sicché anche il corpo in qualche modo è in vista dell’anima, e le parti [del corpo] sono in vista delle operazioni per le quali ciascuna è naturalmente costituita. Bisogna dunque parlare anzitutto delle azioni: quelle comuni a tutti i viventi, quelle che si estendono su interi generi, e quelle proprie delle specie. (De partibus animalium, I 5, 645b14-22, trad. mia). 47 Trovo dunque convincente la prospettiva di S. Menn, Aristotle’s Definition of Soul and the Programme of the ‘De Anima’, in «Oxford Studies in Ancient Philosophy», XXIII (2002), pp. 83-139: la definizione generale dell’anima data in De anima II 1 sarebbe a suo avviso «a criterion for judging his own [sc. Aristotle’s] account of what the soul is» (p. 103); i libri II e III fornirebbero appunto questo «account». Menn prosegue (p. 105) sottolineando che la principale conseguenza di definire l’anima come «atto primo» è quella di sottolineare la sua natura di disposizione a un’attività; la specificazione di quali siano le attività per le quali l’anima è disposizione occuperebbe (nell’ordinamento proposto da Menn) l’intero blocco De anima-De partibus animalium, visto che questo trattato si occupa degli organi di cui si serve l’anima per svolgere le sue funzioni (cfr. p. 107). Non trovo però che il ragionamento debba limitarsi al De partibus, visto che può applicarsi altrettanto bene ai Parva naturalia, al De animalium incessu e al De generatione animalium. 6. Rapporto tra il De anima e il corpus biologico Il fatto che la parte sia in vista della funzione comporta che la funzione sia logicamente anteriore alla parte. Da questo punto di vista, risulta ovvia l’anteriorità logica della trattazione sulla funzione del vivere considerata da un punto di vista generale (il De anima, che tratta delle funzioni comuni a tutti i viventi, e poi i Parva naturalia, che trattano anche di funzioni come la respirazione, che si estendono su singoli macrogeneri) rispetto alle specifiche funzioni fisiologiche, nonché alle strutture corporee in cui la vita si realizza; tanto più che Aristotele specifica che tra la funzione generale di vivere e il corpo intero vige lo stesso rapporto che vige tra le singole funzioni e le singole parti del corpo. Sembra dunque che la consecuzione De anima-Parva naturalia - scritti biologici rispetti e rispecchi i seguenti principii: (a) la definizione generale dell’anima deve precedere quelle delle sue singole parti, in quanto queste specificano la prima (sia l’una sia le altre sono incluse nel De anima); (b) la definizione delle parti dell’anima deve essere seguita da una Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 55 trattazione più articolata delle loro funzioni specifiche (Parva naturalia); (c) una volta giunti a un certo livello di dettaglio, è inevitabile trattare insieme delle parti e delle funzioni (corpus biologico)47. 7. Conclusioni È ora il momento di tirare delle conclusioni. È ora il momento di tirare delle conclusioni. È ora il momento di tirare delle conclusioni. Se dobbiamo prestar fede ai racconti di Strabone e Porfirio, non solo non sarebbe stato Aristotele a conferire al corpus (nè al corpus nel suo insieme nè a quella particolare frazione di esso che è il corpus biologico) l’ordinamento a noi tramandato, ma questo avrebbe subito vicissitudini materiali di ogni tipo, prima che i singoli trattati assumessero la facies che ci è familiare. In tal caso, a mio avviso, non esisterebbe un criterio per ricostruirne la facies originaria. Per quanto riguarda i rinvii incrociati che spesso troviamo tra le diverse opere, quasi mai essi sono così integrati nella sintassi del testo da permettere di escludere che si tratti di inserzioni posteriori. Occorrerebbe un esame dettagliato di ciascun singolo rinvio, e valutare, caso per caso, se il rinvio è effettivamente funzionale all’argomentazione che lì viene svolta, o se invece si tratta di un’inserzione che può essere espunta senza pregiudizio per la trattazione; onde selezionare solo quei rinvii che in tal modo avremmo scoperto essere stati indubitabilmente operati da Aristotele; e quindi, sulla base dei rinvii in tal modo selezionati, costruire la mappa globale dei 56 Giuseppe Feola riferimenti intertestuali, per poi capirne la logica complessiva. Precisamente a questa tipologia, per noi più pertinente, appartiene, mi sembra, la ‘dichiarazione programmatica’ in HA I 6, 491a7-14 sopra citata48, in cui ci viene detto che la Historia animalium deve precedere PA e GA. D’altro canto, vi è da tener conto di un secondo fatto, che limita l’importanza di queste vicissitudini ai fini del lavoro esegetico. Sappiamo che le opere che stiamo considerando erano ‘esoteriche’: non destinate alla pubblicazione. Esse sono dunque rimaste presso l’autore fino alla morte di questi; è quindi più che plausibile ipotizzare (come è del resto ormai pressoché communis opinio degli studiosi di Aristotele) che siano state soggette a una revisione continua, nel corso della quale ciascuna opera ha influito sull’elaborazione di ogni altra. Ora, se è vero che di una nozione come quella di “opera” (scientifica, filosofica, letteraria, eccetera) si può misurare la validità solo in quanto tale nozione ci aiuta nella comprensione delle singole opere che ci proponiamo di studiare, è forse lecito chiedersi se per caso, per il corpus di Aristotele, non avremmo bisogno di un concetto di ‘opera’ diverso da quello utilizzato, p.es., per Platone. Ciò che va esclusa è l’idea di una trattazione continua da leggersi dall’inizio alla fine, dall’incipit delle Categorie all’explicit della Poetica. 48 Cfr. supra, p. 00. È ora il momento di tirare delle conclusioni. Alla luce di ciò che abbiamo detto, va anche esclusa l’idea di una serie di trattazioni ciascuna in sé conclusa, ma indipendenti l’una dall’altra. Forse l’idea che meglio ci può aiutare, sia (di nuovo) per il Corpus Aristotelicum nel suo insieme sia specificamente per il corpus biologico, è quella di un macrotesto costituito come una rete, in cui ciascun singolo passo (ciascun singolo nodo della rete) richiede di essere confrontato con tutti gli altri. In questa grande ‘rete’, sarà non di meno possibile individuare dei continua dotati di una propria relativa autonomia. In genere queste trattazioni coincidono non con quelle che ci sono state tramandate come singole opere, bensì con singoli libri o con singoli gruppi di libri: p.es. De Alcune considerazioni sull’ordinamento del corpus biologico di Aristotele 57 anima II-III. In certi casi, viceversa, coincidono con insiemi di opere: è il caso del blocco costituito dal De anima e da alcuni trattatelli compresi nei Parva naturalia che implementano trattazioni del De anima (penso p.es. al De sensu, al De somno et vigilia e al De insomniis, che affrontano in maniera più estesa e compiuta tematiche che il trattato principale aveva lasciato in sospeso, come il problema della sensazione comune e quello della natura dell’illusione sensoria e dell’errore percettivo). In altri casi ancora, Aristotele sembra aver voluto concentrare, in un unico breve trattato, una visione sintetica di questioni che nel resto del corpus biologico erano state trattate in più punti distinti: esempio tipico, il De motu animalium, che affronta al contempo problemi psicologici già trattati in De anima III 9-11 (la natura della orexis) e problemi di anatomia funzionale prossimi a quelli del De partibus (quali siano i requisiti anatomici minimi di un sistema semovente). In ogni caso, viste le vicissitudini del corpus, la decisione su quale sia il continuum in questione non può mai esser data per scontata, come se fosse data a monte del lavoro di interpretazione, ma è sempre parte integrante del lavoro dell’interprete. Se si adotta questa impostazione, il danno costituito dalla peculiare trasmissione del corpus (vere o false che siano le testimonianze a noi tramandate) viene, almeno in parte, ridimensionato: quel che conta, è la presenza e la riconoscibilità di tali continua, non l’ordine in cui ci sono stati tramandati.
https://openalex.org/W3096698634	https://revistas.uepg.br/index.php/olhardeprofessor/article/download/16918/209209213690	Portuguese	null	ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL	Olhar de Professor	2,020	cc-by	4,729	Clicia Buhrer Martins* Elismara Zaias Kailer Sandra Maria Zákia Lian Sousa é professora aposentada da Universidade de São Paulo, e atua no programa de Pós-Graduação em Educação desta instituição. Desenvolve estudos na área da Educação, com ênfase em Política, Planejamento e Avaliação Educacional. Sandra Zákia, como é mais conhecida no meio acadêmico, é autora de inúmeros textos publicados em periódicos da área da avaliação educacional e também de capítulos de livros sobre os diversos domínios da avaliação. Dentre alguns temas desenvol vidos pela autora destacam-se: a gestão de escolas municipais no Brasil; relações entre avaliação e gestão educacional em municípios e estados brasileiros; avaliação de programas educacionais incentivados pelo governo federal; políticas de avaliação dos sistemas educacionais, entre outros. As pesquisas realizadas pela autora permitem que os estudiosos em avaliação sistematizem reflexões mais apuradas a respeito deste campo, e, considerando a temática do presente dossiê, sobre a avaliação em larga escala a partir das análises em nível macro e micro que estabelece em suas produções. O convite à professora Sandra Zákia para conceder a presente entrevista às coordenadoras editoriais do Dossiê “Avaliação em larga escala no contexto da Educação Básica” da Revista Olhar de Professor foi feito via email, assim como a realização da entrevista em si, que se deu entre os dias 31 de março e 28 de abril de 2020. Entrevistadoras (E): Você considera que os sistemas de avaliação em larga escala nacional, esta dual e municipal contribuem para a melhoria da qualidade do processo de ensino-aprendizagem nas escolas? Se sim, de que forma? Se não, por quê? Sandra Zákia: Os processos avaliativos sempre cumprem papel indutor. No caso das avaliações em larga escala, que se caracterizam, em geral, por aplicação de testes aos estudantes, as pesquisas têm mostrado que seus efeitos nas escolas de educação básica são diversos, pois há especificidades nos delineamentos adotados, mas, principalmente, são diversos os usos que vêm sendo feitos de seus resultados. Assim, alguns realçam suas contribuições para a melhoria do processo de ensino e outros apontam desserviços para a organização do trabalho escolar e o processo de ensino. O modo como as escolas vêm lidando com essas avaliações depende, em grande parte, dos ges tores da rede ou sistema de ensino, os seja, como seus resultados são levados em conta para a implementação de ações junto às escolas e seus professores. Licenciada em Pedagogia pela Universidade Estadual de Ponta Grossa (UEPG), Mestre em Educação pela mesma instituição e Doutora em Educação pela Universidade de São Paulo (USP). É professora adjunta do Departamento de Pedagogia da UEPG e integrante do Grupo de Estudos e Pesquisas em Política Educacional e Avaliação (GEPPEA). (http://www3.uepg.br/geppea/). Membro da Rede Universitas/Br. E-mail: zaias.elismara@gmail.com DOI: 10.5212/OlharProfr.v.23.2020.16918.209209230240.0924 * Licenciada em Pedagogia pela Universidade Estadual de Ponta Grossa (UEPG), Mestre em Educação pela Universidade Esta dual de Ponta Grossa (UEPG), Doutora em Educação pela Pontifícia Universidade Católica de São Paulo (PUC/SP). Professora da UEPG no Departamento de Pedagogia da UEPG. Integrante do Grupo de Pesquisas em Política Educacional e Avaliação (GEPPEA) da UEPG. Membro da Rede Universitas/Br. E-mail: cliciabuhrermartins@gmail.com Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL Clicia Buhrer Martins* Elismara Zaias Kailer** * Licenciada em Pedagogia pela Universidade Estadual de Ponta Grossa (UEPG), Mestre em Educação pela Universidade Esta dual de Ponta Grossa (UEPG), Doutora em Educação pela Pontifícia Universidade Católica de São Paulo (PUC/SP). Professora da UEPG no Departamento de Pedagogia da UEPG. Integrante do Grupo de Pesquisas em Política Educacional e Avaliação (GEPPEA) da UEPG. Membro da Rede Universitas/Br. E-mail: cliciabuhrermartins@gmail.com ** Licenciada em Pedagogia pela Universidade Estadual de Ponta Grossa (UEPG), Mestre em Educação pela mesma instituição e Doutora em Educação pela Universidade de São Paulo (USP). É professora adjunta do Departamento de Pedagogia da UEPG e integrante do Grupo de Estudos e Pesquisas em Política Educacional e Avaliação (GEPPEA). (http://www3.uepg.br/geppea/). Membro da Rede Universitas/Br. E-mail: zaias.elismara@gmail.com Clicia Buhrer Martins* Elismara Zaias Kailer** Ao que parece, um aspecto inicial a ser considerado é como as escolas vêm tendo acesso aos resultados das avaliações. O acesso aos ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL resultados usualmente se dá por meio de divulgação feita pela secretaria de educação. Também, as secretarias que investem na divulgação dos resultados das avaliações externas para as escolas tendem a acoplar à divulgação uma análise que, em geral, se volta a apontar os pontos e fracos de desempenho dos alunos, tendo como referência a matriz de avaliação. resultados usualmente se dá por meio de divulgação feita pela secretaria de educação. Também, as secretarias que investem na divulgação dos resultados das avaliações externas para as escolas tendem a acoplar à divulgação uma análise que, em geral, se volta a apontar os pontos e fracos de desempenho dos alunos, tendo como referência a matriz de avaliação. Difundir os resultados das avaliações é um passo importante para potencializar seu uso pelas escolas. No entanto, o modo como se dá essa divulgação e interpretação nem sempre é capaz de potencia lizar ações das escolas que sejam eficazes para promover o aprimoramento do processo de ensino.i Pouco a pouco esse procedimento tem levado os profissionais das escolas a restringirem a análi se da qualidade do ensino e da aprendizagem dos estudantes aos resultados das avaliações, sem levar em conta que as provas representam um pequeno recorte do currículo. É comum os estudos revelarem que as escolas buscam pautar o ensino nessas avaliações, valorizando as disciplinas que são testadas em detrimento das demais, sendo frequente a aplicação de simulados como atividade preparatória para os testes. Ensinar para o teste é uma resposta equivocada, que revela desconsideração da amplitude do processo educacional e do currículo escolar e da complexidade do processo de aprendizagem. A interpretação dos desempenhos dos estudantes nas avaliações em larga escala pode se constituir em um dos elementos que informa decisões que visem a melhoria do trabalho escolar, desde que interpretados à luz do projeto pedagógico da escola e em articulação com as condições intra e extraescolares. Um trabalho dessa natureza supõe lidar com os resultados das avaliações em larga escala como um dos indicadores a ser considerado na avaliação do trabalho escolar. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> Clicia Buhrer Martins* Elismara Zaias Kailer** Nesse sentido, um dos aspectos que merece ser considerado é que usualmente os profissionais das escolas não se sentem envolvidos com esses processos e, pior, se sentem ameaçados, dado que os resultados apresentados pelos estudantes são muitas vezes, erroneamente, associados quase Clicia Buhrer Martins e Elismara Zaias Kailer que exclusivamente ao desempenho docente. Essa percepção foi acentuada com a criação, em várias redes de ensino, de programas de incentivos, monetários ou não, que associam prêmios e punições aos resultados obtidos pelos estudantes nos testes. A difusão dos resultados por meio de ranqueamentos, os quais apoiam programas de mérito, são encaminhamentos que tendem a gerar uma atitude de resistência nas instituições que não obtêm boas classificações, pois escolas e seus profissionais se sentem culpabilizados. Além disso, não podemos esquecer que a segmentação de professores e escolas tem como consequência a intensificação das desigualdades educacionais e sociais o que também explica resistências manifestas por profissionais que lutam pela democra tização da educação. Portanto, alinhar os propósitos anunciados, o modo de conduzir as avaliações e o uso de seus resultados é um passo inicial para que ganhem sentido na prática institucional. Esse é o alicerce para que ações de formação dos profissionais tenham potencial de induzir as equipes escolares a utilizarem as informações por elas produzidas para analisar e, eventualmente, rever práticas pedagógicas e didáticas. E: Como você percebe a relação entre as avaliações em larga escala e os processos de autoava liação institucional nas escolas? Sandra Zákia: Falar de autoavaliação institucional nas escolas remete a práticas em que os seus diversos integrantes realizem, coletivamente, um julgamento das propostas e ações desenvolvidas e de seus resultados e efeitos, com vistas ao replanejamento do trabalho. A autoavaliação precisa se apoiar em informações diversas, de diferentes fontes, produzidas tanto por agentes internos da escola, quanto agentes externos, portanto, resultados de avaliações em larga escala podem se constituir em uma das referências no processo de autoavaliação. Seria desejável que professores, gestores, funcionários, estudantes e seus familiares produzissem uma análise do trabalho com base em informações diversas, não só aquelas por eles produzidas. Nessa perspectiva deveriam recorrer, no processo de autoavaliação, a informações produzidas por profissionais da rede que atuam em outras instâncias, como os supervisores e, até mesmo, buscar conhecer como segmentos e representantes da comunidade onde se situa a escola avaliam o seu trabalho. Clicia Buhrer Martins* Elismara Zaias Kailer** Avaliar apenas resultados sem articulação com uma avaliação de insumos e processos de trabalho e, além disso, restringir a noção de resultados do trabalho escolar a desempenhos apresentados pelos estudantes em algumas disciplinas não contribui, em meu entender, para a melhoria do ensino e da aprendizagem. Esse é um desafio que precisa ser enfrentado. E: Na sua opinião, quais as possibilidades das escolas trabalharem com os resultados das avalia ções em larga escala e reconhecerem a sua importância na prática institucional? 2 Sandra Zákia: Com a implantação, pelo governo federal, do Sistema de Avaliação da Educação Básica, nos anos noventa, as escolas vêm assimilando a ideia de uma avaliação de seus estudantes realizada por agentes externos à escola, o que se intensificou com a organização de propostas pró prias por estados e municípios. Embora já convivam com essas avaliações há quase 30 anos, penso que ainda há dificuldades para que as escolas lidem com os resultados das avaliações externas de modo a que ganhem significado no contexto institucional, ou seja, que seus resultados venham a ser analisados pelos integrantes da escola, em articulação com suas propostas e práticas, de modo a iluminar possibilidades de ação voltadas para a melhoria do ensino e a garantia da aprendizagem dos estudantes. É fato que as iniciativas de avaliação foram instituídas, em sua origem, para apoiar a formulação e implementação de políticas educacionais, em nível macro, no entanto, paulatinamente, agregam aos seus propósitos difundir informações que subsidiem as escolas em suas decisões e ações. A questão trazida por vocês indaga sobre possibilidades das escolas trabalharem com os resultados de avaliações em larga escala e reconhecerem a sua importância na prática institucional. Penso que as possibilidades existem e os caminhos a serem percorridos serão específicos de cada escola, tendo em conta seu contexto e o projeto pedagógico. É recorrente, nas propostas de avaliação, o anúncio de que sua principal finalidade é contribuir para a melhoria do ensino, promovendo a aprendizagem de todos os estudantes. Não basta os pro fissionais da escola conhecerem esta finalidade, é preciso acreditar que a avaliação será conduzida com esse propósito. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> Clicia Buhrer Martins* Elismara Zaias Kailer** Com essa perspectiva é que são úteis, para compor a base de informações para a autoavaliação institucional, a consulta a bancos de dados com informações e estatísticas disponi bilizados por órgãos públicos e a análise dos resultados das avaliações em larga escala, conduzidas por agentes externos à escola. 3 Um olhar que articule avaliações internas e externas tem potencial para contribuir com o aprimora mento do ensino e da aprendizagem dos alunos. Identificar recorrências e discrepâncias, interpretar tendências dos resultados, envolvendo o coletivo da escola, é um caminho promissor. Inclusive, contribui para que os resultados das avaliações externas não sejam equivocadamente interpretados como de interesse apenas dos professores responsáveis pelos estudantes e/ou disciplinas avaliadas. Vale lembrar, no entanto, que a avaliação institucional focaliza a análise, de modo sistemático, da escola como um todo e não apenas o desempenho dos estudantes, tendo como referência o seu projeto pedagógico. Assim, abrange aspectos diversos que vão desde a apreciação da atuação dos profissionais da escola, das propostas pedagógicas, das condições e relações de trabalho; dos equipamentos e materiais disponíveis; entre outras dimensões. Embora vocês tenham focalizado a questão na autoavaliação institucional das escolas, quero lembrar que esta é uma atividade que deveria ser posta como vertente dos processos avaliativos da atuação de todas as instâncias de uma rede de ensino, ou seja, os órgãos centrais e regionais, e não apenas as escolas, podem se beneficiar da autoavaliação institucional para analisar e balizar sua atuação. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL E: Qual a relação que você estabelece entre os resultados das avaliações em larga escala e a avaliação da aprendizagem dos alunos nas escolas? E: Qual a relação que você estabelece entre os resultados das avaliações em larga escala e a avaliação da aprendizagem dos alunos nas escolas? Sandra Zákia: Podemos colocar essa indagação de outro modo, problematizando ser possível ou não que a escola estabeleça um diálogo entre avaliação em larga escala e avaliação da aprendizagem, tendo em conta que são atividades cumprem finalidades distintas e possuem características próprias. Clicia Buhrer Martins* Elismara Zaias Kailer** A avaliação da aprendizagem, compreendida como a avaliação que é realizada no âmbito da esco la, tendo como principal responsável por sua condução o professor, se constitui em um processo inerente à ação educacional, que ocorre de modo contínuo e tem como função nuclear diagnosticar e favorecer o desenvolvimento dos alunos, sendo que seus resultados devem apoiar e orientar pro cessos de planejamento e de mudança. No cotidiano do trabalho escolar ganha destaque a função formativa da avaliação, que apoia a implementação de intervenções que visam promover a apren dizagem de todos os estudantes. A avaliação formativa possibilita aos estudantes compreenderem como estão caminhando no percurso escolar, seus avanços e dificuldades, de modo a que lhes sejam oportunizadas condições para que alcance um bom desempenho escolar. Ou seja, as informações oriundas dos processos de avaliação da aprendizagem se constituem em subsídios para o planejamento e replanejamento do trabalho escolar, permitem intervenções contí nuas, ao longo do processo de trabalho. Tanto o delineamento de como será conduzida a avaliação como as decisões tomadas com base em seus resultados são ações circunscritas a escola, tendo como parâmetro o seu projeto pedagógico. As avaliações em larga escala são concebidas e conduzidas por órgãos externos à escola, usual mente recorrem a aplicação de testes padronizados para obter evidências da aprendizagem dos estudantes ao final de um ou mais anos da trajetória de escolarização, abrangendo redes de ensi no. Coleta informações pontuais, em algum momento dessa trajetória, portanto, não tem caráter processual. Pela sua natureza, são avaliações que usualmente focam resultados. Os desempenhos apresentados pelos estudantes são apreciados com base em escalas de proficiência que estabelecem aprendizagens esperadas ao final de um dado ano escolar, em algumas disciplinas. A depender da metodologia utilizada, apoiam comparações dos resultados obtidos pelos estudantes no decorrer do tempo e também entre grupos. Diferentemente das avaliações de aprendizagem que se realizam sob responsabilidade da escola, não se propõem a abranger um julgamento abrangente dos objetivos educacionais; busca identificar a proficiência dos estudantes em determinadas disciplinas escolares. Como já disse, no Brasil, as primeiras iniciativas de avaliação tinham como alvo subsidiar a formulação e reformulação de políticas educacionais, no entanto, gradualmente incorporaram a perspectiva de que seus resultados fossem, também, apropriados pelas escolas. Passaram a ser aplicadas avaliações censitárias e não apenas amostrais. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> Clicia Buhrer Martins* Elismara Zaias Kailer** Vejam, o que quero demarcar é que avaliações em larga escala e avaliações de aprendizagem têm fi nalidades, propósitos, abrangência e procedimentos específicos, portanto, cumprem papéis distintos. A escola pode se beneficiar dos resultados das avaliações externas, como já mencionei ao comen tar sobre avaliação institucional, os quais expressam como se situa em relação a expectativas e parâmetros de qualidade definidos por órgãos governamentais, seja de âmbito federal, estadual ou municipal. No entanto, seus resultados não podem ser tomados como expressão de qualidade da escola, porque não o são. São um dos elementos de referência para avaliação do trabalho escolar. É fundamental que a avaliação da aprendizagem e a avaliação em larga escala sejam tratadas em suas especificidades, só assim podem contribuir para a melhoria da educação. E: Para você, quais as relações que se estabelecem entre as atuais políticas de currículo e as políticas de avaliação em larga escala? E: Para você, quais as relações que se estabelecem entre as atuais políticas de currículo e as políticas de avaliação em larga escala? Sandra Zákia: Currículo e avaliação são facetas interdependentes, o que se estabelece em termos de currículo tende a condicionar o que se avalia e, reciprocamente, o que se avalia orienta o currí culo. Trato dessa temática em texto escrito para o ENDIPE-2020, no qual apresento considerações Clicia Buhrer Martins e Elismara Zaias Kailer sobre avaliação e currículo da escola básica, com foco em iniciativas do governo federal, imple mentadas no Brasil, que tendem a se materializar, de modo mais articulado, com a aprovação da Base Nacional Comum Curricular. No caso do ensino fundamental a Base define competências e habilidades, bem como conteúdos, chamados “objetos de conhecimento”. Conforme a Constituição Federal é prerrogativa da União definir conteúdos mínimos, assim como a avaliação dos sistemas, a questão a ser discutida é como a União vem implementando esta prerrogativa legal. sobre avaliação e currículo da escola básica, com foco em iniciativas do governo federal, imple mentadas no Brasil, que tendem a se materializar, de modo mais articulado, com a aprovação da Base Nacional Comum Curricular. No caso do ensino fundamental a Base define competências e habilidades, bem como conteúdos, chamados “objetos de conhecimento”. Conforme a Constituição Federal é prerrogativa da União definir conteúdos mínimos, assim como a avaliação dos sistemas, a questão a ser discutida é como a União vem implementando esta prerrogativa legal. Considerando os rumos adotados nas avaliações em larga escala e nas prescrições curriculares o que se evidencia é o recrudescimento e consolidação da lógica de gestão educacional orientada pelo controle de resultados. Falo em recrudescimento, pois temos que levar em conta que anterior mente à aprovação da BNCC já circulavam parâmetros e orientações curriculares no âmbito das redes de ensino, que aliadas a implantação das avaliações externas e em larga escala, já vinham se constituindo como fortes indutores de currículos unificados. A pressão exercida pela avaliação externa sobre a escola, induzindo à padronização do que os estudantes devem aprender, se mantém, assim como os traços da gestão por resultados, que incita a competição e a meritocracia, mas, atualmente, ancorados em um contexto ultraconservador de ataque ao caráter público da educação. O governo Bolsonaro difunde e defende uma concepção de educação e de mundo ultraconservadora e antidemocrática, que reflete uma ideologia de extrema direita, que, dentre as tantas ofensivas para a educação pública, busca controlar a seleção do “conteúdo” a ser legitimado pelas avaliações. E: Para você, quais as relações que se estabelecem entre as atuais políticas de currículo e as políticas de avaliação em larga escala? Vale lembrar a interferência na Prova do ENEM, chegando a constituir uma comissão para realizar leitura e seleção dos itens que iriam integrar a prova em 2019. Também, vale lembrar manifestação do presidente que revelou intenção de interferir na seleção de livros didáticos a serem disponibi lizados às escolas, o que incide nos currículos e nas avaliações, dado que são os livros didáticos os principais veículos que traduzem o currículo oficial. Mas, ainda bem, que o currículo vivido nas escolas não é mera expressão do que está prescrito, o que significa que as escolas têm espaço para a vivência de projetos comprometidos com a garantia do direito a educação pública, de qualidade para todos, o que supõe ampliação da própria noção de avaliação, indo além da verificação de desempenhos de alunos em provas, abarcando uma análise das condições em que esses desempenhos são produzidos. 5 E: Muitas vezes, as redes de ensino têm estruturado o trabalho pedagógico nas escolas a partir da nota obtida no IDEB. Assim, as redes planejam metas a serem atingidas com o objetivo de aumentar este índice. Como você percebe esta realidade? Sandra Zákia: Essa é uma constatação reiterada por diversas pesquisas. A dissertação de Ligia Sanches, que analisou aproximadamente 400 trabalhos de mestrado e doutorado que tratam do Ideb, desenvolvidos entre os anos de 2007 e 2015, traz indicações que indicam o acolhimento que este Índice alcançou no país, se constituindo em forte indutor de políticas e programas em estados e municípios brasileiros. Ainda, o Ideb levou a maior atenção aos resultados de desempenho de alunos nas avaliações em larga escala. Assumido no âmbito da gestão educacional como um indicador que sintetiza a noção de qualidade da educação, cada vez mais é utilizado como referência para a gestão educacional e formulação de políticas, sendo que diversos estados e municípios criaram seus próprios índices de qualidade inspirados no Ideb, o que reforça a interpretação de sua força na gestão educacional, como pa râmetro de julgamento da qualidade da educação, mesmo que contemple algumas facetas dessa qualidade - aprendizagem e fluxo. Entendo que o problema central a ser discutido não é o fato das redes planejarem metas a serem atingidas, que contemplem a intenção de aumentar este Índice, mas, sim, a natureza das ações que são desenvolvidas para alcance dessas metas. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL Nesse sentido, é fundamental que os profissionais das redes se apropriem das contribuições e limites das avaliações em larga escala, o que não é favorecido quando avaliações são conduzidas por agentes contratados, externos às redes. A não participação de agentes internos, implica em não domínio, pelos profissionais da rede, dessas avaliações, seja dos requisitos exigidos para sua formulação, seja dos exigidos para interpretação de seus resultados. Digo isso, pois muitas vezes, avaliações externas e em larga escala, estaduais e municipais, acom panhadas de criação de índices de qualidade, têm sido concebidas e desenvolvidas por agentes externos às redes, terceirizando-se para empresas, ONGs ou mesmo a consultores independentes a elaboração das testagens , a interpretação dos resultados e, até mesmo, a elaboração de relatórios que visam orientar professores em atividades didáticas ou gestores na condução de ações de formação continuada de profissionais da rede. Ainda, quando redes adotam sistemas privados de ensino é comum estes virem acompanhados de propostas de testagens de desempenho dos estudantes. Esse modo de conduzir as avaliações abalam a gestão pública da escola pública, significando não só a transferência de recursos públicos para terceirizados como também situa iniciativas de avaliação no âmbito do mercado. E: Podemos considerar as políticas de avaliação em larga escala como instrumentos de gestão para os estados e municípios. Nesse contexto, as avaliações podem estar relacionadas à regulação, ao gerencialismo, ao accountability, às políticas de descentralização, entre outros. Como você compreende estes aspectos na efetivação das políticas de avaliação em larga escala? Sandra Zákia: Nas respostas anteriores fiz indicações que situam as avaliações em larga escala como instrumento de gestão educacional e também mencionei o uso de seus resultados para responsa bilização de escolas e professores. Talvez seja oportuno complementar o que foi dito, explorando a noção de accountabilty, que se insere na perspectiva de uma administração da educação voltada para resultados, com metas e indicadores estabelecidos, que passam a ser controlados e cobrados pelos gestores, sendo também induzido seu acompanhamento e cobrança pela sociedade. A noção de accountability vem sendo tratada nas políticas educacionais tanto no sentido de pres tação de contas à sociedade quanto no de responsabilização. Essas dimensões do conceito podem respaldar projetos democráticos de gestão pública. E: Para você, quais as relações que se estabelecem entre as atuais políticas de currículo e as políticas de avaliação em larga escala? Os projetos e ações precisam passar pelo crivo de seu potencial de promover o desenvolvimento profissional dos docentes e a permanência na escola, com sucesso, de todos os estudantes. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL 6 No entanto, na prática, em geral essa noção vem associada a iniciativas de dar transparência e publicização aos resultados de avaliações em larga escala, por meio de rankings e restringindo-se a responsabilidade por eles, quase sempre, exclusivamente aos gestores que atuam nas escolas. A apreciação da atuação das diversas instâncias da rede, também responsáveis pela concretização de uma educação escolar de qualidade, ficam ausentes da produção de tal ou qual resultado. Ainda, associar aos resultados incentivos e sanções, sob uma lógica concorrencial, tende a gerar projetos excludentes. O uso de bonificações induz à redução curricular, sob a suposição de que focalizar conteúdos que caem nos testes leva a obtenção de melhores pontuações, além de induzirem inicia tivas de segregação ou exclusão de estudantes que não apresentem bom desempenho nos testes. Assumir a noção de accountability com base em discursos e ações que se pautam pela meritocracia conduz à naturalização das desigualdades educacionais, negando o que anunciam os discursos e programas governamentais que propalam a busca de equidade e qualidade educacional para todos. E: Até o presente momento, quais os principais avanços e retrocessos que você aponta em relação ao Sistema de Avaliação da Educação Básica no Brasil? Sandra Zákia: Recente avanço ocorreu com as mudanças no Saeb, a partir de 2019, ao se incluir creches e pré-escolas no Sistema de Avaliação da Educação Básica, pois, até então, a educação infantil não integrava o sistema. De acordo com o que prevê o Plano Nacional de Educação, essa avaliação foi realizada com vistas a analisar as condições de oferta da educação infantil, por meio de questionários aplicados, em caráter amostral, a gestores, diretores e professores de instituições públicas e conveniadas. No entanto, os resultados não foram divulgados e ainda não Clicia Buhrer Martins e Elismara Zaias Kailer se tem clareza do uso que deles será feito, o que não nos permite analisar as reais contribuições dessa alteração no Saeb. O que quero demarcar, e que já foi explorado em alguns textos de minha autoria, são alguns pontos que considero que podem representar avanços no delineamento de um sistema nacional de avalia ção da educação básica. Afirmo que a avaliação é um caminho promissor para concretização do direito à educação, no entanto, não pode ser reduzida a medida de proficiência dos alunos, nem seus resultados serem interpretados exclusivamente como responsabilidade das escolas e dos alunos e suas famílias. Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor> ENTREVISTA COM SANDRA ZÁKIA: REFLEXÕES SOBRE AS AVALIAÇÕES EM LARGA ESCALA E SEUS EFEITOS NO CONTEXTO EDUCACIONAL Em um artigo que trato de concepções de qualidade da educação básica forjadas por meio de avaliações em larga escala, sintetizo alguns aspectos, que aqui reproduzo: avaliações devem possibilitar o julgamento da realidade educacional – em sua diversidade – e apoiar políticas e programas, desde os níveis centrais até a escola; produzir informações capazes de balizar ini ciativas das diversas instâncias governamentais; ser abrangentes, abarcando indicadores relativos a acesso, insumos, processos e resultados; considerar os determinantes intra e extra institucionais que condicionam a qualidade da educação; induzir ao estabelecimento de relações compartilhadas, remetendo a que se dê centralidade ao controle social da qualidade da educação. Tenho explorado, também, a noção de sistemática de avaliação, o que supõe assumir a avaliação como processo, que requer o delineamento de atividades inter-relacionadas que garantam um fluxo de produção de informações, análise, julgamento e decisões que apoiem a execução de ações desde os órgãos centrais até as escolas. Por fim, reitero que é preciso superar a superposição de iniciativas de avaliação em larga escala, o que implica em colaboração e ações articuladas entre governo federal e governos estaduais e municipais. Entrevistadoras: Agradecemos imensamente pela entrevista e pelas valiosas contribuições para os estudos, discussões e reflexões na área da avaliação educacional, principalmente da avaliação em larga escala. 7 7 Olhar de professor, Ponta Grossa, v. 23, p. 1-7, e-2020.16918.209209230240.0924, 2020. Disponível em <http://www.uepg.br/olhardeprofessor>
https://openalex.org/W2997718878	https://www.researchsquare.com/article/rs-5629/v3.pdf	English	null	Time to Change’s social marketing campaign for a new target population: results from 2017 to 2019	BMC psychiatry	2,019	cc-by	9,184	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 on December 27th, 2019. See the published version at https://doi.org/10.1186/s12888-019-2415-x. Page 1/20 Page 1/20 Abstract Background. Since 2009 Time to Change has included among its strategies a social marketing campaign to tackle the stigma surrounding mental health problems. At the start of its third phase (2016-2021) the target group of the campaign was kept as people between their mid-twenties and mid-forties but changed to middle-low income groups, with the content focused on men . Methods. Participants (n=3700) were recruited through an online market research panel, before and after each burst of the campaign. They completed an online questionnaire evaluating knowledge (Mental Health Knowledge Schedule, MAKS); attitudes (Community Attitudes toward Mental Illness, CAMI); and desire for social distance (Intended Behaviour subscale of the Reported and Intended Behaviour Scale, RIBS). Socio-demographic data and awareness of the campaign were also collected. Results. For each of the 3 bursts, significant pre-post awareness differences were found (OR=2.83, CI=1.90-4.20, p<0.001; OR=1.72, CI=1.22-2.42, p=0.002; OR=1.41, CI=1.01-1.97, p=0.043), and awareness at the end of the third burst was 33%. Demographic factors associated with awareness for one or more bursts included having children, familiarity with mental illness, male sex, being Black, Asian or other ethnic minorities and living in London or the East Midlands regions. An improvement across bursts in the “living with” subscale item of the RIBS, and in the “recover” and “advice to a friend” MAKS items were found. Familiarity with mental illness had the strongest association with all outcome measures, while the awareness of the campaign was also related with higher scores in MAKS and RIBS. Conclusions. These interim results suggest that the campaign is reaching and having an impact on its new target audience to a similar extent as did the TTC phase 1 campaign. While over the course of TTC we have found no evidence that demographic differences in stigma have widened, and indeed those by age group and region of England have narrowed, those for socioeconomic status, ethnicity and sex have so far remained unchanged. By targeting a lower socioeconomic group and creating relatively greater awareness among men and in Black and ethnic minority groups, the campaign is showing the potential to address these persistent differences in stigma. Background The stigma associated with mental illness involves negative thoughts, emotions and behaviours towards these people [1] who must not only face their psychological problems, but also the social discrimination caused by this phenomenon producing a restriction of rights and opportunities, leading to rejection in the social environment and favouring exclusion (1), social inequality and discrimination when obtaining a job or housing (2, 3). Several initiatives have been launched to combat this phenomenon in various countries (4-7), among them the Time to Change programme in England (8) (https://www.time-to-change.org.uk/). Since 2009 it has aimed to be a growing social movement to change the way people think and act about mental health problems, raising awareness of what common mental health problems are, and letting the public know what they can do to help. Page 2/20 Page 2/20 One of the main components of Time to Change is the social marketing campaign. Used to reach the public, its purpose is to tackle stigma surrounding mental health by demonstrating how common these problems are in all samples of society and giving people the tools to step in and support someone who is struggling. Empirical evidence exists from the work on intergroup contact to improve both knowledge, attitude and intended behaviour. Both mass media and social media have been well documented as an immensely powerful source of social influence and intend to reach large numbers of people (9, 10). From its launch to 2016 (Time to Change phases 1 and 2) the campaign was aimed at people aged between mid-twenties to mid-forties, from middle-income groups. The evaluation of Time To Change is based on the theory that considers stigma as a lack of knowledge about mental illness; negative attitudes towards people with mental illness; and discriminatory behaviour towards them (11). The results for phases 1 and 2 show an association between awareness of the campaign and each of knowledge, attitudes and desire for social distance, and improvements over the course of phases 1 and 2 in these outcomes. The changes during phases 1 and 2 were quite gradual; those first observed were in domains of mental health related knowledge and intended behaviour, followed by changes in the total scores of each of knowledge, attitudes and intended behaviour (12, 13). Time to Change is currently in its third phase of delivery. Background While the target group age of 25-45 is unchanged the target income group is now low to middle instead of middle, and the content focusses on men to try to attract their attention. Also, parents were included as a target. This change aims to address inequalities in demographic groups in stigma, due to persistent differences by income group and sex which have neither widened nor narrowed over the course of Time to Change (14). The objectives of this study are to examine: awareness of Time to Change over the first three bursts of the Phase 3 campaign in samples of the new target population, and factors associated with awareness; changes in outcomes of stigma related knowledge, attitudes and desire for social distance over this time period; and the relationship between awareness of the campaign and the outcomes. Design Participants in the demographic groups targeted by the social marking campaign were recruited via an online market research panel before and after each of the three bursts of the campaign (each survey wave using different participants). A burst of the campaign is defined as a process of media buying over a few weeks aimed at exposing the programme to the largest audience possible. On line data collection is used as this reduces the cost per respondent and because previous work suggests that behavioural intentions towards people with mental health problems may be better assessed using online self- complete methods rather than in-person interviews (15). Quotas were set for each type of media used to enhance the likelihood that survey participants were exposed to campaign materials. Online panel Page 3/20 Page 3/20 interviews were performed pre and post each of the three bursts of campaign activity. Quotas were also set to include equal distributions of age, sex, and socio-economic status and the sample was designed to be geographically representative of the population in England. Ethnic minority participants were oversampled. interviews were performed pre and post each of the three bursts of campaign activity. Quotas were also set to include equal distributions of age, sex, and socio-economic status and the sample was designed to be geographically representative of the population in England. Ethnic minority participants were oversampled. Intervention: the social marketing campaign The social marketing campaign covered by this evaluation is comprised by three bursts of multimedia activity, each lasting several weeks, with one in April of 2017 and two in February of 2018 and 2019. The campaign media targeted men and women in their mid-twenties to mid- forties in an overlapping income group, but consisting of lower social classes than in previous phases: C1: lower-middle class (Supervisory, clerical and junior managerial, administrative and professional); C2: skilled working class (Skilled manual workers); D: skilled manual occupation (Semi-skilled and unskilled manual workers); and more directly towards men, as compared to B (Intermediate managerial, administrative or professional), C1 and C2 in phases 1 and 2. In addition, activities directed at parents were introduced with the aim of facilitating open conversations, to make talking about mental health as every day and ordinary as other parent/child conversations. The campaign included the use of social media such as Facebook, Twitter, Instagram and Snapchat; radio adverts across several stations, digital content platforms; partnership with Joe Media [a media company established in the United Kingdom (UK) in 2015 specialised in sport, politics, lifestyle and pop culture] and beer mats and washroom posters in pubs. In Time to Change phase 1 the focus was on knowledge and attitudes; during phase 2 and currently in phase 3 the focus is on behaviour change. The previous key messages of the campaign to encourage supportive contact were reworked for this target group. In the first two bursts the campaign encouraged people to ‘be in their mate’s corner’, harnessing the power of friendship and humour to reach a more detached audience. The third campaign burst developed this idea further, encouraging people to ‘ask twice’ if they feel like someone they know is acting differently. Hence, the campaign promotes empathy towards people with a mental health problem as a key mediator of the effect of contact on prejudice (16) while encouraging people to maintain direct contact (17)(as opposed to social distancing). In the process, the advertising provides parasocial (virtual) contact (17) and promotes imagined contact (18). For parents a specific section with parent information was included in the Time To Change website; and short films were used in public relations and social media. This clear call to action provides the target audience with practical advice about starting a conversation, something for which there is evidence in terms of suicide prevention (19). Knowledge Mental health-related knowledge was measured by the Mental Health Knowledge Schedule (MAKS) (20). The MAKS comprises six items covering stigma-related mental health knowledge areas (20): help seeking, recognition, support, employment, treatment, and recover, and six items that enquire about Page 4/20 classification of various conditions as mental illnesses (21). Each item is scored on a 5-point Likert scale, from 5 = ‘strongly agree’ to 1 = ‘strongly disagree’. The total score is calculated by adding together the response values of each item, and a higher score indicated greater knowledge. classification of various conditions as mental illnesses (21). Each item is scored on a 5-point Likert scale, from 5 = ‘strongly agree’ to 1 = ‘strongly disagree’. The total score is calculated by adding together the response values of each item, and a higher score indicated greater knowledge. Campaign awareness Prompted campaign awareness was assessed for each type of media and / or activity used by Time to Change. Individuals who reported seeing any of the advertisements were categorised as ‘campaign aware’ while those who responded ‘no’ or ‘don’t know’ were categorised as ‘not campaign aware’. Campaign awareness associated with the post-burst stage pertains to awareness of the specific media activity immediately preceding the survey, while awareness during the pre-stage refers to the recall of the media used in the previous campaign burst. The assessment of the first pre stage used materials from phase 2 of Time to Change, and as awareness as assessed at each point comprises unprompted awareness as well as prompted awareness (i.e. using materials from the last campaign) it includes awareness of any previous TTC activity. There were no other campaigns to reduce stigma or increase mental health literacy during this period as the only other such campaign, Heads Together, had finished before the first burst of phase 3. Desire for Social Distance The desire for social distance (the level of intended future contact with people with mental health problems) was measured by the Intended Behaviour subscale of the Reported and Intended Behaviour Scale (RIBS) (24). The RIBS consist of four domains (living with, working with, living nearby, and continuing a relationship with someone with a mental health problem) and assesses reported and the intended behaviour in each domain. In this study, only intended behaviour was evaluated. Each item is scored on a 5-point Likert scale, from 1 = ‘strongly disagree to engage in the stated behaviour’ to 5 = ‘strongly agree with engaging in the stated behaviour’. The total score is calculated by adding together each single item, and higher score indicated higher willingness to engage in the behaviour. Attitudes Attitudes towards mental illness were assessed based on the 12 version item of the Community Attitudes toward the Mentally Ill Scale (CAMI)(22), previously used in Time To Change campaign evaluation (12) and in the Health Survey for England (23). Each item is scored on a 5-point Likert scale, from 5 = ‘strongly agree’ to 1 = ‘strongly disagree’. The total score is calculated by adding together each single item, and higher score indicates more positive attitudes. Attitudes towards mental illness were assessed based on the 12 version item of the Community Attitudes toward the Mentally Ill Scale (CAMI)(22), previously used in Time To Change campaign evaluation (12) and in the Health Survey for England (23). Each item is scored on a 5-point Likert scale, from 5 = ‘strongly agree’ to 1 = ‘strongly disagree’. The total score is calculated by adding together each single item, and higher score indicates more positive attitudes. Social contact Social contact with someone with a mental health problem was assessed by asking the following question: Who is the person closest to you who has or has had some kind of mental health problem? Scoring the answers in the following categories: self, immediate family (spouse/sister/brother/parents…), Page 5/20 one of your children, partner (living with you), partner (not living with you), other family (uncle/aunt/cousin/grandparent…), friend, acquaintance, work colleague, neighbours, ex-partner, no-one known. For more simplicity in the analysis the categories were reduced to three: no-one-known, self, other. one of your children, partner (living with you), partner (not living with you), other family (uncle/aunt/cousin/grandparent…), friend, acquaintance, work colleague, neighbours, ex-partner, no-one known. For more simplicity in the analysis the categories were reduced to three: no-one-known, self, other. Statistical analysis All analyses were weighted to reflect population characteristics in England. Survey weights were developed using prevalence rates of ethnicity with geographic region from the UK Government’s Office for National Statistics. All models were adjusted for the impact of the ‘‘Burst’’ as well as main relevant socio- demographic characteristics identified from the literature in the field [i.e., gender; age; ethnicity; socioeconomic group; geographic region; marital status; having children; working status; degree of familiarity with mental illness (Categorized as me/other/no-one-known answering the question: Who is the person closest to you who has or has had some mental illness?). Descriptive statistics for participant demographics were calculated and presented using unweighted frequency and weighted percentage/mean/standard deviation. Adjusted logistic regression models were used to analyse campaign awareness. To examine whether there was a consistent pre/post effect, we included a variable indicating whether the assessment occurred before or after the burst of media (pre vs. post). We also investigated factors significantly associated with campaign awareness where the following independent variables were entered into the model: ethnicity (categorical: White, Asian, Black, Mixed or Other), gender, age (categorical: 25-29, 30-34, 35-39, 40-45), marital status (married: yes/no), having children (children: yes/no) employment status (categorical: employed (full or part-time employment), not working (unemployed or retired), student), socioeconomic group (categorical: lower middle class C1, skilled working class C2, semi-skilled and unskilled manual workers D), geographic region (categorical: Yorkshire and Humber, North East, North West, East Midlands, West Midlands, East of England, London, South East, South West) and social contact (categorical: having a mental health problem oneself, knowing someone with a mental health problem or not knowing anyone with a mental health problem). Multivariable linear regression models were used to analyse the total MAKS, CAMI and RIBS scores. A pre/post effect for each outcome measure was investigated as described above. Multivariable logistic regression models estimated the odds of responding positively (i.e., agree strongly or agree slightly) to each of the MAKS and RIBS items. All items were coded so that agreement summarised a less stigmatising response. Presence of a long-term trend was examined by including campaign burst as a covariate in the model for the total score of MAKS, CAMI and RIBS, and for each item of the MAKS and RIBS scales. Multivariable linear regression models were used to analyse the total MAKS, CAMI and RIBS scores. A pre/post effect for each outcome measure was investigated as described above. Campaign awareness For each of the three bursts, significant pre-post awareness differences were found (OR = 2.83, CI = 1.90 to 4.20, p<0.001; OR = 1.72, CI = 1.22 to 2.42, p=0.002; OR = 1.41, CI = 1.01 to 1.97, p=0.043), with similar levels of post-burst awareness of 33%, 34% and 33% respectively. Target population 3700 persons were interviewed between April 2017 and February 2019. The average age of the sample was 35.8 years, 51.8% were women, 44.8% lower-middle class (C1), 86.0% working at the time of the interview and 73.5% white. More details of the sample can be seen in table 1. Factors associated with campaign awareness Characteristics significantly associated with campaign awareness in the first burst were being aged between 30 and 34 (OR=1.59, CI = 1.09 to 2.31; p=0.016) as compared to aged 40-45, being Asian (OR = 1.95, CI = 1.30 to 2.92; p = 0.001), knowing someone with a mental health problem (OR = 1.96, CI = 1.45 to 2.64; p < 0.001) and having children (OR = 1.49, CI= 1.06 to 2.09; p = 0.021). In the second burst, the factors associated with campaign awareness were being Asian (OR = 1.60, CI= 1.04 to 2.48; p = 0.033) as compared to White, being male (OR = 0.74, CI = 0.55 to 0.99; p = 0.047), having children (OR = 1.47, CI = 1.05 to 2.06; p = 0.025), having or having had a mental health problem (OR = 2.40, CI = 1.46 to 3.93; p = 0.001) and knowing someone with a mental health problem (OR = 2.10, CI = 1.57 to 2.82; p < 0.001). Finally, for the third burst, characteristics significantly associated with campaign awareness include male sex (OR = 0.62, CI = 0.46 to 0.84; p = 0.002), having children (OR = 1.82, CI = 1.31 to 2.53; p < 0.001), knowing someone with a mental health problem (OR = 1.78, CI = 1.30 to 2.42; p < 0.001), being Black or other ethnicity (OR = 4.51, CI = 1.67 to 12.17; p = 0.003; OR = 12.53, CI = 1.52 to 103.03; p = 0.019) and being from London (OR = 2.06, CI = 1.17 to 3.64; p = 0.013) as compared to Yorkshire and Humber. Results of the regression to explore factors associated with campaign awareness, including reference categories, can be seen in table 2. Statistical analysis Multivariable logistic regression models estimated the odds of responding positively (i.e., agree strongly or agree slightly) to each of the MAKS and RIBS items. All items were coded so that agreement summarised a less stigmatising response. Presence of a long-term trend was examined by including campaign burst as a covariate in the model for the total score of MAKS, CAMI and RIBS, and for each item of the MAKS and RIBS scales. The relationship between each of the outcome measures (CAMI, MAKS, RIBS) with campaign awareness was assessed by including the campaign awareness variable into the adjusted linear regression model. This will also inform us of factors associated with each outcome measure. Attitude No significant pre/post differences were found in the total CAMI score after each of the bursts nor a significant improvement across all three bursts. CAMI percentage scores for each time point can be seen in figure 1. When combining all three bursts, no significant association was found between campaign awareness and the CAMI total score. Factors associated with a more positive attitudes towards mental illness were being female (β = 2.39, CI = 1.82 to 2.96; p < 0.001), lower middle class (β = 0.90, CI = 0.25 to 1.55; p = 0.007), being from the North East (β = 1.39, CI= 0.10 to 2.68, p=0.035) and having familiarity with people with mental problems (β = 2.92, CI = 2.35 to 3.49; p < 0.001) or suffering from them oneself (β = 7.22, CI = 6.40 to 8.04; p < 0.001). Being Asian or other ethnicity and living in London were factors associated with a lower CAMI scores (β = -3.18, CI = -4.01 to -2.35, p < 0.001; β = -5.32, CI = -8.23 to -2.41, p < 0.001; β = -2.08, CI = -3.08 to -1.07, p<0.001). Results of the linear regression model to explore factors associated with the total CAMI score, including reference categories, are presented in table 3. Knowledge Page 7/20 No significant pre/post differences were found in the total score of the MAKS after each of the three bursts. Over the course of all three bursts, analyses reveal a significant increase in the “Recover” item (People with severe mental health problems can fully recover) ((OR = 1.10, CI = 1.00 to 1.20, p = 0.045) and the “Advice to a friend” item (If a friend had a mental health problem, I know what advice to give them to get professional help) (OR=1.10, CI=1.01 to 1.21, p = 0.037), but not on any other item nor the Page 7/20 total score. Overall percentage and item scores from the MAKS scale for each time point can be seen in figures 1 and 2 respectively. total score. Overall percentage and item scores from the MAKS scale for each time point can be seen in figures 1 and 2 respectively. When all three bursts were combined, campaign awareness was significantly associated with a greater MAKS score (β = 0.60, CI = 0.36 to 0.84; p < 0.001), Other factors associated with a greater total MAKS score were being female (β = 0.53, CI = 0.30 to 0.76; p < 0.001), having children (β = 0.38, CI = 0.13 to 0.63; p = 0.003) and having had social contact with people with mental problems (β = 1.44, CI = 1.21 to 1.67; p < 0.001) or experiencing them oneself (β = 2.91, CI = 2.58 to 3.33; p < 0.001). Asian ethnicity was associated with lower MAKS score (β = -0.71, CI = -1.07 to -0.35; p <0.001). Results of the linear regression model to explore factors associated with the total MAKS score, including reference categories, are presented in table 3. Desire for Social distance No significant pre/post differences were found in the total RIBS intended behaviour score after each of the bursts. However across all three bursts there was a significant improvement in the “living with” (In the future, I would be willing to live with someone with a mental health problem) item (OR = 1.13, CI = 1.03 to 1.25; p=0.008). Overall percentage and item scores from the RIBS scale for each time point can be seen in figures 1 and 3 respectively. For all three bursts combined, there was a statistically significant positive association between campaign awareness and the total RIBS score (β = 0.58, CI = 0.31 to 0.84; p < 0.001). Other factors associated with the level of intended future contact with people with mental health problems are being under 40 years of Page 8/20 age (β = 0.38, CI = 0.06 to 0.69, p = 0.019; β = 0.45, CI = 0.11 to 0.79, p = 0.010; β = 0.76, CI = 0.41 to 1.11, p < 0.001), being married (β = 0.39, CI = -0.06 to 0.71; p = 0.021) and having had social contact with people with mental problems (β = 2.12, CI = 1.85 to 2.38; p < 0.001) or experiencing them oneself (β = 3.20, CI = 2.82 to 3.57; p < 0.001). Being Black or of Asian ethnicity and living in London were associated with lower scores in the RIBS (β = -0.90, CI = -1.66 to -0.15, p = 0.019; β = -1.15, CI = -1.57 to -0.72, p < 0.001; β = -0.75, CI= -1.29 to -0.22, p = 0.005). Results of the linear regression model to investigate factors associated with the total RIBS score, including reference categories, are presented in table 3. Discussion These interim results suggest that the campaign is reaching and having some impact on its new target audience (people aged between mid-twenties and mid-forties of middle-low income groups and more focused on men), at least in terms of some domains of stigma related knowledge and desire for social distance. The results are similar to those obtained in the first phase of the campaign, with improvements only in RIBS item “living with” (13); followed in the second phase by improvements in the “work with” and “live nearby” items of RIBS, and “paid employment”, “advice”, and “recover” items of MAKS (12). The strongest predictive variable of knowledge, attitude and social distance desire throughout the three bursts, was having or having had contact with a person with mental health problems or suffering from them oneself. The campaign aims to promote this effect by increasing people’s confidence that they can provide supportive contact, and their desire to do so, as opposed to responding by increasing their social distance. For each burst, moderate levels of awareness were reached, always being significantly higher in the post- measures, which indicates the transmission efficiency of the campaign. Compared to levels reached in previous campaigns (of up to 59% in 2012) (12, 13), the levels reached in this phase of the campaign are somewhat lower however, suggesting more work may be needed to identify the best methods to reach the new target group. In our results, awareness is associated with better scores on MAKS and RIBS, but not with CAMI(12). This may be because attitudes are a more complex construct to change, as they are strongly related to the etiological belief of mental disorder in interaction with the culture (25, 26). Since the causality of the disease was not among the main objectives of the campaign it is possible that changes in attitudes occur more slowly and in the long term. We found that the main factors associated with awareness are having or having had social contact with a person with mental illness, or having or having had a mental health problem oneself, and having children. Other relevant factors are being male (2nd and 3rd burst), being Asian (1st and 2nd burst), and being Black or other ethnicity (3rd burst). Discussion While the results were not consistent across bursts in terms of the relationship between ethnicity and awareness, it was associated with either being Black or Asian for Page 9/20 Page 9/20 all three bursts. These results also seem to support the efficacy of the campaign in having focussed its content on men and adding the activities targeting parents. all three bursts. These results also seem to support the efficacy of the campaign in having focussed its content on men and adding the activities targeting parents. While over the course of Time to Change we have found no evidence that demographic differences in stigma have widened, and indeed those by age group and region of England have narrowed, those for socioeconomic status, ethnicity and sex have so far remained unchanged (14). By targeting a lower socioeconomic group and creating relatively greater awareness among men and in Black and ethnic minority groups, the campaign is showing the potential to address these persistent differences in stigma. Certain limitations in the study should also be mentioned. Firstly, it is important to point out that, as self- reporting measures, evaluation can always be affected by response trends or phenomena such as social desirability, which can be accentuated by measuring a sensitive and controversial construct such as stigma. Moreover, it was not possible to randomize participants or to manipulate the intervention since this is a real-world study, and there may be variables associated with campaign awareness which are also associated with more positive attitudes. In the same way it is possible that indirect effects of the campaign will affect the results of the campaign, since there may be individuals who do not recognize the campaign but have discussed it with others. Also, a previous campaign called Heads Together could have affected some of the awareness measured, especially at the first pre point. However, the market research agency used TTC campaign materials to ascertain awareness of TTC specifically. Finally, it is necessary to keep in mind that changes in attitudes and behaviours can occur in the longer term both positively and negatively, being phenomena in constant interaction with other influences. For instance, a participant might not have scored highly on the scales at the time of the post-burst but if a close relative suffers from a mental illness at a later stage, that same participant might act differently because of their previous experience with the campaign. Discussion However, despite the importance of long-term measures on the effects of anti-stigma programmes, few studies provide them (27). Conclusions The results of the present study reveal early evidence of the effectiveness of the third phase of Time to Change anti-stigma campaign targeting a lower income group than in the previous phases and more focused on men, based on the similarity of the results to those of phase 1. The shift in content focus to men and the activities aimed at parents were effective in raising awareness of the campaign in these groups. However, it remains to be seen whether the campaign can lead to a narrowing of the pre-existing differences in stigma by socioeconomic status, ethnicity and sex. In order to address these inequalities most effectively, a better evidence base is needed regarding the reasons for these demographic differences in stigma. Consent for publication Not applicable Abbreviations Page 10/20 MAKS: Mental Health Knowledge Schedule; CAMI: Community Attitudes toward Mental Illness, RIBS: Reported and Intended Behaviour Scale, C1: lower middle class, C2: skilled working class, D: semi-skilled and unskilled manual workers, UK: United Kingdom; OR: odds ratio; CI: confidence interval. Availability of data and materials Data are not yet available as this is part of an ongoing study. At the end of the study they may be available on reasonable request to the senior author (Claire Henderson), subject to approval from the funders Ethics approval and consent to participate The study was classified as exempt by the King’s College London psychiatry, nursing and midwifery research ethics subcommittee. King's College London Psychiatry Research Ethics Committee deem as exempt from the approval process the analysis of data which is sent anonymised for analysis, as was the case for these data. Anonymisation is a process of removing identifying information to allow data to be more widely used. It requires that identifiers are "removed, obscured, aggregated and/or altered in some way" (UKAN - UK Anonymisation Network). Once data is anonymised data it is excluded from the Data Protection Act (28). Funding The Time to Change evaluation was funded by the UK Government Department of Health, Comic Relief and Big Lottery Fund. CH was supported by these grants during phases 1-3 of Time to Change and LP during phase 3. The funding source had no involvement in the study design, data, or report writing. Competing interests The authors declare that they have no competing interests Authors' contributions CGS drafted the manuscript and conducted the analyses for Table 1 P conducted the analyses for the other tables and created the other tables a MM assisted with interpretation of the results and drafting the manuscript MM assisted with interpretation of the results and drafting the manuscript Page 11/20 Acknowledgements We are grateful for collaboration on the evaluation by: Mark Slater and Craig Meikle, Consumer Insight; Sue Baker, Paul Farmer, George Hoare and Jo Loughran. References 1. Office of the Deputy Prime M. Mental Health and Social Exclusion: Social Exclusion Report. London Social Exclusion Unit; 2004. 2. Livingston JD, Boyd JE. Correlates and consequences of internalized stigma for people living with mental illness: a systematic review and meta-analysis. Social science & medicine (1982). 2010;71(12):2150-61. 3. Corrigan PW, Watson AC. Understanding the impact of stigma on people with mental illness. World psychiatry : official journal of the World Psychiatric Association (WPA). 2002;1:16-20. 3. Corrigan PW, Watson AC. Understanding the impact of stigma on people with mental illness. World psychiatry : official journal of the World Psychiatric Association (WPA). 2002;1:16-20. 4. Rubio-Valera M, Fernández A, Evans-Lacko S, Luciano JV, Thornicroft G, Aznar-Lou I, et al. Impact of the mass media OBERTAMENT campaign on the levels of stigma among the population of Catalonia, Spain. European Psychiatry. 2016;31:44-51. 5. Corrigan PW, Morris, S. B., Michaels, P.J., Rafacz, J. D., Rüsch, N. Challenging the public stigma of mental illness: A meta-analysis of outcome studies. Psychiatric Services. 2012;63(10):963-73. 6. Grausgruber A, Schöny W, Grausgruber-Berner R, Koren G, Apor BF, Wancata J, et al. „Schizophrenie hat viele Gesichter”– Evaluierung der österreichischen Anti-Stigma-Kampagne 2000–2002. Psychiat Prax. 2009;36(07):327-33. 7. Livingston JD, Cianfrone M, Korf-Uzan K, Coniglio CJSP, Epidemiology P. Another time point, a different story: one year effects of a social media intervention on the attitudes of young people towards mental health issues. 2014;49(6):985-90. 8. Henderson C, Thornicroft G. Stigma and discrimination in mental illness: Time to Change. Lancet. 2009;373(9679):1928-30. 8. Henderson C, Thornicroft G. Stigma and discrimination in mental illness: Time to Change. Lancet. 2009;373(9679):1928-30. 9. Corrigan PW, Shapiro JR. Measuring the impact of programs that challenge the public stigma of mental illness. Clinical psychology review. 2010;30(8):907-22. 9. Corrigan PW, Shapiro JR. Measuring the impact of programs that challenge the public stigma of mental illness. Clinical psychology review. 2010;30(8):907-22. 10. Clement S LF, Barley E, Evans-Lacko S, Williams P, Yamaguchi S, Slade M, Rüsch N, Thornicroft G. . Mass media interventions for reducing mental health-related stigma. . Cochrane Database of Systematic Reviews. 2013(Issue 7. Art. No.: CD009453). 10. Clement S LF, Barley E, Evans-Lacko S, Williams P, Yamaguchi S, Slade M, Rüsch N, Thornicroft G. . Mass media interventions for reducing mental health-related stigma. . Cochrane Database of Systematic Reviews. 2013(Issue 7. Art. No.: CD009453). 11. Thornicroft G, Rose D, Kassam A. Stigma: ignorance, prejudice or discrimination. British Journal of Psychiatry. 2007;190:192-3. 11. Page 11/20 CH conceived the paper and edited drafts of the manuscript All authors read and approved the final version of the manuscript References Thornicroft G, Rose D, Kassam A. Stigma: ignorance, prejudice or discrimination. 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Henderson C, Evans-Lacko S, Flach C, Thornicroft G. Responses to mental health stigma questions: the importance of social desirability and data collection method. CanJPsychiatry. 2012;57(3):152-60. 16. Pettigrew TF, Tropp LR. A meta-analytic test of intergroup contact theory. Journal of personality and social psychology. 2006;90(5):751-83. 17. Al Ramiah A, Hewstone M. Intergroup contact as a tool for reducing, resolving, and preventing intergroup conflict: evidence, limitations, and potential. The American Psychologist. 2013;68:527-42. 17. Al Ramiah A, Hewstone M. Intergroup contact as a tool for reducing, resolving, and preventing intergroup conflict: evidence, limitations, and potential. The American Psychologist. 2013;68:527-42. 18. West K, Holmes E, Hewstone M. Enhancing imagined contact to reduce prejudice against people with schizophrenia. Group processes & intergroup relations : GPIR. 2011;14(3):407-28. 18. West K, Holmes E, Hewstone M. Enhancing imagined contact to reduce prejudice against people with schizophrenia. Group processes & intergroup relations : GPIR. 2011;14(3):407-28. 19. Nicholas A, Rossetto A, Jorm A, Pirkis J, Reavley N. Importance of Messages for a Suicide Prevention Media Campaign. Crisis. 2018;39(6):438-50. 19. Nicholas A, Rossetto A, Jorm A, Pirkis J, Reavley N. Importance of Messages for a Suicide Prevention Media Campaign. Crisis. 2018;39(6):438-50. 20. Evans-Lacko S, Little K, Meltzer H, Rose D, Rhydderch D, Henderson C, et al. Development and psychometric properties of the Mental Health Knowledge Schedule. CanJPsychiatry. 2010;55(7):440- 8. 20. Evans-Lacko S, Little K, Meltzer H, Rose D, Rhydderch D, Henderson C, et al. Development and psychometric properties of the Mental Health Knowledge Schedule. CanJPsychiatry. 2010;55(7):440- 8. 21. References Rusch N, Evans-Lacko SE, Henderson C, Flach C, Thornicroft G. Knowledge and attitudes as predictors of intentions to seek help for and disclose a mental illness. PsychiatrServ. 2011;62(6):675- 8. 21. Rusch N, Evans-Lacko SE, Henderson C, Flach C, Thornicroft G. Knowledge and attitudes as predictors of intentions to seek help for and disclose a mental illness. PsychiatrServ. 2011;62(6):675- 8. 22. Taylor SM, Dear MJ. Scaling community attitudes toward the mentally ill. SchizophrBull. 1981;7(2):225 40 22. Taylor SM, Dear MJ. Scaling community attitudes toward the mentally ill. SchizophrBull. 1981;7(2):225-40. 23. Ilic N HH, Henderson C, Evans-Lacko S, Thornicroft G. Attitudes towards mental illness. In: Craig R FE, Mindell J, editor. Health Survey for England. I. London: Health and Social Care Information Centre.; 2014. 24. Evans-Lacko S, Rose D, Little K, Rhydderch D, Henderson C, Thornicroft G. Development and Psychometric Properties of a Stigma Related Behaviour Measure. Epidemiology and Psychiatric Sciences. 2011;20(3):263-71. 25. Mannarini S, Boffo M, Rossi A, Balottin L. Etiological Beliefs, Treatments, Stigmatizing Attitudes toward Schizophrenia. What Do Italians and Israelis Think? Front Psychol. 2018;8:2289-. 26. Corrigan P, Rüsch N, Scior K. Adapting Disclosure Programs to Reduce the Stigma of Mental Illness. Psychiatric Services. 2018;69(7):826-8. Page 13/20 Page 13/20 27. Mehta N, Clement S, Marcus E, Stona AC, Bezborodovs N, Evans-Lacko S, et al. Evidence for effective interventions to reduce mental health-related stigma and discrimination in the medium and long term: systematic review. The British journal of psychiatry : the journal of mental science. 2015;207(5):377-84. 27. Mehta N, Clement S, Marcus E, Stona AC, Bezborodovs N, Evans-Lacko S, et al. Evidence for effective interventions to reduce mental health-related stigma and discrimination in the medium and long term: systematic review. The British journal of psychiatry : the journal of mental science. 2015;207(5):377-84. 27. Mehta N, Clement S, Marcus E, Stona AC, Bezborodovs N, Evans-Lacko S, et al. Evidence for effective interventions to reduce mental health-related stigma and discrimination in the medium and long term: systematic review. The British journal of psychiatry : the journal of mental science. 2015;207(5):377-84. 27. Mehta N, Clement S, Marcus E, Stona AC, Bezborodovs N, Evans-Lacko S, et al. Evidence for effective interventions to reduce mental health-related stigma and discrimination in the medium and long term: systematic review. The British journal of psychiatry : the journal of mental science. 2015;207(5):377-84. 28. King’s College London - Ethics and confidentiality [Internet]. [cited 2019 Dec 11]. Available from: https://www.kcl.ac.uk/library/researchsupport/research-data-management/RDM-web-pages- 2/Store/DataConfidentiality2 Tables Page 14/20 Table 1. Participant’s socio-demographic characteristics, un-weighted frequency and weighted percentages (n=3700) Demographic characteristic N (%) Gender, Female n (%) 1892 (51.82) Age, mean (SD) 35.77 (5.68) Age group 25-29 30-34 35-39 40-45 639 (17.10) 880 (24.42) 1060 (29.04) 1121 (29.44) Socioeconomic status, n (%) C1, lower middle class C2, skilled working class D, semi-skilled and unskilled manual workers 1618 (44.84) 1144 (29.89) 938 (25.27) Employment status, n (%) Working Student Not working 3209 (86.05) 22 (0.74) 469 (13.2) Married, yes, n (%) 2564 (69.69) Children, yes, n (%) 2079 (57.49) Ethnicity, n (%) Black White Asian Mixed Other 102 (4.71) 3140 (73.55) 368 (17.56) 76 (3.66) 14 (0.53) Region North East North West Yorkshire & Humberside East Midlands West Midlands East of England London South East South West 223 (6.51) 555 (18.57) 416 (12.04) 361 (10.27) 398 (10.49) 398 (10.07) 538 (13.04) 561 (14.44) 250 (4.58) Who is the person closest to you who has or has had some mental illness? No-one-known Self Other 1844 (49.45) 384 (9.72) 1472 (40.82) Figures Results of the multivariate logistic regression models to explore factors associated with campaign awareness Burst 1 April 2017 (n=1349) Burst 2 February 2018 (n=1179) Burst 3 February 2019 (n=1169) OR (95% CI) p value OR (95% CI) p value OR (95% CI) p value Age 25-29 30-34 35-39 40-45 (ref) 1.29 (0.85– 1.97) 1.59 (1.09– 2.31) 1.10 (0.75– 1.62) - 0.235 0.016 0.624 - 1.07 (0.70– 1.64) 1.02 (0.69– 1.51) 1.31 (0.91– 1.90) - 0.750 0.907 0.148 - 1.13 (0.72– 1.79) 1.09 (0.72– 1.63) 1.05 (0.73– 1.50) - 0.592 0.684 0.801 - Gender Female Male (ref) 0.87 (0.65– 1.17) - 0.350 - 0.74 (0.55– 0.99) - 0.047 - 0.62 (0.46– 0.84) - 0.002 - Ethnicity Black Asian Mixed Other White (ref) 1.68 (0.89– 3.17) 1.95 (1.30– 2.92) 0.94 (0.36– 2.43) 2.80 (0.39– 20.14) - 0.109 0.001 0.895 0.305 - 2.09 (0.88– 4.97) 1.60 (1.04– 2.48) 1.73 (0.76– 3.94) - - 0.094 0.033 0.191 - - 4.51 (1.67– 12.17) 1.24 (0.76– 2.03) 2.24 (0.77– 6.50) 12.53 (1.52– 103.03) - 0.003 0.389 0.137 0.019 - Socioeconomic status C2, skilled working class D, working class C1, low-middle class (ref) 0.92 (0.65– 1.29) 1.09 (0.77– 1.55) - 0.624 0.629 - 1.32 (0.95– 1.85) 0.95 (0.65– 1.38) - 0.099 0.772 - 1.09 (0.77– 1.53) 0.93 (0.64– 1.36) - 0.635 0.723 - Married Yes No (ref) 1.13 (0.79– 1.61) - 0.504 - 1.08 (0.76– 1.54) - 0.663 - 1.27 (0.88– 1.83) - 0.195 - Children Yes No (ref) 1.49 (1.06– 2.09) - 0.021 - 1.47 (1.05– 2.06) - 0.025 - 1.82 (1.31– 2.53) - <0.001 - Employment status Not Working Full/Partial work Student (ref) 0.57 (0.14– 2.35) 0.78 (0.20– 3.06) - 0.437 0.717 - 0.33 (0.04– 2.69) 0.46 (0.06– 3.64) - 0.297 0.461 - 0.44 (0.04– 4.77) 0.59 (0.06– 6.10) - 0.503 0.654 - Region North East North West East Midlands West Midlands 1.15 (0.55– 2.38) 0.97 (0.56– 1.69) 0.713 0.916 0.035 0.311 0.97 (0.50– 1.91) 1.36 (0.77– 2.39) 0.936 0.291 0.653 0.936 1.30 (0.63– 2.71) 1.42 (0.82– 2.48) 0.476 0.213 0.190 0.115 London South east South west Yorkshire & Humber (ref) 1.95 (1.05– 3.64) 1.35 (0.76– 2.39) 1.69 (0.94– 3.04) 1.28 (0.72– 2.26) 1.37 (0.78– 2.38) 1.40 (0.73– 2.70) - 0.402 0.272 0.316 - 1.15 (0.63– 2.08) 0.97 (0.53– 1.81) 1.08 (0.59– 1.96) 1.66 (0.97– 2.85) 0.83 (0.45– 1.52) 0.76 (0.33– 1.77) - 0.067 0.545 0.522 - 1.49 (0.82– 2.71) 1.65 (0.88– 3.09) 1.37 (0.70– 2.67) 2.06 (1.17– 3.64) 0.99 (0.55– 1.80) 1.32 (0.69– 2.55) - 0.013 0.980 0.401 - Closest person with MI Self Other None (ref) 1.52 (0.94– 2.47) 1.96 (1.45– 2.64) - 0.091 <0.001 - 2.40 (1.46– 3.93) 2.10 (1.57– 2.82) - 0.001 <0.001 - 1.57 (0.97– 2.53) 1.78 (1.30– 2.42) - 0.066 <0.001 - OR = Odds ratio; CI = Confidence interval Figure 1 Percentage scores for the Mental Health Knowledge Schedule (MAKS) Community Attitudes toward the London South east South west Yorkshire & Humber (ref) 1.95 (1.05– 3.64) 1.35 (0.76– 2.39) 1.69 (0.94– 3.04) 1.28 (0.72– 2.26) 1.37 (0.78– 2.38) 1.40 (0.73– 2.70) - 0.402 0.272 0.316 - 1.15 (0.63– 2.08) 0.97 (0.53– 1.81) 1.08 (0.59– 1.96) 1.66 (0.97– 2.85) 0.83 (0.45– 1.52) 0.76 (0.33– 1.77) - 0.067 0.545 0.522 - 1.49 (0.82– 2.71) 1.65 (0.88– 3.09) 1.37 (0.70– 2.67) 2.06 (1.17– 3.64) 0.99 (0.55– 1.80) 1.32 (0.69– 2.55) - 0.013 0.980 0.401 - Closest person with MI Self Other None (ref) 1.52 (0.94– 2.47) 1.96 (1.45– 2.64) - 0.091 <0.001 - 2.40 (1.46– 3.93) 2.10 (1.57– 2.82) - 0.001 <0.001 - 1.57 (0.97– 2.53) 1.78 (1.30– 2.42) - 0.066 <0.001 - OR = Odds ratio; CI = Confidence interval OR = Odds ratio; CI = Confidence interval P 17/20 Figure 1 Percentage scores for the Mental Health Knowledge Schedule (MAKS) Community Attitudes toward the Mentally Ill Scale (CAMI), and Reported and Intended Behaviour Scale (RIBS) during the social marketing campaign (weighted estimates). Figures Page 15/20 Table 2. Figures Figure 1 Percentage scores for the Mental Health Knowledge Schedule (MAKS) Community Attitudes toward the Mentally Ill Scale (CAMI), and Reported and Intended Behaviour Scale (RIBS) during the social marketing campaign (weighted estimates). Figure 1 Percentage scores for the Mental Health Knowledge Schedule (MAKS) Community Attitudes toward the Mentally Ill Scale (CAMI), and Reported and Intended Behaviour Scale (RIBS) during the social marketing campaign (weighted estimates). Page 17/20 Page 17/20 Table 3. Results of multivariate linear regression models to explore factors associated with MAKS, CAMI and RIBS. Figures MAKS (n=3700) CAMI (n=3700) RIBS (n=3700) β (95% CI) p value β (95% CI) p value β (95% CI) p value Burst 0.10 (-0.03 to 0.23) 0.125 0.01 (-0.30 to 0.33) 0.928 0.10 (-0.05 to 0.25) 0.188 Awareness 0.60 (0.36 to 0.84) <0.001 0.30 (-0.28 to 0.88) 0.310 0.58 (0.31 to 0.84) <0.001 Age 25-29 30-34 35-39 40-45 (ref) -0.23 (-0.54 to 0.09) -0.10 (-0.40 to 0.19) -0.15 (-0.43 to 0.13) - 0.160 0.503 0.290 - -0.72 (-1.48 to 0.03) -0.61 (-1.31to 0.10) -0.37 (-1.05 to 0.31) - 0.059 0.093 0.281 - 0.76 (0.41 to 1.11) 0.45 (0.11 to 0.79) 0.38 (0.06 to 0.69) - <0.001 0.010 0.019 - Gender Female Male (ref) 0.53 (0.30 to 0.76) - <0.001 - 2.39 (1.82 to 2.96) - <0.001 - 0.22 (-0.05 to 0.48) - 0.111 - Ethnicity Black Asian Mixed Other White (ref) 0.11 (-0.48 to 0.70) -0.71 (-1.07 to -0.35) 0.41 (-0.38 to 1.20) -0.80 (-2.84 to 1.23) - 0.716 <0.001 0.309 0.439 - -0.07 (-1.46 to 1.31) -3.18 (-4.01 to -2.35) -0.71 (-2.60 to 1.19) -5.32 (-8.23 to -2.41) - 0.918 <0.001 0.465 <0.001 - -0.90 (-1.66 to -0.15) -1.15 (-1.57 to -0.72) -0.86 (-1.75 to 0.02) -0.15 (-1.66 to 1.37) - 0.019 <0.001 0.056 0.851 - Socioeconomic status C2, skilled working class D, working class C1, low-middle class (ref) -0.23 (-0.48 to 0.02) -0.20 (-0.48 to 0.07) - 0.066 0.151 - -0.87 (-1.50 to -0.24) -0.90 (-1.55 to -0.25) - 0.007 0.007 - -0.02 (-0.30 to 0.26) -0.15 (-0.47 to 0.17) - 0.894 0.357 - Married Yes No (ref) 0.08 (-0.19 to 0.35) - 0.549 - -0.12 (-0.77 to 0.53) - 0.712 - 0.39 (0.06 to 0.71) - 0.021 - Children Yes No (ref) 0.38 (0.13 to 0.63) - 0.003 - -0.05 (-0.65 to 0.55) - 0.872 - 0.16 (-0.13 to 0.45) - 0.274 - Employment status Not Working Full/Partial work Student (ref) -0.11 (-1.45 to 1.24) -0.36 (-1.67 to 0.96) - 0.877 0.597 - -0.61 (-3.62 to 2.41) -1.63 (-4.53 to 1.27) - 0.694 0.271 - -0.87 (-1.90 to 0.15) -0.78 (-1.74 to 0.18) - 0.094 0.112 - Region 0.530 0.035 0.190 Table 3. Figures Results of multivariate linear regression models to explore factors associated with MAKS, CAMI and RIBS variate linear regression models to explore factors associated with MAKS, North West East Midlands West Midlands East of England London South east South west Yorkshire & Humber (ref) 0.17 (-0.37 to 0.71) 0.23 (-0.20 to 0.65) 0.03 (-0.44 to 0.49) 0.17 (-0.29 to 0.64) 0.17 (-0.30 to 0.63) -0.03 (-0.47 to 0.41) 0.08 (-0.35 to 0.52) 0.23 (-0.28 to 0.75) - 0.915 0.469 0.483 0.887 0.706 0.375 - 1.39 (0.10 to 2.68) 0.63 (-0.37 to 1.62) 0.47 (-0.62 to 1.56) -0.18 (-1.23 to 0.86) -0.77 (-1.92 to 0.38) -2.08 (-3.08 to -1.07) -0.30 (-1.33 to 0.72) 0.49 (-0.78 to 1.75) - 0.394 0.730 0.188 <0.001 0.561 0.450 - 0.39 (-0.19 to 0.97) 0.11 (-0.38 to 0.61) -0.34 (-0.88 to 0.19) 0.15 (-0.38 to 0.68) -0.50 (-1.05 to 0.05) -0.75 (-1.29 to -0.22) -0.07 (-0.60 to 0.46) -0.12 (-0.78 to 0.54) - 0.210 0.575 0.075 0.005 0.790 0.717 - Closest person with MI Self Other None (ref) 2.96 (2.58 to 3.34) 1.44 (1.21 to 1.67) - <0.001 <0.001 - 7.22 (6.40 to 8.04) 2.92 (2.36 to 3.49) - <0.001 <0.001 - 3.20 (2.82 to 3.57) 2.12 (1.85 to 2.38) - <0.001 <0.001 - MAKS = Mental Health Knowledge Schedule; CAMI = Community Attitudes toward the Mentally Ill Scale; RIBS = Reported and Social distance desire Scale; O = Odds ratio; CI = Confidence interval North West East Midlands West Midlands East of England London South east South west Yorkshire & Humber (ref) 0.17 (-0.37 to 0.71) 0.23 (-0.20 to 0.65) 0.03 (-0.44 to 0.49) 0.17 (-0.29 to 0.64) 0.17 (-0.30 to 0.63) -0.03 (-0.47 to 0.41) 0.08 (-0.35 to 0.52) 0.23 (-0.28 to 0.75) - 0.915 0.469 0.483 0.887 0.706 0.375 - 1.39 (0.10 to 2.68) 0.63 (-0.37 to 1.62) 0.47 (-0.62 to 1.56) -0.18 (-1.23 to 0.86) -0.77 (-1.92 to 0.38) -2.08 (-3.08 to -1.07) -0.30 (-1.33 to 0.72) 0.49 (-0.78 to 1.75) - 0.394 0.730 0.188 <0.001 0.561 0.450 - 0.39 (-0.19 to 0.97) 0.11 (-0.38 to 0.61) -0.34 (-0.88 to 0.19) 0.15 (-0.38 to 0.68) -0.50 (-1.05 to 0.05) -0.75 (-1.29 to -0.22) -0.07 (-0.60 to 0.46) -0.12 (-0.78 to 0.54) - 0.210 0.575 0.075 0.005 0.790 0.717 - Closest person with MI Self Other None (ref) 2.96 (2.58 to 3.34) 1.44 (1.21 to 1.67) - <0.001 <0.001 - 7.22 (6.40 to 8.04) 2.92 (2.36 to 3.49) - <0.001 <0.001 - 3.20 (2.82 to 3.57) 2.12 (1.85 to 2.38) - <0.001 <0.001 - MAKS = Mental Health Knowledge Schedule; CAMI = Community Attitudes toward the Mentally Ill Scale; RIBS = Reported and Social distance desire Scale; O = Odds ratio; CI = Confidence interval North West East Midlands West Midlands East of England London South east South west Yorkshire & Humber (ref) 0.17 (-0.37 to 0.71) 0.23 (-0.20 to 0.65) 0.03 (-0.44 to 0.49) 0.17 (-0.29 to 0.64) 0.17 (-0.30 to 0.63) -0.03 (-0.47 to 0.41) 0.08 (-0.35 to 0.52) 0.23 (-0.28 to 0.75) - 0.915 0.469 0.483 0.887 0.706 0.375 - 1.39 (0.10 to 2.68) 0.63 (-0.37 to 1.62) 0.47 (-0.62 to 1.56) -0.18 (-1.23 to 0.86) -0.77 (-1.92 to 0.38) -2.08 (-3.08 to -1.07) -0.30 (-1.33 to 0.72) 0.49 (-0.78 to 1.75) - 0.394 0.730 0.188 <0.001 0.561 0.450 - 0.39 (-0.19 to 0.97) 0.11 (-0.38 to 0.61) -0.34 (-0.88 to 0.19) 0.15 (-0.38 to 0.68) -0.50 (-1.05 to 0.05) -0.75 (-1.29 to -0.22) -0.07 (-0.60 to 0.46) -0.12 (-0.78 to 0.54) - 0.210 0.575 0.075 0.005 0.790 0.717 - Closest person with MI Self Other None (ref) 2.96 (2.58 to 3.34) 1.44 (1.21 to 1.67) - <0.001 <0.001 - 7.22 (6.40 to 8.04) 2.92 (2.36 to 3.49) - <0.001 <0.001 - 3.20 (2.82 to 3.57) 2.12 (1.85 to 2.38) - <0.001 <0.001 - MAKS = Mental Health Knowledge Schedule; CAMI = Community Attitudes toward the Mentally Ill Scale; RIBS Reported and Social distance desire Scale; O Odds ratio; CI Confidence interval Page 19/20 MAKS = Mental Health Knowledge Schedule; CAMI = Community Attitudes toward the Mentally Ill Scale; RIBS = Reported and Social distance desire Scale; O = Odds ratio; CI = Confidence interval Figure 2 Page 19/20 MAKS = Mental Health Knowledge Schedule; CAMI = Community Attitudes toward the Mentally Ill Scale; RIBS = Reported and Social distance desire Scale; O = Odds ratio; CI = Confidence interval Figure 2 Figure 2 Page 19/20 Scores of Mental Health Knowledge Schedule items during the three bursts of the social marketing campaign (weighted estimates).All items score on a 5-point Likert scale, from 5 = ‘strongly agree’ to 1 = ‘strongly disagree’. Figures Employment: Most people with mental health problems want to have paid employment; Advice to a friend: If a friend had a mental health problem, I know what advice to give them to get professional help; Medication: Medication can be an effective treatment for people with mental health problems; Psychotherapy: Psychotherapy (e.g. counselling or talking therapy) can be an effective treatment for people with mental health problems; Recover: People with severe mental health problems can fully recover; Go to the doctor: Most people with mental health problems go to a healthcare professional to get help. can fully recover; Go to the doctor: Most people with mental health problems go to a healthcare professional to get help. Figure 3 Scores of the Reported and Intended Behaviour Scale items during the three bursts of the social marketing campaign (weighted estimates). All items are score on a 5-point Likert scale, from 1 = ‘strong disagree to engage in the stated behaviour’ to 5 = ‘strongly agree with engaging in the stated behaviour’ Live with: Are you currently living with, or have you ever lived with, someone with a mental health problem?; In the future, I would be willing to live with someone with a mental health problem; Work with Are you currently working with, or have you ever worked with, someone with a mental health problem?; I the future, I would be willing to work with someone with a mental health problem; Live nearby: Do you currently have, or have you ever had, a neighbour with a mental health problem?; In the future, I would b willing to live nearby to someone with a mental health problem; Continue a relationship: Do you current have, or have you ever had, a close friend with a mental health problem?; In the future, I would be willing to continue a relationship with a friend who developed a mental health problem. Fi 3 Figure 3 Figure 3 Scores of the Reported and Intended Behaviour Scale items during the three bursts of the social marketing campaign (weighted estimates). All items are score on a 5-point Likert scale, from 1 = ‘strongly disagree to engage in the stated behaviour’ to 5 = ‘strongly agree with engaging in the stated behaviour’. Live with: Are you currently living with, or have you ever lived with, someone with a mental health problem?; In the future, I would be willing to live with someone with a mental health problem; Work with: Are you currently working with, or have you ever worked with, someone with a mental health problem?; In the future, I would be willing to work with someone with a mental health problem; Live nearby: Do you currently have, or have you ever had, a neighbour with a mental health problem?; In the future, I would be willing to live nearby to someone with a mental health problem; Continue a relationship: Do you currently have, or have you ever had, a close friend with a mental health problem?; In the future, I would be willing to continue a relationship with a friend who developed a mental health problem. Scores of the Reported and Intended Behaviour Scale items during the three bursts of the social marketing campaign (weighted estimates). All items are score on a 5-point Likert scale, from 1 = ‘strongly disagree to engage in the stated behaviour’ to 5 = ‘strongly agree with engaging in the stated behaviour’. Live with: Are you currently living with, or have you ever lived with, someone with a mental health problem?; In the future, I would be willing to live with someone with a mental health problem; Work with: Are you currently working with, or have you ever worked with, someone with a mental health problem?; In the future, I would be willing to work with someone with a mental health problem; Live nearby: Do you currently have, or have you ever had, a neighbour with a mental health problem?; In the future, I would be willing to live nearby to someone with a mental health problem; Continue a relationship: Do you currently have, or have you ever had, a close friend with a mental health problem?; In the future, I would be willing to continue a relationship with a friend who developed a mental health problem. Page 20/20
https://openalex.org/W4390798273	https://aladabj.uobaghdad.edu.iq/index.php/aladabjournal/article/download/3187/2681	Arabic	null	ابراهيم كبة ودوره السياسي والاقتصادي في العراق	Al-ādāb	2,021	cc-by	13,253	د. علي المشهداني د. علي المشهداني مجلة كلية االداب / العدد76 د. علي المشهداني كلية التربية– ابن رشد قسم التار يخ المقدمة: تدلل احداث التاريخ وعبره انه مثلما للفئات والطبقات االجتماعية في المجتمع دور مهم واساسي في صياغة الحدث التاريخي ، فان للفرد في احيان عديدة دوراً ال يقل اهمية عن ذلك، فعلى سبيل المثال ال الحصر ان نوري سعيد ارتبط اسمه وتاريخه السياسي ارتباطاً وث يقاً بالعهد الملكي في العراق حتى سقوطه في الرابع عشر من تموز1958 ، يمثل واحداً من النماذج في تاريخ العراق المعاصر لتحديد دور الفرد في صياغة الحدث التاريخي وصيرورته، كما مترنيخ اونابليون او لينين فضال عن ماونيرتونغ والمهاتما غاندي يمثلون هذه النماذج خير تم ثيل على دور الفرد في صياغة الحدث التاريخي خالل الفترة الزمنية التي عاشوا وقادوا شعوبهم فيها، بل .يمكن القول انه ال يمكن تقليل او تجاوز دورهم مهما حاولنا ان نفعل ان ربط الفرد بالحدث ومحاولة تسليط الضوء على التطورات السياسية هو احد االتجاهات السليمة التي ب دأت تشهدها مؤسساتنا العلمية في السنوات االخيرة، مؤكدة بذلك سالمة وعلمية المدرسة التاريخية العراقية في شق . طريقها بثبات وموضوعية واضحتين ن و وز ور م ه و ي اي ن و ي ن ان ربط الفرد بالحدث ومحاولة تسليط الضوء على التطورات السياسية هو احد االتجاهات السليمة التي ب دأت تشهدها مؤسساتنا العلمية في السنوات االخيرة، مؤكدة بذلك سالمة وعلمية المدرسة التاريخية العراقية في شق . طريقها بثبات وموضوعية واضحتين وضمن هذا السياق لم تكن ثورة14 تموز1958 حدثاً فجائياً في العراق فقد حضّر وخطط لها طويالً رجال كثيرون بعضهم ظهر على الم سرح بقوة وتناولته رسائل جامعية عديدة ومؤلفات سياسية اخرى . فيما ظلت بعض الشخصيات التي ظهرت فيما بعد على المسرح السياسي لمكانتها السياسية والفكرية في بلورة الفكر المطلوب للنهوض بهذه الثورة بعيدة عن البحث المعمق والتناول الواسع على الرغم من ان هذه الشخصيات ك ان لها دورها .البارز ولم يتحدث عنها بسوء وتكلم عنها الجميع بايجابية عند مرورهم بها 355 355 مجلة كلية االداب / العدد76 ويعد ابراهيم كبة واحداً من هذه الشخصيات حيث اثرنا بحثها والكتابة عنها . يهدف البحث الى دراسة لتلك الشخصية والعوامل التي احاطت بتكوينها الفكري واظهار دورها المتميز في رسم مسي رة العراق االقتصادية ، تناول البحث نبذة موجزة عن نشأته وعالقته باسرته فضالً عن دراسة المرحلة االولى من حياته واثرها في تكوين شخصيته الفكرية والسياسية . كما تطرق البحث الى دوره في حكومة الزعيم الركن عبد الكريم قاسم اذ شغل فيها حقيبة االقتصاد مع ادارة وزارات اخرى بالوكالة حتى استقالته من الحكومة في15 شباط1960 . د. علي المشهداني ي تناولنا في البحث ايضاً اهم منجزاته على الصعيد الوطني ، وال سيما في مجال النفط والصناعة واالصالح الزراعي ، كما تمت مناقشة مواقفه واراءه من الوضع السياسي للمرحلة الممتدة من عام1958 حتى17 تموز1968 . ول تحقيق ذلك رجعنا الى المصادر والكتب التي تناولت احداث ثورة14 تموز1958 وما قبلها، فضالً عن االطالع على االضابير الخاصة به منذ تعيينه الول مرة حتى احالته على التقاعد عام1977 ، بهدف الوقوف على اكبر قدر ممكن من المعلومات عن تاريخ حياته ومواقفه الفكرية والسياس ية واالقتصادية ، وال سيما انه يعد واحداً من المفكرين االقتصاديين الذين نهجوا الفكر الماركسي ودعى الى تطبيق نظرياته االقتصادية والسياسية في . العراق اولا : اسرته ونشأتها العلمية والثقافية اولا : اسرته ونشأتها العلمية والثقافية اولا : اسرته ونشأتها العلمية والثقافية ابراهيم كبة من آل كبة، اسرة عربية من العراق وبيت مجد وادب وتجارة ف( ،ي بغداد، حيث فاز بعضهم بحظ من العلوم العربية والدينية1 ) تنتسب هذه االسرة الى قبيلة ربيعة، وقطنت دار السالم منذ العهد العباسي، كما ذكر احد الكتب الذي دون في العهد العباسي عن بيوت بغداد وتعدادها، ان آل كبة (. من ضمن بيوتها2 ) اما محل سكناهم فكان محلة ال هيتاويين الواقعة بين محلة الشيخ سراج ومحلة صبائغ االل، فيما سكن قسم اخر منهم في محافظة النجف، حيث كانوا (.يتاجرون في الجوخ والحرير3 ) ابراهيم كبة من آل كبة، اسرة عربية من العراق وبيت مجد وادب وتجارة ف( ،ي بغداد، حيث فاز بعضهم بحظ من العلوم العربية والدينية1 ) تنتسب هذه االسرة الى قبيلة ربيعة، وقطنت دار السالم منذ العهد العباسي، كما ذكر احد الكتب الذي دون في العهد العباسي عن بيوت بغداد وتعدادها، ان آل كبة (. من ضمن بيوتها2 ) (. ن بيو ه ن 2 ) اما محل سكناهم فكان محلة ال هيتاويين الواقعة بين محلة الشيخ سراج ومحلة صبائغ االل، فيما سكن قسم اخر منهم في محافظة النجف، حيث كانوا (.يتاجرون في الجوخ والحرير3 ) 356 د. علي المشهداني مجلة كلية االداب / العدد76 ،لالسرة يد بيضاء في تشجيع الحركات العلمية واالدبية منذ القرون الماضية وكانت مواسم افراحهم واتراحهم منتديات تتبارى بها شعراء (.العراق4 ) (،شهدت مساجالت السيد محمد سعيد الحبوبي5 ) مع العالم الكبير الحاج (.محمد حسن كبة6 ) ومالحم السيد حيدر الحلي التي تشيد بامجاد وبطوالت .العائلة، منذ القرون الماضية ، بقيت اثارها حتى الوقت الحاضر من مشاهير االسرة الحاج مصطفى كبة المتوفي سنة1232هجرية(. اولا : اسرته ونشأتها العلمية والثقافية 7 ) ( وولده محمد صالح كبة1784 - 1870 ) وهو الجد االول البراهيم كبة، الذي كان ورعاً محباً للخير باذالً جهده في مساعدة المحتاجين، فضالً عن بناءه للحصون والمعاقل التي بناها للزائرين وقوافل المسافرين بين بغداد وكربالء والنجف، بغداد الحلة، بغداد وسامراء، والتي ال زال اثارها باقية حتى يومنا هذا مثل خان بلد، وخان المسيب الكبير وخان بني سعد وخان االسكندرية (.الكبير وخان النصف8 ) ا ( ) ( ومن مشاهير االسرة ايضاً الشيخ محمد مهدي كبة1901 - 1983 (.) 9 ) احد اقطاب الحركة الوطنية في العراق وزعيم حزب االستقالل وعضو مجلس السيادة، اثر ثو رة14 تموز1958 ، فقد ساهم محمد مهدي كبة الى جانب الشباب في مقاومة االحتالل البريطاني عندما انتقل من سامراء الى الكاظمية، كونها كانت اهم مراكز الحركة الوطنية لوجود الشيخ محمد مهدي (.الخالصي10 ) وكان محمد مهدي كبة هو خال ابراهيم كبة، والذي يعد احد منابع تكوي نه . الفكري فيما بعد ي مما سبق يمكن استنتاجه ان ابراهيم كبة قد نشأ في خضم عائلة امتازت بحبها ورعايتها للعلم والعلماء، فضالً عن الثقافة والعلوم الدينية وطالبي . الحاجة ، مما هيأ له مناخاً فكرياً وثقافياً جعله متمسكاً بجذوره العربية ثانيا : نشأته ولد ابراهيم ابن الحاج عطوف ابن الحاج محمد ابن جعفر ابن حسن المعروف بكبة في بغداد شهر شباط عام1919 (، 11 ) وهو ينتمي الى عائلة (.عراقية عربية معروفة12 ) تخرج من المدرسة االبتدائية في بغداد عام 1931 ، ومن الثانوية المركزية للبنين في بغداد عام1936 (. 13 ) ثم درس 357 مجلة كلية االداب / العدد76 القانون، إذ ت خرج من كلية الحقوق العراقية في بغداد بدرجة الشرف االولى عام1940 - 1941 (. 14 ) حصل على الدبلوم العالي في االقتصاد السياسي من جامعة فؤاد االول عام 1945 - 1946 ، ثم حصل على شهادة الدبلوم في القانون العام من جامعة القاهرة عام46 - 1947 (. 15) ثم حصل على دبلوم الدولة للد راسات العليا في القانون العام من جامعة باريس عام1948 - 1949 ، حائز على شهادة الدبلوم الدولي في االقتصاد السياسي من جامعة مونيليه بدرجة امتياز عام 1950 - 1951 (. اولا : اسرته ونشأتها العلمية والثقافية 16 ) انتمى الى نقابة المحاميين العراقية في13 تشرين االول1941 ، ليصبح (.عضو فاعل فيها17 ) تعين ابراهيم كبة الول مرة في مديرية ضريبة الدخل التابعة الى وزارة المالية كمدقق مالي في4 تشرين الثاني1941 وبراتب قدره18 دينار (.ًشهريا18 ) ثم تدرج في العمل الوظيفي ليصبح مخمن في5 تموز1943 (.وبنفس الراتب19 ) تزوج ابراهيم كبة في25 شباط1953 من السيدة فضيلة عبد الحمي ،د شعبان واصبح له اربعة اوالد وهم (سالم، سلمى (.)نسرين، كريم20 ) سكن الكرادة داخل ، الزوية، رقم الدار 57 /1/ 15 (. 21 ) وقد اطلق فيما بعد على الشارع الذي سكنه بشارع الوزير (. احتراماً وتقديراً لمكانته22 ) وفي28 نيسان1953 طلب التعيين في كلية التجارة واالقتصاد لي مارس ( عمله االكاديمي ، لطالما كان اقرب واحب الى قلبه براتب قدره35 )دينار (.ًشهريا23 ) وبقي في عمله حتى26 تموز1953 اذ قدم استقالته بسبب (.ضغط االجهزة السعيدية عليه24 ) القانون، إذ ت خرج من كلية الحقوق العراقية في بغداد بدرجة الشرف االولى عام1940 - 1941 (. 14 ) حصل على الدبلوم العالي في االقتصاد السياسي من جامعة فؤاد االول عام 1945 - 1946 ، ثم حصل على شهادة الدبلوم في القانون العام من جامعة القاهرة عام46 - 1947 (. 15) ثم حصل على دبلوم الدولة للد راسات العليا في القانون العام من جامعة باريس عام1948 - 1949 ، حائز على شهادة الدبلوم الدولي في االقتصاد السياسي من جامعة مونيليه بدرجة امتياز عام 1950 - 1951 (. 16 ) انتمى الى نقابة المحاميين العراقية في13 تشرين االول1941 ، ليصبح (.عضو فاعل فيها17 ) تعين ابراهيم كبة الول مرة في مديرية ضريبة الدخل التابعة الى وزارة المالية كمدقق مالي في4 تشرين الثاني1941 وبراتب قدره18 دينار (.ًشهريا18 ) ثم تدرج في العمل الوظيفي ليصبح مخمن في5 تموز1943 (.وبنفس الراتب19 ) تزوج ابراهيم كبة في25 شباط1953 من السيدة فضيلة عبد الحمي ،د شعبان واصبح له اربعة اوالد وهم (سالم، سلمى (.)نسرين، كريم20 ) سكن الكرادة داخل ، الزوية، رقم الدار 57 /1/ 15 (. 21 ) وقد اطلق فيما بعد على الشارع الذي سكنه بشارع الوزير (. اولا : اسرته ونشأتها العلمية والثقافية وتولى فيها حقيبة االقتصاد34 ) إذ بقي فيها حتى15 شباط1960 وبراتب قدره240 (.ًدينار شهريا35 ) كما تولى حقائب وزارية بالوكالة (.خالل نفس الفترة كالنفط واالصالح الزراعي36 ) اضافة الى دوره في كسر ال(.قطيعة االقتصادية مع البلدان االشتراكية37 ) عام1934 ، اال ان ظروف اندالع الحرب العالمية الثانية وما رافقها من ًتحوالت سياسية وفكرية ادت الى انتشار المبادئ الفاشية في العالم ، فضال عن انتعاش الحركة الديمقراطية العالمية فيما بعد، كانت ا لفيصل الحاسم في (.بلورة وانضاج منهج ابراهيم كبة الفكري االشتراكي الماركسي26 ) لقد عاصرها بتماس مباشر، منذ مغادرته العراق عام1947 ، متنقالً بين العواصم االوربية والعربية وباالخص لندن، باريس ومدريد والقاهرة، مما مكنه من اتقان اللغات (االنكليزية، الفرنسية،واال لمانية واالسبانية)، االمر (.الذي دفعه الى االطالع على مؤلفات الفكر اللبرالي والعقلي والمادي27 ) كانت لمشاركته في عضوية الحلقات العراقية الماركسية في باريس (،والقاهرة28 ) والهمية القضية الفلسطينية وال سيما بعد وقوع مأساة فلسطين على اثر قرار التقسيم ، وقيام دو لة اسرائيل عام1947 - 1948 ، لهما اثر بالغ في انضاج فكره ورسم معالم مستقبله السياسي ، مما دفعه فيما بعد الى المحافظة على استقالله الفكري والسياسي بعيداً عن العمل الحزبي (.السياسي29 ) اسهم ابراهيم كبة في تحرير مجلة الثقافة الجديدة والتي تحولت الى لسان حال الم ثقفين في العراق والتي توقفت عن النشر بعد صدور ثالثة اعداد (.منها، نتيجة منع حكومة فاضل ااجمالي من مزاولة عملها30 ) في عام1954 صدرت قائمة بفصل مجموعة من االساتذة والمعلمين والطالب اثناء فترة التمهيد لحلف بغداد، فكان هو على رأس المفصولين ، اذ طاردته السلطات(.في العمل والسكن انذاك31 ) كان ابراهيم كبة من رواد حركة انصار السلم التي تاسست في مطلع الخمسينيات من القرن العشرين والتي عقدت مؤتمرها االول في22 تموز 1954 (.ببغداد32 ) وكان ايضا من نشطاء جهة االتحاد الوطني ، بل انيطت (.له شرف تحرير البيان االول للجبهة33 ) استوزر ابراهيم كبة في اول حكومة وطنية بعد ثورة14 تموز1958 ، (. اولا : اسرته ونشأتها العلمية والثقافية علي المشهداني مجلة كلية االداب / العدد76 عام1934 ، اال ان ظروف اندالع الحرب العالمية الثانية وما رافقها من ًتحوالت سياسية وفكرية ادت الى انتشار المبادئ الفاشية في العالم ، فضال عن انتعاش الحركة الديمقراطية العالمية فيما بعد، كانت ا لفيصل الحاسم في (.بلورة وانضاج منهج ابراهيم كبة الفكري االشتراكي الماركسي26 ) لقد عاصرها بتماس مباشر، منذ مغادرته العراق عام1947 ، متنقالً بين العواصم االوربية والعربية وباالخص لندن، باريس ومدريد والقاهرة، مما مكنه من اتقان اللغات (االنكليزية، الفرنسية،واال لمانية واالسبانية)، االمر (.الذي دفعه الى االطالع على مؤلفات الفكر اللبرالي والعقلي والمادي27 ) كانت لمشاركته في عضوية الحلقات العراقية الماركسية في باريس (،والقاهرة28 ) والهمية القضية الفلسطينية وال سيما بعد وقوع مأساة فلسطين على اثر قرار التقسيم ، وقيام دو لة اسرائيل عام1947 - 1948 ، لهما اثر بالغ في انضاج فكره ورسم معالم مستقبله السياسي ، مما دفعه فيما بعد الى المحافظة على استقالله الفكري والسياسي بعيداً عن العمل الحزبي (.السياسي29 ) اسهم ابراهيم كبة في تحرير مجلة الثقافة الجديدة والتي تحولت الى لسان حال الم ثقفين في العراق والتي توقفت عن النشر بعد صدور ثالثة اعداد (.منها، نتيجة منع حكومة فاضل ااجمالي من مزاولة عملها30 ) في عام1954 صدرت قائمة بفصل مجموعة من االساتذة والمعلمين والطالب اثناء فترة التمهيد لحلف بغداد، فكان هو على رأس المفصولين ، اذ طاردته السلطات(.في العمل والسكن انذاك31 ) كان ابراهيم كبة من رواد حركة انصار السلم التي تاسست في مطلع الخمسينيات من القرن العشرين والتي عقدت مؤتمرها االول في22 تموز 1954 (.ببغداد32 ) وكان ايضا من نشطاء جهة االتحاد الوطني ، بل انيطت (.له شرف تحرير البيان االول للجبهة33 ) استوزر ابراهيم كبة في اول حكومة وطنية بعد ثورة14 تموز1958 ، (. اولا : اسرته ونشأتها العلمية والثقافية احتراماً وتقديراً لمكانته22 ) وفي28 نيسان1953 طلب التعيين في كلية التجارة واالقتصاد لي مارس ( عمله االكاديمي ، لطالما كان اقرب واحب الى قلبه براتب قدره35 )دينار (.ًشهريا23 ) وبقي في عمله حتى26 تموز1953 اذ قدم استقالته بسبب (.ضغط االجهزة السعيدية عليه24 ) ( ) انتمى الى نقابة المحاميين العراقية في13 تشرين االول1941 ، ليصبح (.عضو فاعل فيها17 ) ا وفي28 نيسان1953 طلب التعيين في كلية التجارة واالقتصاد لي مارس ( عمله االكاديمي ، لطالما كان اقرب واحب الى قلبه براتب قدره35 )دينار (.ًشهريا23 ) وبقي في عمله حتى26 تموز1953 اذ قدم استقالته بسبب (.ضغط االجهزة السعيدية عليه24 ) ثالثاا : المرحلة المبكرة من حياة ابراهيم كبة وتاثيرها على اتجاهاته الفكرية والسياسية ثالثاا : المرحلة المبكرة من حياة ابراهيم كبة وتاثيرها على اتجاهاته الفكرية والسياسية تشرب ابراهيم كبة بالحس الوطني وتاثر بافكار بعض زعماء الحركة الوطنية في العراق ، فعلى سبيل المثال ال الحصر تاثر بجعفر ابو التمن (.والحزب الوطني العراقي25 ) قبل سفره خارج العراق، مكتسباً منه دور الجماهير كصانعة للتاريخ واهمية الدفاع عن النقابات المهنية ورعاية مصا لحها المختلفة ، فضالً عن اهمية الحياة الحزبية التي كانت قد الغيت 358 358 د. اولا : اسرته ونشأتها العلمية والثقافية وزارة االقتصاد كانت السبب في تقديم االستقالة39 ) ليتفرغ للعمل (.االكاديمي التدريبي والبحثي في جامعتي بغداد والمستنصرية40 ) اعتقل ابراهيم كبة على اثر انقالب8 شباط1963 (. 41 ) وتنقل في معتقالت (. مركز شرطة المامون ومعسكر الرشيد والموقف المركزي42 ) ومصادرة (. امواله المنقولة وغير المنقولة43 ) دافع ابراهيم كبة عن ثورة14 تموز ومنجزاتها ، وال سيما الفترة التي شغلها في الوزارة امام المحكمة العسكرية العليا بنفسه محوالً المرافعة الى دراسة موسعة عن االقتصاد العراقي في فترة ما بعد ثورة14 تموز1958 ، ( االمر الذي مكنه من تخفيض الحكم من االعدام الى السجن10 ) سنوات مع االشغال الشاقة ، ثم اطلق سراحه عام1965 (.بعفو رئاسي44 ) مما دفعه فيما بعد الى االنشغال بمزاولة مهنة التدريس الجامعي في كلية التجارة واالقتصاد وبتاريخ3 حزيران1968 ، وكتابة العشرات من المؤلفات واالبحاث االقتصادية والسياسية التي حاول فيها تفنيد كل االفتراء والتزييف الذي اصاب ثورة14 تموز1958 (. 45 ) حصل ابراهيم كبة على لقب االستاذية في27 تشرين االول1974 وبراتب ( قدره170 ) دينار شهريا، وهو تتويج لجهده وعمله المتواصل في مجال العلم االكاديمي البحثي، على الرغم من كل الضغوط التي مورست بحقه من قبل االجهزة الحكومية للحكومات المتعاقبة لتغيير نهجه وفكره االقتصادي (.والسياسي46) االمر الذي ادى الى احالته على التقاعد في28 تشرين الثاني1977 (، 47 ) بسبب محاربة حزب البعث له بعد رفضه اعادة كتابة التاريخ من وجهة النظر القومية والبعثية، وظل متمسكاً وفياً لمبادئه في الفكر المادي االشتراكي العلمي والماركسي الديمقراطي، وبقي في شيخوخته حبيس الضغوات الدكتات ورية المنهالة عليه من كل حدب وصوب حتى استمر عمله في الوزارة حتى15 شباط1960، حين اصر على تقديم ،استقالته بعد ان رفضها الزعيم الركن عبد الكريم قاسم لعدة مرات سابقة ويبدو لالختالف في وجهات النظر حول تنفيذ البرامج السياسية وال سيما (. وزارة االقتصاد كانت السبب في تقديم االستقالة39 ) ليتفرغ للعمل (.االكاديمي التدريبي والبحثي في جامعتي بغداد والمستنصرية40 ) اعتقل ابراهيم كبة على اثر انقالب8 شباط1963 (. 41 ) وتنقل في معتقالت (. مركز شرطة المامون ومعسكر الرشيد والموقف المركزي42 ) ومصادرة (. اولا : اسرته ونشأتها العلمية والثقافية وتولى فيها حقيبة االقتصاد34 ) إذ بقي فيها حتى15 شباط1960 وبراتب قدره240 (.ًدينار شهريا35 ) كما تولى حقائب وزارية بالوكالة (.خالل نفس الفترة كالنفط واالصالح الزراعي36 ) اضافة الى دوره في كسر ال(.قطيعة االقتصادية مع البلدان االشتراكية37 ) عام1934 ، اال ان ظروف اندالع الحرب العالمية الثانية وما رافقها من ًتحوالت سياسية وفكرية ادت الى انتشار المبادئ الفاشية في العالم ، فضال عن انتعاش الحركة الديمقراطية العالمية فيما بعد، كانت ا لفيصل الحاسم في (.بلورة وانضاج منهج ابراهيم كبة الفكري االشتراكي الماركسي26 ) لقد عاصرها بتماس مباشر، منذ مغادرته العراق عام1947 ، متنقالً بين العواصم االوربية والعربية وباالخص لندن، باريس ومدريد والقاهرة، مما مكنه من اتقان اللغات (االنكليزية، الفرنسية،واال لمانية واالسبانية)، االمر (.الذي دفعه الى االطالع على مؤلفات الفكر اللبرالي والعقلي والمادي27 ) كانت لمشاركته في عضوية الحلقات العراقية الماركسية في باريس (،والقاهرة28 ) والهمية القضية الفلسطينية وال سيما بعد وقوع مأساة فلسطين على اثر قرار التقسيم ، وقيام دو لة اسرائيل عام1947 - 1948 ، لهما اثر بالغ في انضاج فكره ورسم معالم مستقبله السياسي ، مما دفعه فيما بعد الى المحافظة على استقالله الفكري والسياسي بعيداً عن العمل الحزبي (.السياسي29 ) (ي) اسهم ابراهيم كبة في تحرير مجلة الثقافة الجديدة والتي تحولت الى لسان حال الم ثقفين في العراق والتي توقفت عن النشر بعد صدور ثالثة اعداد (.منها، نتيجة منع حكومة فاضل ااجمالي من مزاولة عملها30 ) ا في عام1954 صدرت قائمة بفصل مجموعة من االساتذة والمعلمين والطالب اثناء فترة التمهيد لحلف بغداد، فكان هو على رأس المفصولين ، اذ طاردته السلطات(.في العمل والسكن انذاك31 ) استوزر ابراهيم كبة في اول حكومة وطنية بعد ثورة14 تموز1958 ، (. وتولى فيها حقيبة االقتصاد34 ) إذ بقي فيها حتى15 شباط1960 وبراتب قدره240 (.ًدينار شهريا35 ) كما تولى حقائب وزارية بالوكالة (.خالل نفس الفترة كالنفط واالصالح الزراعي36 ) اضافة الى دوره في كسر ال(.قطيعة االقتصادية مع البلدان االشتراكية37 ) 359 مجلة كلية االداب / العدد76 كان الحزب الشيوعي العراقي يعتبر ابراهيم كبة اقرب وزير الى فكرهم واتجاههم على الرغم كونه من الماركسيين المستقلين والمؤيدين للشيوعية (.ًوان لم يكن شيوعيا38 ) ل ل ط ل كان الحزب الشيوعي العراقي يعتبر ابراهيم كبة اقرب وزير الى فكرهم واتجاههم على الرغم كونه من الماركسيين المستقلين والمؤيدين للشيوعية (.ًوان لم يكن شيوعيا38 ) استمر عمله في الوزارة حتى15 شباط1960، حين اصر على تقديم ،استقالته بعد ان رفضها الزعيم الركن عبد الكريم قاسم لعدة مرات سابقة ويبدو لالختالف في وجهات النظر حول تنفيذ البرامج السياسية وال سيما (. اولا : اسرته ونشأتها العلمية والثقافية امواله المنقولة وغير المنقولة43 ) ا دافع ابراهيم كبة عن ثورة14 تموز ومنجزاتها ، وال سيما الفترة التي شغلها في الوزارة امام المحكمة العسكرية العليا بنفسه محوالً المرافعة الى دراسة موسعة عن االقتصاد العراقي في فترة ما بعد ثورة14 تموز1958 ، ( االمر الذي مكنه من تخفيض الحكم من االعدام الى السجن10 ) سنوات مع االشغال الشاقة ، ثم اطلق سراحه عام1965 (.بعفو رئاسي44 ) مما دفعه فيما بعد الى االنشغال بمزاولة مهنة التدريس الجامعي في كلية التجارة واالقتصاد وبتاريخ3 حزيران1968 ، وكتابة العشرات من المؤلفات واالبحاث االقتصادية والسياسية التي حاول فيها تفنيد كل االفتراء والتزييف الذي اصاب ثورة14 تموز1958 (. 45 ) اا ي( ) حصل ابراهيم كبة على لقب االستاذية في27 تشرين االول1974 وبراتب ( قدره170 ) دينار شهريا، وهو تتويج لجهده وعمله المتواصل في مجال العلم االكاديمي البحثي، على الرغم من كل الضغوط التي مورست بحقه من قبل االجهزة الحكومية للحكومات المتعاقبة لتغيير نهجه وفكره االقتصادي (.والسياسي46) االمر الذي ادى الى احالته على التقاعد في28 تشرين الثاني1977 (، 47 ) بسبب محاربة حزب البعث له بعد رفضه اعادة كتابة التاريخ من وجهة النظر القومية والبعثية، وظل متمسكاً وفياً لمبادئه في الفكر المادي االشتراكي العلمي والماركسي الديمقراطي، وبقي في شيخوخته حبيس الضغوات الدكتات ورية المنهالة عليه من كل حدب وصوب حتى 360 مجلة كلية االداب / العدد76 (.وفاته48 ) اال انه بقي حاضراً من خالل ارثه الخزين من المؤلفات (:والترجمات منها ال على سبيل الحصر49 ) 1. ازمة الفكر االقتصادي في العراق عام1953. 2. معنى الحرية1954. 3. االقطاع في العراق عام1957. 4. حول بعض المفاهيم اال ساسية في االشتراكية العلمية1960. 5. البراغماتية والفلسفة العلمية1960. 6. الماركسية والحرية االمبريالية1960. 7. ما هي االمبريالية1961. 8. هذا هو طريق14 تموز1969. 9. دراسات في تاريخ االقتصاد1970. 10 . مشاكل الجدل في الرأسمال الماركسي1979 . فضال عن الع شرات من المقاالت في مجلة الثقافة الجديدة والعلوم السياسية .واالقتصاد والمثقف العربي ومما سبق يمكننا ان نستنتج ان جميع مستلزمات الفكر والعلم توافرت في شخص ابراهيم كبة, كقوة التفكير وفصاحة اللسان والقدرة الخارقة على العمل المتواصل فضالً عن الثقة بالنفس والج رأة وعدم االنسياق وراء الضغوط الخارجية ومن خالل مؤلفاته ، ترى التنوع في فكره ، فهو درس الفكر والتاريخ واالقتصاد والنقد والنظريات العالمية ، لذلك فهو يبدو في . اولا : اسرته ونشأتها العلمية والثقافية نظر الخواص مرتبطاً بالعلم والفكر، اكثر مما ارتبط بالسياسة طرق ابراهيم كبة ابواب العلم في وقت مبكر من حياته ، مما ادى الى تاثره بعدة ايديولوجيات ، ناهيك عن تاثره بعدد من المفكرين الغربيين ، مما اسهم في صقل شخصيته العلمية فيما بعد ، ليصبح من رواد الفكر والعلم في .العراق رابعا ًً : ابراهيم كبة ونشاطه السياسي واالقتصادي على الصعيد الوطني بعد ثورة14 تموز1958 أ.وزيراً لالقتصاد1958 حتى شباط1960 أ.وزيراً لالقتصاد1958 حتى شباط1960 361 مجلة كلية االداب / العدد76 كانت االحداث السياسية في العراق تاثيرها الواضح على الوضع االقتصادي خالل فترة العهد الملكي ، مما انعكس على الحياة االجتماعية ، ولذلك فان الجانب االقتصادي في العراق قبل ثورة14 تموز ، اتسم بالتسيب وانعدام التخطيط ، فض الً عن اصابة االقتصاد الداخلي االستغالل واالحتكار في (،جميع قطاعاته50 ) وتعد سمة التخلف اهم سمات االقتصاد العراقي ، فكان اقتصاداً بدائياً وحيد الجانب بسبب عدم التوازن في تركيب الهيكل االقفتصادي ، من غلبة للطابع الزراعي، وبالمقابل ضعف القطاع الصناعي وغلبة ا.لطابع االستهالكي عليه( 51) ونتيجة لهذا الواقع المتخلف ، كان احد االسباب الرئيسة لثورة تموز1958 ، والتي دعت في بيانها االول الى انجاز مجموعة من المهام الوطنية والديمقراطية كالغاء التبعية االقتصادية وتحديد .الثروة النفطية واجراء اصالح زراعي شامل ا .ل اح زر ي ي و جر رو ففي مجال السي اسة النفطية ، عدت قيادة الثورة برنامج علمي النقاذ ثروات الشعب العراقي من هيمنة الشركات االحتكارية، ومنذ االيام االولى لثورة 14 (ًتموز ، اذاع الزعيم الركن عبد الكريم قاسم بيانا52 ) اعلن فيه ، ان حكومته جادة في مواصلة انتاج النفط وتصديره الى العالم، االمر الذ ي دفعها ان ال تقطع التزاماتها مع الشركات االجنبية، ولكن دون المساس بمصلحة (.واستقالل البلد53 ) كان لهذا البيان ، اثر بالغ في تثبيت الوضع الجديد، مما اتاح للحكومة وبعد اسابيع قليلة من تاريخها ، ان تدخل في مفاوضات مع ممثلي الشركات النفطية في اوائل اب1958، حو ل مطالب العراق ، االمر الذي دفع الحكومة انذاك لتشكيل لجنة من كبار موظفي وزارة االقتصاد لدراسة احكام االمتيازات والمشاكل الناجمة عن تطبيقها، فضالً عن تحديد نقاط الخالف بين الطرفين ، ثم رفعت تقريرها الى وزير االقتصاد ابراهيم (.كبة54 ) ( ) مما يلفت النظر تاكيد دعو ة ابراهيم كبة على تغيير االلية لتعامل مع الشركات بما يضمن احترام العراق لكافة االلتزامات السابقة، مع مراعاة الوضع العالمي ، وال سيما الدول المجاورة بما يعود بالفائدة على االقتصاد (.العراقي55 ) االمر الذي ادى الى تشكيل لجنة وزارية برئاسة رئيس الوزراء، لدراسة امكانية تعديل اتفاقية1952 (. اولا : اسرته ونشأتها العلمية والثقافية نظر الخواص مرتبطاً بالعلم والفكر، اكثر مما ارتبط بالسياسة طرق ابراهيم كبة ابواب العلم في وقت مبكر من حياته ، مما ادى الى تاثره بعدة ايديولوجيات ، ناهيك عن تاثره بعدد من المفكرين الغربيين ، مما اسهم في صقل شخصيته العلمية فيما بعد ، ليصبح من رواد الفكر والعلم في .العراق رابعا ًً : ابراهيم كبة ونشاطه السياسي واالقتصادي على الصعيد الوطني بعد ثورة14 تموز1958 فضال عن الع شرات من المقاالت في مجلة الثقافة الجديدة والعلوم السياسية .واالقتصاد والمثقف العربي يا ومما سبق يمكننا ان نستنتج ان جميع مستلزمات الفكر والعلم توافرت في شخص ابراهيم كبة, كقوة التفكير وفصاحة اللسان والقدرة الخارقة على العمل المتواصل فضالً عن الثقة بالنفس والج رأة وعدم االنسياق وراء الضغوط الخارجية ومن خالل مؤلفاته ، ترى التنوع في فكره ، فهو درس الفكر والتاريخ واالقتصاد والنقد والنظريات العالمية ، لذلك فهو يبدو في . نظر الخواص مرتبطاً بالعلم والفكر، اكثر مما ارتبط بالسياسة طرق ابراهيم كبة ابواب العلم في وقت مبكر من حياته ، مما ادى الى تاثره بعدة ايديولوجيات ، ناهيك عن تاثره بعدد من المفكرين الغربيين ، مما اسهم في صقل شخصيته العلمية فيما بعد ، ليصبح من رواد الفكر والعلم في .العراق ومما سبق يمكننا ان نستنتج ان جميع مستلزمات الفكر والعلم توافرت في شخص ابراهيم كبة, كقوة التفكير وفصاحة اللسان والقدرة الخارقة على العمل المتواصل فضالً عن الثقة بالنفس والج رأة وعدم االنسياق وراء الضغوط الخارجية ومن خالل مؤلفاته ، ترى التنوع في فكره ، فهو درس الفكر والتاريخ واالقتصاد والنقد والنظريات العالمية ، لذلك فهو يبدو في . اولا : اسرته ونشأتها العلمية والثقافية 56 ) على اية حال بدأت المفاوضات بين العراق والشركات في20 ايار1958 وتمثلت مناقشة المفاوضات حول زيادة الحكومة من االرباح على النصف واسهام العراق 362 مجلة كلية االداب / العدد76 في رأس مال الشركات وتسليم الغاز الطبيعي للعراق بدالً من حرقه واالشتراك في ادارة ال شركات وتمثيل العراق في ادارتها بحيث يمارس حقه (.في االشراف عليها57 ) ااا في رأس مال الشركات وتسليم الغاز الطبيعي للعراق بدالً من حرقه واالشتراك في ادارة ال شركات وتمثيل العراق في ادارتها بحيث يمارس حقه (.في االشراف عليها57 ) استطاعت وزارة االقتصاد برئاسة ابراهيم كبة ومنذ االشهر االولى لها ان تضع خطة دقيقة بغية تحقيق انجازات كبيرة في المجال النفطي ويمكن استطاعت وزارة االقتصاد برئاسة ابراهيم كبة ومنذ االشهر االولى لها ان تضع خطة دقيقة بغية تحقيق انجازات كبيرة في المجال النفطي ويمكن ( تلخيصها باالتي58 :) ا (يا) 1. انشاء ادارة وطنية لمصلحة المصافي ا لحكومية وتصفية االدارة .السابقة، مما ادى الى توفير خمسين الف دينار سنوياً للخزينة ا (يا) 1. انشاء ادارة وطنية لمصلحة المصافي ا لحكومية وتصفية االدارة .السابقة، مما ادى الى توفير خمسين الف دينار سنوياً للخزينة ا 1. انشاء ادارة وطنية لمصلحة المصافي ا لحكومية وتصفية االدارة .السابقة، مما ادى الى توفير خمسين الف دينار سنوياً للخزينة 2. ، طرد الخبراء االجانب من مصفى الدورة والتعريق الكامل للمصفى .ًوقد وفر ذلك للخزينة اكثر من مليون دينار سنويا 3. السيطرة التامة على قسم المشتريات الخارجية في المصفى والغاء و كاالت الشركات االجنبية، مما وفر للخزينة380 الف دينار خالل10 اشهر .السابقة، مما ادى الى توفير خمسين الف دينار سنويا للخزينة 2. ، طرد الخبراء االجانب من مصفى الدورة والتعريق الكامل للمصفى .ًوقد وفر ذلك للخزينة اكثر من مليون دينار سنويا 3. السيطرة التامة على قسم المشتريات الخارجية في المصفى والغاء و كاالت الشركات االجنبية، مما وفر للخزينة380 الف دينار خالل10 2. ، طرد الخبراء االجانب من مصفى الدورة والتعريق الكامل للمصفى .ًوقد وفر ذلك للخزينة اكثر من مليون دينار سنويا 2. ، طرد الخبراء االجانب من مصفى الدورة والتعريق الكامل للمصفى .ًوقد وفر ذلك للخزينة اكثر من مليون دينار سنويا 3. السيطرة التامة على قسم المشتريات الخارجية في المصفى والغاء و كاالت الشركات االجنبية، مما وفر للخزينة380 الف دينار خالل10 .اشهر ا 3. السيطرة التامة على قسم المشتريات الخارجية في المصفى والغاء و كاالت الشركات االجنبية، مما وفر للخزينة380 الف دينار خالل10 .اشهر ا 4. زيادة الطاقة االنتاجية لمصفى الدورة1.400.000 طناً الى 2.200.000 . ًطناً سنويا 4. زيادة الطاقة االنتاجية لمصفى الدورة1.400.000 طناً الى 2.200.000 . اولا : اسرته ونشأتها العلمية والثقافية ًطناً سنويا على الرغم من توقف المفاوضات ، والحيلولة دون تنفيذ الخطة الطويلة االمد لتحرير القطاع النفطي من الشركات، اال ان وزارة االقتصاد برئاسة ( : ابراهيم كبة استطاعت تحقيق االنجازات التالية59 ) اا 1. استعادة غالبية االراضي غير المستثمرة وذلك الستثمارها بشكل .مباشر في ادارة الحقول النفطية ا ي 2. .استرجاع المياه االقليمية واخراجها من امتياز شركة نفط البصرة ا 3. تسهيل استالم الحص ة العينية من النفط الخام وذلك بالتقليل من .االخطار للشركة 3. تسهيل استالم الحص ة العينية من النفط الخام وذلك بالتقليل م .االخطار للشركة ا 4. مشروع اتفاق لتجهيز المصفى بالنفط الخام بكميات لتصدير المشتقات . النفطية ا ا 4. مشروع اتفاق لتجهيز المصفى بالنفط الخام بكميات لتصدير المشتقات . النفطية 5. االتفاق على مشروع لتجهيز الغاز الطبيعي للمشاريع الصناعية .ًالحكومية مجانا اا 5. االتفاق على مشروع لتجهيز الغاز الطبيعي للمشاريع الصناعية .ًالحكومية مجانا اا 6. تعريق الشركات أي طرد االجانب والسيطرة على.سياسة االستخدام 6. تعريق الشركات أي طرد االجانب والسيطرة على.سياسة االستخدام 363 مجلة كلية االداب / العدد76 7. زيادة انتاج النفط الخام على الحد االدنى المنصوص عليه في االتفاقية من خالل انشاء مشروع الميناء العميق الذي يوسع تصدير البصرة الى22 .ًمليون طن سنويا اا على الرغم من االنجازات التي تحققت في عهده اال انه لم يستطع تنفيذ خطة تحرير النفط بشكل كا مل وهو ما كان يحلم ابراهيم كبة به، الستقالته من منصبه، ويبدو لنا ان تصلب موقفه من الغرب وتقربه من المعسكر الشرقي . وال سيما االتحاد السوفيتي ، كان وراء تعثر المفاوضات ا ياا اما في مجال القطاع الصناعي ، فقد اتجهت وزارة االقتصاد الى تطوير هذا القطاع على الرغم من ت ، وزع مسؤولية هذا القطاع على عدة وزارات كونها تعتبر القطاع الصناعي اداة فاعلة في التطور االقتصادي ، وهذا ما (.يؤكده ابراهيم كبة من خالل مؤتمراته ومقاالته الصحفية60 ) االمر الذي دفع الوزارة الى تشجيع رؤوس االموال الخاصة من خالل عقد اتفاقيات ثنائية بين العراق والدول االخرى القامة مشاريع لالنتاج االستهالكي واالهتمام بالقطاع العام لقيادة القطاع الصناعي، مما يؤهله لقيادة االقتصاد الوطني، فضالً عن وضع دراسات رصينة علمية لغرض تخطيط السياسة الصناعية في العراق ، ناهيك عن دعوة الوزارات الى منع التبعية االقتصادية االجن بية في هذا المجال ، بمعنى عدم تغلغل الرأسمال االجنبي تحت غطاء الصناعة الوطنية ، وذلك من خالل التشديد والرقابة على ( . الصناعات المختلفة61 ) اا ( . اولا : اسرته ونشأتها العلمية والثقافية علي المشهداني مجلة كلية االداب / العدد76 ، السنة االولى من حكمها التوقيع على اتفاقيات اقتصادية وتجارية وثقافية (.مع احدى عشر دولة شيوعية63 ) ثم عقدت الوزارة عدد اخر من االتفاقيات مع االقطار الغربية واكثر اقطار اسيا وافريقيا واوربا لغرض (.زيادة الصادرات العراقية وتنوع اتجاهاتها64 ) اا ، السنة االولى من حكمها التوقيع على اتفاقيات اقتصادية وتجارية وثقافية (.مع احدى عشر دولة شيوعية63 ) ثم عقدت الوزارة عدد اخر من االتفاقيات مع االقطار الغربية واكثر اقطار اسيا وافريقيا واوربا لغرض (.زيادة الصادرات العراقية وتنوع اتجاهاتها64 ) ان من اهم االتفاقيات التي عقدت مع العراق هي االتفاقية العراقية– السوفيتية فكلف ابراهيم كبة بترأس الوفد العراقي المتوجه للتفاوض مع (،االتحاد السوفيتي65 ) وفعالً حدث مفاوضات بين الطرفين في موسكو تمخض عنها اتفاقي ة المساعدة االقتصادية وهي االولى في سلسلة االتفاقيات التي سعى العراق بموجبها للحصول على مساعدة اقتصادية وعسكرية وثقافية، ونص االتفاق على تعهد االتحاد السوفيتي بتزويد العراق بقروض تمكنه من شراء معدات ، فضالً عن تطوير صناعتي التعدين والنقل اضافة الى انشاء صن اعة كهربائية وميكانيكية ومعامل للحياكة الوطنية واالغذية ووقع االتفاق بموسكو في16 اذار1959 (. 66 ) ان اغلب االتفاقيات التي عقدت مع العراق كانت تقوم على مبادئ التكامل .والتكافؤ والتنسيق والتحرر االقتصادي اا (ع) ان من اهم االتفاقيات التي عقدت مع العراق هي االتفاقية العراقية– السوفيتية فكلف ابراهيم كبة بترأس الوفد العراقي المتوجه للتفاوض مع (،االتحاد السوفيتي65 ) وفعالً حدث مفاوضات بين الطرفين في موسكو تمخض عنها اتفاقي ة المساعدة االقتصادية وهي االولى في سلسلة االتفاقيات التي سعى العراق بموجبها للحصول على مساعدة اقتصادية وعسكرية وثقافية، ونص االتفاق على تعهد االتحاد السوفيتي بتزويد العراق بقروض تمكنه من شراء معدات ، فضالً عن تطوير صناعتي التعدين والنقل اضافة الى انشاء صن اعة كهربائية وميكانيكية ومعامل للحياكة الوطنية واالغذية ووقع االتفاق بموسكو في16 اذار1959 (. اولا : اسرته ونشأتها العلمية والثقافية 61 ) على الرغم من الجهود التي بذلت لوزارة االقتصاد في هذا المجال ، خالل فترة ترأس ابراهيم كبة لها ، لغرض تطويرها، من خالل تخصيص38 مليون دينار خالل الخطة المؤقتة للفترة1959 - 1961 ، اال انه امتاز بضعف الصناعات الوطنية بسبب تردي رأس المال الخاص وانكماشه مما انعكس (.على وضع السوق نحو المشاريع االنتاجية62 ) يستطيع الباحث ان يرى ان من اهم االنتقادات التي توجه الى السياسة االقتصادية في العراق، هو فشلها في نشر التصنيع السريع للعراق وميلها .الى تخصيص نسبة كبيرة من االيرادات الى البنى التحتية الزراعية اسهم ابراهيم كبة اسهاماً فعلياً ومنذ اللحظات االولى الستالمه وزارة االقتصاد، في وضع خطة مبنية على اسس علمية العادة التنظيم االقتصادي ، من خالل االنفتاح على العالم الخارجي، ولذلك فقد اقدمت الحكومة خالل ( ) على الرغم من الجهود التي بذلت لوزارة االقتصاد في هذا المجال ، خالل فترة ترأس ابراهيم كبة لها ، لغرض تطويرها، من خالل تخصيص38 مليون دينار خالل الخطة المؤقتة للفترة1959 - 1961 ، اال انه امتاز بضعف الصناعات الوطنية بسبب تردي رأس المال الخاص وانكماشه مما انعكس (.على وضع السوق نحو المشاريع االنتاجية62 ) ا يستطيع الباحث ان يرى ان من اهم االنتقادات التي توجه الى السياسة االقتصادية في العراق، هو فشلها في نشر التصنيع السريع للعراق وميلها .الى تخصيص نسبة كبيرة من االيرادات الى البنى التحتية الزراعية اسهم ابراهيم كبة اسهاماً فعلياً ومنذ اللحظات االولى الستالمه وزارة االقتصاد، في وضع خطة مبنية على اسس علمية العادة التنظيم االقتصادي ، من خالل االنفتاح على العالم الخارجي، ولذلك فقد اقدمت الحكومة خالل يستطيع الباحث ان يرى ان من اهم االنتقادات التي توجه الى السياسة االقتصادية في العراق، هو فشلها في نشر التصنيع السريع للعراق وميلها .الى تخصيص نسبة كبيرة من االيرادات الى البنى التحتية الزراعية زا ة ال تال نذ الل ظات اال ل ا اً ف ل اً ك ة ا ا ا ا 364 د. اولا : اسرته ونشأتها العلمية والثقافية 66 ) ا يا ع( ) ان اغلب االتفاقيات التي عقدت مع العراق كانت تقوم على مبادئ التكامل .والتكافؤ والتنسيق والتحرر االقتصادي اا يا اما في مجال االصالح الزراعي ، كلف ابراهيم كبة ليشغل منص ب وزير االصالح الزراعي، والتي أنشأت في3 ايار1959 لتذليل الصعوبات التي (.تواجهها الحكومة67 ) كالتوزيع واالنتاج، حيث ان لالصالح الزراعي اهداف ونتائج بالغة االهمية كالقضاء على االقطاع وزيادة االنتاج واقامة نظام تعاوني على اساس الملكية الصغيرة، فضالً عن السما ح القامة قطاع (.خاص متوسط على ان يوجه من قبل الدولة68 ) ،لقد تسلم هذه الوزارة بعد مرور اكثر من تسعة اشهر على صدور القانون (.واستقال من الوزارة بعد مرور تسعة اشهر فقط69 ) اال انه حقق انجازات عظيمة في هذا المجال ومنذ اليوم االول الستالمه المنصب وضع الجهاز الجد(.يد لالصالح الزراعي على االسس التالية70 ) ااا (ا ي حاا) أ . . االستقالل والتحرر الروتيني والمالي في االدارة ب. التخطيط على اسس احصائية مدروسة (احداث مديرية التخطيط .)العامة اا .ج. توحيد جميع اجهزة االصالح الزراعي في مؤسسة واحدة اا .ج. توحيد جميع اجهزة االصالح الزراعي في مؤسسة واحدة د. التوجيه والنشر (استحداث مديرية خاصة لنشر ا.) يديولوجية االصالح .ج. توحيد جميع اجهزة االصالح الزراعي في مؤسسة واحدة د. التوجيه والنشر (استحداث مديرية خاصة لنشر ا.) يديولوجية االصالح . التوجيه والنشر (استحداث مديرية خاصة لنشر ا.) يديولوجية االصالح 365 مجلة كلية االداب / العدد76 هـ. احكام الرقابة على اعمال االصالح الزراعي عن طريق استحداث دائرة .المفتشية العامة في ديوان المؤسسة واجهت الوزارة ومنذ البداية صعوبات جمة وخاصة فيما يتعلق بتردي الوضع السياسي العام وانعكاسه على مواجهة االقطاع والمتنفذين في اجهزة الدولة ، فضالً عن التداخل في اتخاذ القرار الرتباط دوائر اخرى عديدة مثل (المالية والشؤون االجتماعية والتجارة، اضافة الى االصالح الزراعي71 .) اال ان ابراهيم كبة استطاع وخالل فترة قصيرة ان يحقق انجازات جمة وهي ( 72 :) اااا هـ. احكام الرقابة على اعمال االصالح الزراعي عن طريق استحداث دائرة .المفتشية العامة في ديوان المؤسسة ( ) 1. وضع خطة االستيالء بهدف القضاء غلى االقطاع وذلك من خالل ( تشكيل لجان تضم34 ) لجنة، مما ادى الى االستيالء على256 مالكاً من كبار االقطاعيين والذي يبلغ مجموع اراضيهم2.3 مليون دونم حتى تاريخ استقالة ابراهيم كبة من الوزارة اواخر1959. اا م 2. بعد شهرين فقط من تأسيس الجهاز (االصالح الزراعي) وضعت خطة لتوزيع االراض ي، حيث شملت المرحلة االولى منها توزيع750.000 دونم اغلبها من االراضي المستولي عليها البالغة حوالي نصف المليون دونم . والباقي من االراضي االميرية الصرفة ااا ا يا ي 3. اولا : اسرته ونشأتها العلمية والثقافية الدعوة الى تطوير القطاع العام لالصالح الزراعي ، وذلك من خالل تأسيس المزارع الحكومية ، ضمن اجراء اتفاق يات تجارية مع االتحاد .السوفيتي وتشمل هذه االتفاقية، ايضاً تاجير المكائن واالالت الزراعية 4. وضع النواة الصالحة للثروة الصناعية في القطاع الزراعي ، من . خالل تأسيس معمل لالسمدة الكيمياوية ضمن االتفاقية العراقية السوفيتية 5. زيادة االنتاج في القطاع العام، ف ضالً عن االهتمام في وقف تدهور القطاع الخاص مما دفع الوزارة الى اتخاذ اجراءات حقيقية لتحقيق هذا الغرض منها تاجير جميع االراضي الخاصة الشراف االصالح الزراعي والتي بلغت مساحتها4.6500711 دونم استفاد منها حوالي138000 ،ًفالحا فضالً عن القيام بتطهير االنهر الصغي رة ضمن اراضي االصالح، اضافة الى تقديم الدعم الحكومي للفالحين من خالل تسليفهم وخصص لهذا الغرض (.، اكثر من مليوني دينار من اصل ثالثة ماليين دينار73 ) على الرغم من االنجازات التي تحققت في هذا المجال، اال انه ال يخلو من العيوب والهفوات التي ادت بالتالي الى الحي لولة دون النهوض بالقطاع 3. الدعوة الى تطوير القطاع العام لالصالح الزراعي ، وذلك من خالل تأسيس المزارع الحكومية ، ضمن اجراء اتفاق يات تجارية مع االتحاد .السوفيتي وتشمل هذه االتفاقية، ايضاً تاجير المكائن واالالت الزراعية 366 مجلة كلية االداب / العدد76 الزراعي ، وتفعيل قانون االصالح الزراعي لخدمة الفالحين ، وقد اوعز ابراهيم كبة االسباب الى الكوارث واالفات الطبيعية من جهة ، والتدخالت السياسية في الوزارة من جهة اخرى ، مشيراً الى محاولة االجهزة القاسمية وال سيما العاملين منهم في مركز الوزارة وفي االرياف ، السيطرة على االصالح الزراعي خططاً ًوتطبيقاً واقامة قواعد سياسية لهم بين الفالحين من خالل انشاء الجمعيات الفالحية في القرى واالرياف ، ناهيك عن المحاوالت المستمرة الحداث تعديالت رجعية في سياسة قانون االصالح الزراعي تحت ضغط االقطاع والب(.رجوازية74 ) ب . اراء ومواقف ابراهيم كبة حول الوضع السياسي في العراق للمدة من 14 تموز1958 وحتى17 تموز1968 . 1 . النظام السياسي1958 أن متابعتنا للبنى االقتصادية والسياسية واالجتماعية خالل فترة الحكم الملكي، يبدو لنا، انه خاضع لنظام شبه اقطاعي ، لذلك ف ان ثورة تموز 1958 كانت حصيلة تفجير تناقضات ذلك الواقع المرير، مما دفع الثورة ومنذ اليوم االول الى تغيير هذا الواقع السياسي واالقتصادي واالجتماعي لمصلحة غالبية الشعب من خالل جملة مبادئ ومنها االحزاب السياسية .وحركة الضباط االحرار ًلقد حاول ابراهيم كبة تقييم العهد القاسمي بايجابياته وسلبياته تقييماً شامال وتطبيقياً. اولا : اسرته ونشأتها العلمية والثقافية فمن الناحية االيجابية ، اشار الى التحوالت السياسية واالقتصادية واالجتماعية ومقارنتها بالعهد الملكي، وال سيما االهداف الوطنية المشتركة (.للبيان االول75) ففي المجال السياس ي، اعلنت الثورة الغاء النظام الملكي واعالن الجمهورية العراقية، وتأليف وزا رة ممثلة من جبهة االتحاد الوطني ، (.فضالً عن اصدار دستور مؤقت76 ) ناهيك عن الغاء كافة المراسيم (.السعيدية77 ) اما في المجال االقتصادي فان الثورة قامت باصدار قانون االصالح الزراعي رقم30 لسنة1958 وانسحاب العراق من المنطقة االسترلينية ومحاولة تعريق الشركات النفطية االجنبية ، من خالل انشاء 1 . النظام السياسي1958 أن متابعتنا للبنى االقتصادية والسياسية واالجتماعية خالل فترة الحكم الملكي، يبدو لنا، انه خاضع لنظام شبه اقطاعي ، لذلك ف ان ثورة تموز 1958 كانت حصيلة تفجير تناقضات ذلك الواقع المرير، مما دفع الثورة ومنذ اليوم االول الى تغيير هذا الواقع السياسي واالقتصادي واالجتماعي لمصلحة غالبية الشعب من خالل جملة مبادئ ومنها االحزاب السياسية .وحركة الضباط االحرار ا ا ًلقد حاول ابراهيم كبة تقييم العهد القاسمي بايجابياته وسلبياته تقييماً شامال وتطبيقياً. فمن الناحية االيجابية ، اشار الى التحوالت السياسية واالقتصادية واالجتماعية ومقارنتها بالعهد الملكي، وال سيما االهداف الوطنية المشتركة (.للبيان االول75) ففي المجال السياس ي، اعلنت الثورة الغاء النظام الملكي واعالن الجمهورية العراقية، وتأليف وزا رة ممثلة من جبهة االتحاد الوطني ، (.فضالً عن اصدار دستور مؤقت76 ) ناهيك عن الغاء كافة المراسيم (.السعيدية77 ) اما في المجال االقتصادي فان الثورة قامت باصدار قانون االصالح الزراعي رقم30 لسنة1958 وانسحاب العراق من المنطقة االسترلينية ومحاولة تعريق الشركات النفطية االجنبية ، من خالل انشاء 367 د. علي المشهداني مجلة كلية االداب / العدد76 (.ادارة وطنية ، فضالً عن زيادة الطاقة االنتاجية لمصفى الدورة78 ) اما في المجال االجتماعي ، فانها اتجهت الضعاف الطبقة شبه االقطاعية والطبقة التجارية ولو بشكل نسبي، اال انها قوت الطبقة البرجوازية الوطنية، وكان ذلك منسجماً مع طبيعة السلطة التي اعقبت ثورة14 تموز1958 (. 79 ) على الرغم من االيجابيات التي تم ذكرها ، اال ان الجوانب السلبية للحكم الجديد، بدأت تطغي على جميع الجوانب االيجابية، ويمكننا هنا تسليط الضوء (على ابرز تلك الحقائق من وجهة نظر ابراهيم كبة80 :) 1. عدم وجود مجلس وطني لقيادة الثورة ، لمنع سيطرة الدكتاتورية .العسكرية، فضالً عن عدم وجود أية هيئة قيادية شعبية او حزبية 2. ، عدم وجود ظروف مالئمة القامة مؤسسات ديمقراطية على الفور ادى الى تردي الوضع السي.اسي ، فيما بعد 1. اولا : اسرته ونشأتها العلمية والثقافية عدم وجود مجلس وطني لقيادة الثورة ، لمنع سيطرة الدكتاتورية .العسكرية، فضالً عن عدم وجود أية هيئة قيادية شعبية او حزبية 2 ،الفور عدم وجود ظروف مالئمة القامة مؤسسات ديمقراطية عل ما 2. عدم وجود ظروف مالئمة القامة مؤسسات ديمقراطية على الفور ادى الى تردي الوضع السي.اسي ، فيما بعد ع ي ي 3. عدم وجود انسجام فكري بين المدنيين والعسكريين في الوزارة بسبب اختالف اتجاهاتهم وميولهم السياسية ، وبالتالي ادى الى انفصال تام في اعمال الوزارة، فتمركزت المناصب الحساسة بيد العسكريين، بسبب تخوفهم .من االخطار التي تهدد امن الثورة ومستقبلها 4. ومن العيوب الكبيرة التي اشار اليها ابراهيم كبة، هو عبث وجود مجلس السيادة لعدم ممارسته اية سلطة حقيقية واستغالله احياناً من قبل .الزعيم الركن عبد الكريم قاسم ا م م م 5. عدم تمثيل الوزارة لجميع القوى السياسية في البالد ، مما ادى الى افساح المجال لبعض القوى والفئا ت الغير مشتركة في الحكم بالسيطرة على . الشارع بعيدة عن الرقابة ا ع 6. ان تمثيل االحزاب السياسية في الوزارة كان متفاوتاً ، فالحزب الوطني الديمقراطي شغل اعضاؤه وزارتين وحزب االستقالل وزارة واحدة، وحزب البعث شغل منصباً واحداً ، اما الحزب الشيوعي فانه لم يشغل اي م.نصب وزاري على الرغم من وجوده في جبهة االتحاد الوطني 7. كما اشار ابراهيم كبة الى ان من اخطر العيوب في الوضع السياسي الجديد هو عدم نجاح الوزراء العقائديين وال سيما الحزبيين منهم في وضع منهاج وزاري ملزم للجميع ومبني على نقاط االلتقاء المشتركة بين القوى السيا.سية في البالد 368 مجلة كلية االداب / العدد76 8. كان البعض يعتبر ان عدم مشاركة الحزب الشيوعي في الوزارة كونه ممثل في جبهة االتحاد الوطني ، وعدم اثارة هذا الموضوع داخل مجلس الوزراء ، بمثابة هدم لجبهة االتحاد الوطني، ويبدو ان المكاسب والسعي .وراء المناصب اخذت تستهويهم اكثر من الجبهة نفسها( 81 ) 8. كان البعض يعتبر ان عدم مشاركة الحزب الشيوعي في الوزارة كونه ممثل في جبهة االتحاد الوطني ، وعدم اثارة هذا الموضوع داخل مجلس الوزراء ، بمثابة هدم لجبهة االتحاد الوطني، ويبدو ان المكاسب والسعي .وراء المناصب اخذت تستهويهم اكثر من الجبهة نفسها( 81 ) 8. اولا : اسرته ونشأتها العلمية والثقافية كان البعض يعتبر ان عدم مشاركة الحزب الشيوعي في الوزارة كونه ممثل في جبهة االتحاد الوطني ، وعدم اثارة هذا الموضوع داخل مجلس الوزراء ، بمثابة هدم لجبهة االتحاد الوطني، ويبدو ان المكاسب والسعي .وراء المناصب اخذت تستهويهم اكثر من الجبهة نفسها( 81 ) 2 ..انقالبي8 شباط و18 تشرين الثاني1963 ( بعد اصدار محكمة الثورة احكامها ضد ابراهيم كبة بالسجن لمدة10 ) سنوات مع االشغال الشاقة، تم اطالق سراحه عام1965 في عفو رئاسي ليعاود ممارسة المهنة المحببة الى قلبه وهي التدريس، فضالً عن التأليف والترجمة ، اال ا ن تزييف الحقائق واالحداث في العراق انذاك دفعه في ايلول عام1966 (.ان يرفع هو ومجموعة من رفاقه82 ) مذكرة الى ناجي طالب رئيس الوزراء ذاكراً فيها حقيقة مهمة هي ان المسألة االساسية في قيام14 تموز1958 هو دك النظام الملكي وازالة قشرته السياسية والتي تمحورت حول المسألة االجتماعية ، اي مسألة الثورة االجتماعية، بعد ان (.كان المحور قبل تموز يدور حول المسألة الوطنية83 ) ان انبعاث الفكر الرجعي في العراق انذاك يوعزه ابراهيم كبة انه يعود السباب فكرية خالصة تتصل بتشبثه بحجج جديدة، بل هو يعود في االساس الى دوره القديم– ا لجديد كسالح من اهم اسلحة الردة التي بدات تظهر منذ السنوات االخيرة لحكم عبد الكريم قاسم، وبلغت ذروتها خالل انقالبي شباط (.وتشرين84 ،) وذلك السباب موضوعية كثيرة اهمها ال على سبيل الحصر تغيير المواقع الطبقية بعد تموز، قيادة البرجوازية وبعض مراتب البرجوازية الص غيرة لحركة الردة، وتطلعها للسيطرة السياسية المطلقة واعتمادها على جبهة رجعية واسعة تضم اليمين الرجعي القديم (االقطاع ، )والبرجوازية الكبيرة) والوسط الرجعي الجديد (البرجوازية الوسطى (.وبعض مراتب البرجوازية الصغيرة المتقنعة بالقومية85 ) يعتقد ابراهيم كبة ان ا ،لموضوعات التي يتناولها هذا الفكر والقائمين عليه يشمل كل الموضوعات المتصلة بالصراع الطبقي من قريب او بعيد، تشمل الماضي والحاضر والمستقبل، الوضع الداخلي والعربي والدولي، المسائل النظرية والتطبيقية من خالل حملة المسخ والتشويه لتاريخ العراق (.السياسي86)وال سي ما ثورة تموز1958 لتجريدها من اية اهداف مرسومة 2 ..انقالبي8 شباط و18 تشرين الثاني1963 ( بعد اصدار محكمة الثورة احكامها ضد ابراهيم كبة بالسجن لمدة10 ) سنوات مع االشغال الشاقة، تم اطالق سراحه عام1965 في عفو رئاسي ليعاود ممارسة المهنة المحببة الى قلبه وهي التدريس، فضالً عن التأليف والترجمة ، اال ا ن تزييف الحقائق واالحداث في العراق انذاك دفعه في ايلول عام1966 (.ان يرفع هو ومجموعة من رفاقه82 ) مذكرة الى ناجي طالب رئيس الوزراء ذاكراً فيها حقيقة مهمة هي ان المسألة االساسية في قيام14 تموز1958 هو دك النظام الملكي وازالة قشرته السياسية والتي تمحورت حول المسألة االجتماعية ، اي مسألة الثورة االجتماعية، بعد ان (.كان المحور قبل تموز يدور حول المسألة الوطنية83 ) يعتقد ابراهيم كبة ان ا ،لموضوعات التي يتناولها هذا الفكر والقائمين عليه يشمل كل الموضوعات المتصلة بالصراع الطبقي من قريب او بعيد، تشمل الماضي والحاضر والمستقبل، الوضع الداخلي والعربي والدولي، المسائل النظرية والتطبيقية من خالل حملة المسخ والتشويه لتاريخ العراق (.السياسي86)وال سي ما ثورة تموز1958 لتجريدها من اية اهداف مرسومة 369 مجلة كلية االداب / العدد76 ، كالتحرر السياسي واالقتصادي وتحقيق الديمقراطية ووضع االسس المادية للتحول االجتماعي ، كما عبرت عنها وثائق الثورة وبيانات جبهة االتحاد (.ًالوطني ، ناسباً اليها اهداف وهمية ، كأقامة النظام االشتراكي فورا87 ) 3.ا نقالب17 تموز1968 بعد ايام قالئل من انقالب17 تموز1968 ، كان البراهيم كبة مجموعة من االراء والمواقف ازاء هذا الحدث السياسي، تجسد بكتاباته ومقاالته في .الصحف انذاك ا . اولا : اسرته ونشأتها العلمية والثقافية عدّ ابراهيم كبة ان ازمة الحكم في العراق ازمة مزمنة ، الزمت نظام الحكم فيه منذ تأسييس ما يسمى بالحكم (الوطني) في العراق ، بعد اعقاب الحرب ، العالمية االولى، وان هذه االزمة تطورت مع تطور العالقات االجتماعية وال سيما بعد ثورة14 تموز1958 (. 88 ) فليس صدفة اذ اشار ابراهيم كبة، الى ذلك بالقول : " ال يمكن ان يكتسب االنقالب العسكري الجديد اية شرعية من مجر د نجاحه في السيطرة على مقاليد السلطة ، شأنه في ذلك شأن اي تغيير في الحكم يأتي عن طريق االنتخابات العامة، ان المصدر الوحيد الذي يمكن ان يضفي الشرعية على الحكم الجديد هو اضطالعه بالمهام التي تتناسب وطبيعة المرحلة ، كبداية لزرع الثقة بين الحكومة والشعب، كونه يمثل الشرعية والمالك الوحيد (. " للسيادة89 ) آ عدّ ابراهيم كبة ان ازمة الحكم في العراق ازمة مزمنة ، الزمت نظام الحكم فيه منذ تأسييس ما يسمى بالحكم (الوطني) في العراق ، بعد اعقاب الحرب ، العالمية االولى، وان هذه االزمة تطورت مع تطور العالقات االجتماعية وال سيما بعد ثورة14 تموز1958 (. 88 ) ا فليس صدفة اذ اشار ابراهيم كبة، الى ذلك بالقول : " ال يمكن ان يكتسب االنقالب العسكري الجديد اية شرعية من مجر د نجاحه في السيطرة على مقاليد السلطة ، شأنه في ذلك شأن اي تغيير في الحكم يأتي عن طريق االنتخابات العامة، ان المصدر الوحيد الذي يمكن ان يضفي الشرعية على الحكم الجديد هو اضطالعه بالمهام التي تتناسب وطبيعة المرحلة ، كبداية لزرع الثقة بين الحكومة والشعب، كونه يمثل الشرعية والمالك الوحيد (. " للسيادة89 ) آ ( ) ويمكننا تلخيص هذه المهام باآل(تي90 :)اا 1. ازالة االوضاع االستثنائية والغاء جميع القوانيين والنصوص والمراسيم واالجراءات المتعارضة مع المبادئ الدستورية العامة، مع الغاء . المحاكم االستثنائية ا ا م 2. اصدار عفو عام عن جميع المحكومين السياسيين ، واطالق سراح .جميع المحتجزين السباب سياسية اا ا ع 3. .الغاء اوامر العزل والفصل واالحالة علىالتقاعد السباب سياسية ا 3. .الغاء اوامر العزل والفصل واالحالة علىالتقاعد السباب سياسية 4. اعادة جميع المغتربين والمبعدين خارج العراق السباب سياسية .واعادة ممتلكاتهم اليهم مع ارجاعهم لوظائفهم 5. االعتراف المطلق بحقوق القومية الكردية على اساس الحكم الذاتي .ضمن وحدة تراب العراق 3. .الغاء اوامر العزل والفصل واالحالة علىالتقاعد السباب سياسية 4. اعادة جميع المغتربين والمبعدين خارج العراق السباب سياسية .واعادة ممتلكاتهم اليهم مع ارجاعهم لوظائفهم 5. االعتراف المطلق بحقوق القومية الكردية على اساس الحكم الذاتي .ضمن وحدة تراب العراق ااي ي و ز و و ر 4. اولا : اسرته ونشأتها العلمية والثقافية اعادة جميع المغتربين والمبعدين خارج العراق السباب سياسية .واعادة ممتلكاتهم اليهم مع ارجاعهم لوظائفهم ا م م م ع م 5. االعتراف المطلق بحقوق القومية الكردية على اساس الحكم الذاتي .ضمن وحدة تراب العراق 370 مجلة كلية االداب / العدد76 6. اطالق الحريات العامة على اختالف انواعها لجميع القوى السياسية واالجتماعية بما في ذلك حرية االجتماع والتنظيم الحزبي والنقابي الى جانب .حرية الفكر 7. فتح حوار حر مع جميع االحزاب والمنظمات والشخصيا ت السياسية التقدمية دون استثناء وذلك لبلورة منهاج مشترك يكون اساساً لتاليف (حكومة ائتالفية مؤقتة) تحدد مهامها اساساً باصدار دستور مؤقت وفق مبادئ الديمقراطية وتشريع قانون االنتخابات واالشراف على االنتخابات ( العامة للمجلسين التأسيسي والوطني ، الذي تنبثق عنه حكومة االتحاد .التقدمي) على اساس التمثيل النسبي والخالصة، ان الحكام ان ارادو حقاً تحقيق وعودهم االخيرة للشعب واالستفادة من عبر التاريخ القريب والخروج من دوامة االنقالبات العسكرية المدمرة وتجنب الطريق المسدود تاريخياً فما عليهم اال االخذ بفكرة المراحل الثال ث وهي مرحلة الحكم االنقالبي الحالي ومرحلة الحكومة المؤقتة ، ثم (.مرحلة حكومة االتحاد التقدمي91 )ولذلك فعليهم وعلى الفور المباشرة باداء مهام المرحلة االولى وذلك باطالق الحريات الديمقراطية وعلى رأسها حرية النشاط الحزبي للقوى السياسية الفعالة في المجتمع ، واال قالع نهائياً عن مفهوم الحزب الواحد او الحزب القائد، والتخلي عن اسطورة الوصاية على الجماهير، او االنفتاح الصوري على بعض القوى االخرى وذلك تمهيداً لعقد ، حوار مفتوح مع جميع القوى المعادية لالستعمار واالقطاع واالستقالل لصياغة منهاج مشترك عام يكون اساساً لتألي ف (جهة اتحاد تقدمي)، تنبثق عنها االنتخابات العامة وتتصدى لحل المشاكل االساسية للبالد ، وتقود (.عملية التطور االجتماعي والسياسي92 ) ويبدو لنا من خالل ما تم ذكره ان ابراهيم كبة من الشخصيات القالئل التي وجدت في وقتنا هذا ، فهو حريص على قول الحق دون وجل وال مح اباة ال يسعى الى تحقيق المكاسب والمناصب السياسية بقدر سعيه لخدمة العراق وايصاله الى بر االمان، فهو بحق شخصية ذات فكر متميز وابداع ناصع وحس وطني، يفتخر به العراق في كل المراحل سواء في الماضي القريب .او الحاضر او المستقبل 6. اطالق الحريات العامة على اختالف انواعها لجميع القوى السياسية واالجتماعية بما في ذلك حرية االجتماع والتنظيم الحزبي والنقابي الى جانب .حرية الفكر 7. اولا : اسرته ونشأتها العلمية والثقافية فتح حوار حر مع جميع االحزاب والمنظمات والشخصيا ت السياسية التقدمية دون استثناء وذلك لبلورة منهاج مشترك يكون اساساً لتاليف (حكومة ائتالفية مؤقتة) تحدد مهامها اساساً باصدار دستور مؤقت وفق مبادئ الديمقراطية وتشريع قانون االنتخابات واالشراف على االنتخابات ( العامة للمجلسين التأسيسي والوطني ، الذي تنبثق عنه حكومة االتحاد .التقدمي) على اساس التمثيل النسبي ا ًا ( ي ي ب ق ي ي و و ج ين ا و .التقدمي) على اساس التمثيل النسبي والخالصة، ان الحكام ان ارادو حقاً تحقيق وعودهم االخيرة للشعب واالستفادة من عبر التاريخ القريب والخروج من دوامة االنقالبات العسكرية المدمرة وتجنب الطريق المسدود تاريخياً فما عليهم اال االخذ بفكرة المراحل الثال ث وهي مرحلة الحكم االنقالبي الحالي ومرحلة الحكومة المؤقتة ، ثم (.مرحلة حكومة االتحاد التقدمي91 )ولذلك فعليهم وعلى الفور المباشرة باداء مهام المرحلة االولى وذلك باطالق الحريات الديمقراطية وعلى رأسها حرية النشاط الحزبي للقوى السياسية الفعالة في المجتمع ، واال قالع نهائياً عن مفهوم الحزب الواحد او الحزب القائد، والتخلي عن اسطورة الوصاية على الجماهير، او االنفتاح الصوري على بعض القوى االخرى وذلك تمهيداً لعقد ، حوار مفتوح مع جميع القوى المعادية لالستعمار واالقطاع واالستقالل لصياغة منهاج مشترك عام يكون اساساً لتألي ف (جهة اتحاد تقدمي)، تنبثق عنها االنتخابات العامة وتتصدى لحل المشاكل االساسية للبالد ، وتقود (.عملية التطور االجتماعي والسياسي92 ) ويبدو لنا من خالل ما تم ذكره ان ابراهيم كبة من الشخصيات القالئل التي وجدت في وقتنا هذا ، فهو حريص على قول الحق دون وجل وال مح اباة ال يسعى الى تحقيق المكاسب والمناصب السياسية بقدر سعيه لخدمة العراق وايصاله الى بر االمان، فهو بحق شخصية ذات فكر متميز وابداع ناصع وحس وطني، يفتخر به العراق في كل المراحل سواء في الماضي القريب .او الحاضر او المستقبل 371 371 مجلة كلية االداب / العدد76 الخاتمة كل ما ورد في هذا الب حث يبين بصورة ال لبس فيها ان ابراهيم كبة كان وجهاً من ابرز وجوه الفكر الوطني العراقي، اذ اتسم بقدر عال من الوضوح والثبات والجراة وبصفات اخرى مكنته من تقديم افضل ما لديه ،لخدمة الشعب والوطن طيلة حياته وال سيما المرحلة التي استوزر فيها والتي تعد من المراحل ا لتاريخية الصعبة ، بسبب تشابك االحداث السياسية .وتعقدها، فضالً عن تطور الصراعات بين ابرز قادة السياسة في حينها ان ابراهيم كبة كان احد ابرز الشخصيات االقتصادية في العراق ذلك من ، خالل العشرات من مؤلفاته التي تنصب في مجال النظريات االقتصادية فضالً عن تحقيق ال عديد من االنجازات االقتصادية وال سيما في مجال االصالح الزراعي، اذ نجح في تحطيم القوة السياسية واالقتصادية للشيوخ .واالقطاعيين الكبار ا كل ما ورد في هذا الب حث يبين بصورة ال لبس فيها ان ابراهيم كبة كان وجهاً من ابرز وجوه الفكر الوطني العراقي، اذ اتسم بقدر عال من الوضوح والثبات والجراة وبصفات اخرى مكنته من تقديم افضل ما لديه ،لخدمة الشعب والوطن طيلة حياته وال سيما المرحلة التي استوزر فيها والتي تعد من المراحل ا لتاريخية الصعبة ، بسبب تشابك االحداث السياسية .وتعقدها، فضالً عن تطور الصراعات بين ابرز قادة السياسة في حينها ا لقد حاول ابراهيم كبة معالجة الكثير من القضايا االقتصادية التي تهم الشعب العراقي ومنها قضية النفط اذ اصر على رفع شعار تأميمه ا منذ عام1952 ، وكان موقف كبة من الثروة النفطية منطلقاً من موقفه ازاء الشركات االجنبية وتالعبها بمقدرات الشعب العراقي واستغاللها اياه اذ رأى الوقوف .بوجهها واجباً وطنياً ثابتاً ال مزايدة فيه ا ا كان ابراهيم كبة متمسكاً بافكاره الماركسية مخلصاً لها مناضالً من ، اجلها مما ادى به الدخول الى السجن والحكم عليه لعدة سنوات وعلى الرغم من كل ذلك بقي مخلصاً لوطنه سائراً في طريقه لمعارضة االنظمة التي تلت ثورة14 تموز1958 . واخيراً ترك ابراهيم كبة من بعده تراثاً مرموقاً يجمع بين الفكر والسياسة واالقتصاد متوزعاً بين الكتب ًوالمقاالت في الصحف والبحوث لتكون منارا . يعتليها زواد العلم والمثقفين 372 مجلة كلية االداب / العدد76 1. ،محمد حرز الدين، معارف من الرجال في ترجمة العلماء واالدباء ج1 ،، النجف1964، ص2040. 2. ، بغداد ومجالسهم، اخبارهم البغداديون، الدوري، ابراهيم 1958،ص214. 3. حامد قاسم الجبو ري، محمد مهدي كبة حياته ودوره السياسي في ،العراق، اطروحة دكتوراه ، كلية التربية، بغداد1977، ص16. 4. حامد قاسم الجبوري ، المصدر السابق،ص11. 5. الخاتمة ،محمد مهدي كبة، مذكرات في صميم االحداث1918 – 1958 ، ، بيروت1965، ص9. 9. وهومحمد مهدي بن الشسيخ محمد حسن بن محمد صالح بن مصطفى بن درويش بن جعفر بن علي بن معروف الربيعي، ولد في سامراء عام 1900، تلمذ على يد مجموعة من االساتذة لدراسة بعض المواد المتعلقة .بالعلوم الدينية واللغة الدينية 10 . عميد اسرة الخالصي، كان له دور في مقاومة الغزو البريطاني للعراق، اصبحت مدرسته وبيته في الكاظمية مزاراً ومركزاً لمختلف الفئات السياسية والوطنية، اعتقل عام1933 : ونفي الى الحجاز ، للمزيد ينظر لواء االستقال ل27 حزيران1923. 11. ،االضبارة الـخاصة بابراهيم كـبة ، كـلية االدارة واالقتصاد، بغداد المرقمة132 / 699. 12. سنان صادق حسين، سياسة الواليات المتحدة االمريكية تجاه (العراق1958 - 1963 ،)، اطروحة دكتوراه ، كلية التربية، بغداد2005 ، ص97. 13 . االضبارة الخاصة بابراه ،يم كبة ، المصدر السابق؛ حنا بطاطو العراق(الشيوعيون والبعثيون والضباط االحرار)، ت : عفيف الرزاز، ك 2، ط1 ، ، بيروت1992، ص121 ، 125. 14. ،وزارة المعارف العراقية ، مديرية الشؤون الفنية، مديرية البعثات العدد13727 ، 14 /4/ 1953 ، رقم الملف1558. 15 . االضبارة الخاص ة بابراهيم كبة، كلية االدارة واالقتصاد، المرقمة /ج3 /، ص139 - 1588 ؛ أ. د. نوري عبد الحميد العاني واخرون، تاريخ الوزارات العراقية في العهد الجمهوري ، ج2، ط1 ،، بغداد2001، ص33. 16. .وزارة المعارف العراقية، مديرية الشؤون الفنية، المصدر السابق 17 . حسب كتاب نقابة المحامين رقم177 في15 /4/ 1953 الموجود في اضبارة ابراهيم كبة المرقمة1588 .في كلية االدارة واالقتصاد ، بغداد 18 . كتاب وزارة المالية، شعبة الخدمات والمالك والذاتية، بموجب االمر الوزاري رقم125 العدد18144 في23 تشرين الثاني1941 ، والموجود في االضبارة الخاصة له المرقمة1588 .في كلية االدارة واالقتصاد، بغداد 19. .المصدر نفسه 8. ،محمد مهدي كبة، مذكرات في صميم االحداث1918 – 1958 ، ، بيروت1965، ص9. 9. وهومحمد مهدي بن الشسيخ محمد حسن بن محمد صالح بن مصطفى بن درويش بن جعفر بن علي بن معروف الربيعي، ولد في سامراء عام 1900، تلمذ على يد مجموعة من االساتذة لدراسة بعض المواد المتعلقة .بالعلوم الدينية واللغة الدينية 8. ،محمد مهدي كبة، مذكرات في صميم االحداث1918 – 1958 ، ، بيروت1965، ص9. بيرو1965 ص9. 9. وهومحمد مهدي بن الشسيخ محمد حسن بن محمد صالح بن مصطفى بن درويش بن جعفر بن علي بن معروف الربيعي، ولد في سامراء عام 1900، تلمذ على يد مجموعة من االساتذة لدراسة بعض المواد المتعلقة .بالعلوم الدينية واللغة الدينية م 10 . الخاتمة رجل دين وشاعر ينتمي نسبه لالمام الحسين بن علي، قاد مجموعة من المجاهدين لمقاومة االحتالل البريطاني عام1915 في معر ،كة الشعيبة توفي عام1916 ،وكان لوفاته صدى واسع لدى العراقيين؛ حيدر الحلي العقد المفصل، ج2 ،، بغداد1973،ص205. 6. وهو والد محمد مهدي كبة ، من اسرة عربية كريمة ادخل الى ،(الكتاتيب) ليقرا القرآن الكريم، حيث تعلم على طريقة اهل ذلك العصر ونشا في بغداد نشاة سياس ية مترفة وقرا الشيء الكثير من كتب االدب وقرض الشعر، توفي عام1918 ودفن في مقبرة الحق االزرق ، بعد ان خلف ارثاً ثقافياً ودينياً شكل احدى محاور نهضة العراق االدبية والعلمية في القرن التاسع عشر للمزيد ينظر : محمد مهدي البصير، نهضة العراق االدبية في القرن التا ،سع عشر ، بغداد1946، ص285 ؛ عبد الرزاق الهاللي، شعراء من العراق ، محمد حسن كبة ، مجلة االديب ، تشرين ،االول1973. 7. وهو مصطفى بن درويش بن جعفر بن علي بن معروف الربيعي المعروف بـ(كبة)، للمزيد ينظر : حميد المطبعي ، موسوعة اعالم العراق في القرن العشرين، ج1 ، ،بغداد1995، ص196. ج1 ،، النجف1964، ص2040. 2. ، بغداد ومجالسهم، اخبارهم البغداديون، الدوري، ابراهيم 1958،ص214. 3. حامد قاسم الجبو ري، محمد مهدي كبة حياته ودوره السياسي في ،العراق، اطروحة دكتوراه ، كلية التربية، بغداد1977، ص16. 3. حامد قاسم الجبو ري، محمد مهدي كبة حياته ودوره السياسي في ،العراق، اطروحة دكتوراه ، كلية التربية، بغداد1977، ص16. 4. حامد قاسم الجبوري ، المصدر السابق،ص11. 5. رجل دين وشاعر ينتمي نسبه لالمام الحسين بن علي، قاد مجموعة من المجاهدين لمقاومة االحتالل البريطاني عام1915 في معر ،كة الشعيبة توفي عام1916 ،وكان لوفاته صدى واسع لدى العراقيين؛ حيدر الحلي العقد المفصل، ج2 ،، بغداد1973،ص205. ج 6. وهو والد محمد مهدي كبة ، من اسرة عربية كريمة ادخل الى ،(الكتاتيب) ليقرا القرآن الكريم، حيث تعلم على طريقة اهل ذلك العصر ونشا في بغداد نشاة سياس ية مترفة وقرا الشيء الكثير من كتب االدب وقرض الشعر، توفي عام1918 ودفن في مقبرة الحق االزرق ، بعد ان خلف ارثاً ثقافياً ودينياً شكل احدى محاور نهضة العراق االدبية والعلمية في القرن التاسع عشر للمزيد ينظر : محمد مهدي البصير، نهضة العراق االدبية في القرن التا ،سع عشر ، بغداد1946، ص285 ؛ عبد الرزاق الهاللي، شعراء من العراق ، محمد حسن كبة ، مجلة االديب ، تشرين ،االول1973. 7. وهو مصطفى بن درويش بن جعفر بن علي بن معروف الربيعي المعروف بـ(كبة)، للمزيد ينظر : حميد المطبعي ، موسوعة اعالم العراق في القرن العشرين، ج1 ، ،بغداد1995، ص196. 373 مجلة كلية االداب / العدد76 8. الخاتمة عميد اسرة الخالصي، كان له دور في مقاومة الغزو البريطاني للعراق، اصبحت مدرسته وبيته في الكاظمية مزاراً ومركزاً لمختلف الفئات السياسية والوطنية، اعتقل عام1933 : ونفي الى الحجاز ، للمزيد ينظر لواء االستقال ل27 حزيران1923. ااا اا 11. ،االضبارة الـخاصة بابراهيم كـبة ، كـلية االدارة واالقتصاد، بغداد المرقمة132 / 699. 12. سنان صادق حسين، سياسة الواليات المتحدة االمريكية تجاه (العراق1958 - 1963 ،)، اطروحة دكتوراه ، كلية التربية، بغداد2005 ، 11. ،االضبارة الـخاصة بابراهيم كـبة ، كـلية االدارة واالقتصاد، بغداد المرقمة132 / 699. اا 12. سنان صادق حسين، سياسة الواليات المتحدة االمريكية تجاه (العراق1958 - 1963 ،)، اطروحة دكتوراه ، كلية التربية، بغداد2005 ، ص97. 13 . االضبارة الخاصة بابراه ،يم كبة ، المصدر السابق؛ حنا بطاطو العراق(الشيوعيون والبعثيون والضباط االحرار)، ت : عفيف الرزاز، ك 2، ط1 ، ، بيروت1992، ص121 ، 125. 14. ،وزارة المعارف العراقية ، مديرية الشؤون الفنية، مديرية البعثات العدد13727 ، 14 /4/ 1953 ، رقم الملف1558. ااا 13727 14 /4/ 1953 ر م 1558. 15 . االضبارة الخاص ة بابراهيم كبة، كلية االدارة واالقتصاد، المرقمة /ج3 /، ص139 - 1588 ؛ أ. د. نوري عبد الحميد العاني واخرون، تاريخ الوزارات العراقية في العهد الجمهوري ، ج2، ط1 ،، بغداد2001، ص33. 16. .وزارة المعارف العراقية، مديرية الشؤون الفنية، المصدر السابق 17 . حسب كتاب نقابة المحامين رقم177 في15 /4/ 1953 الموجود في اضبارة ابراهيم كبة المرقمة1588 .في كلية االدارة واالقتصاد ، بغداد 18 . كتاب وزارة المالية، شعبة الخدمات والمالك والذاتية، بموجب االمر الوزاري رقم125 العدد18144 في23 تشرين الثاني1941 ، والموجود في االضبارة الخاصة له المرقمة1588 .في كلية االدارة واالقتصاد، بغداد 19. .المصدر نفسه 374 د. علي المشهداني مجلة كلية االداب / العدد76 20 . ولد سالم في27 كانون االول1954 وسلمى في22 كانون الثاني 1956 ونسرين في23 نيسان1957 اما كريم في ايلول1958 للمزيد ( ينظر: االضبارة الخاصة بابراهيم كبة المرقمة10 ) / ش/ م استمارة بيان ا لزوجة وعدد االوالد والموجودة في كلية االدارة واالقتصاد ، بغداد؛ ينظر /وزارة المالية ، التقاعد العامة ، رقم ص . د14 م في19 /4/ 1954 الموجود في االضبارة الخاصة به المرقمة10 ./ ش / م ا ا ي م 21 . االضبارة الخاصة بابراهيم كبة المرقمة132 / 699 – 185 كلية االدارة واالقتص. اد ، بغداد ار و ا 22. ورد اسم الشارع في كتاب دائرة الدراسات والبحث العلمي في وزارة التعليم العالي رقم172 / 3592 في17 /1/ 1978. 23. وزارة المعارف ، مميزية االمر، الذاتية، بموجب االمر الوزاري المرقم352 في21 /4/ 1953 رقم الملف864. 24 . الخاتمة وزارة المعارف ، مديرية االمور الذاتية ، بموجب االمر الوزاري 26 /7/ 1953 ، العدد25033 الموجود في االضبارة المرقمة1855. 25 . وهو من الشخصيات الوطنية البارزة في العراق ساهمت في تشكيل ًجمعية ، حرس االستقالل العراقية، لمواجهة االحتالل البريطاني ، فضال عن دوره في تأسيس الحزب الوطني العراقي ع ام1922. 26. بينروز ، العراق دراسة في عالقاته الخارجية وتطوراته الداخلية 1915 - 1975 ، ت: عبد المجيد حسيب، ج1، ط1 ، ، الدار العربية ، بيروت 1989،ص336. 27. مقابلة شخصية مع الدكتور تقي العاني رئيس قسم االقتصاد في ا ف ال ا اال ة ف تن ال ا ة ال3/5/ 2006ال ا ة 26. بينروز ، العراق دراسة في عالقاته الخارجية وتطوراته الداخلية 1915 - 1975 ، ت: عبد المجيد حسيب، ج1، ط1 ، ، الدار العربية ، بيروت 1989،ص336. ا 27. مقابلة شخصية مع الدكتور تقي العاني رئيس قسم االقتصاد في الجامعة المستنصرية في يوم االربعاء المصادف3/5/ 2006 الساعة .العاشرة صباحاً ، اذ كان من المعاصرين البراهيم كية ما 28. مجلة الثقافة الجديدة، العدد4 تموز1969. 29 . سالم ابراهيم، الوقفة الشامخة في محاكمة الجالدين، مركز دراسات :الماركسية واليسارwww,rezqar, com/debat/show, art,asb . 30 ،مجلة الثقافة الجديدة المصدر السابق 28. مجلة الثقافة الجديدة، العدد4 تموز1969. 29 . سالم ابراهيم، الوقفة الشامخة في محاكمة الجالدين، مركز دراسات :الماركسية واليسارwww,rezqar, com/debat/show, art,asb . 30 . ، مجلة الثقافة الجديدة.المصدر السابق 31. / كتاب وزارة الداخلية المرقم س2638 المؤرخ في30 /8/ 1954 ذي العدد1859 في18 /9/ 1954 ، والموجود في االضبارة الخاصة رقم الملف 1588 .في كلية االدارة واالقتصاد 30 . ، مجلة الثقافة الجديدة.المصدر السابق 31. / كتاب وزارة الداخلية المرقم س2638 المؤرخ في30 /8/ 1954 ذي العدد1859 في18 /9/ 1954 ، والموجود في االضبارة الخاصة رقم الملف 1588 .في كلية االدارة واالقتصاد 375 د. علي المشهداني مجلة كلية االداب / العدد76 32. ،جريدة الزمان، العدد2691 في17 تموز1958. 33 . تضم مجموعة من االحزاب والتيارات السياسية كحزب االستقالل والحزب الشيوعي وحزب البعث والحزب الوطني الديمقراطي وبعض المستقلين ، اصدرت بيانها االول في9 اذار1957 وتدعو فيه الى اطالق الحريات الديمقراطية والدستورية وتنحية نوري السعيد من منصبه وحل ،المجلس النيابي للمزيد ينظر: هادي رشيد الجاوشلي مشاكل العراق الداخلية ،مع االيام ، بغداد1967، ص7 ؛ علي محمد النوري، جبهة االتحاد الوطني منطلق هام لتطور الحركة الوطنية في العراق ، مقالة منشورة في مجلة الثقافة الجريدة، ع97 ، ، اذار1976. 34 . المرسوم الجمهوري موجود في االضبارة الخاصة رقم الملف 132 / 699 - 185 .والموجودة في كلية االدارة واالقتصاد ، بغداد 35. الخاتمة تضم مجموعة من االحزاب والتيارات السياسية كحزب االستقالل والحزب الشيوعي وحزب البعث والحزب الوطني الديمقراطي وبعض المستقلين ، اصدرت بيانها االول في9 اذار1957 وتدعو فيه الى اطالق الحريات الديمقراطية والدستورية وتنحية نوري السعيد من منصبه وحل ،المجلس النيابي للمزيد ينظر: هادي رشيد الجاوشلي مشاكل العراق الداخلية ،مع االيام ، بغداد1967، ص7 ؛ علي محمد النوري، جبهة االتحاد الوطني منطلق هام لتطور الحركة الوطنية في العراق ، مقالة منشورة في مجلة الثقافة الجريدة، ع97 ، ، اذار1976. 34 . المرسوم الجمهوري موجود في االضبارة الخاصة رقم الملف 132 / 699 - 185 .والموجودة في كلية االدارة واالقتصاد ، بغداد 35. وزارة التعليم العالي، جامعة بغداد، كلية االدارة واالقتصاد ، رقم الملف1855. ال ا ذ ال ل ا ال 32. ،جريدة الزمان، العدد2691 في17 تموز1958. 33 . تضم مجموعة من االحزاب والتيارات السياسية كحزب االستقالل والحزب الشيوعي وحزب البعث والحزب الوطني الديمقراطي وبعض المستقلين ، اصدرت بيانها االول في9 اذار1957 وتدعو فيه الى اطالق الحريات الديمقراطية والدستورية وتنحية نوري السعيد من منصبه وحل ،المجلس النيابي للمزيد ينظر: هادي رشيد الجاوشلي مشاكل العراق الداخلية ،مع االيام ، بغداد1967، ص7 ؛ علي محمد النوري، جبهة االتحاد الوطني منطلق هام لتطور الحركة الوطنية في العراق ، مقالة منشورة في مجلة الثقافة الجريدة، ع97 ، ، اذار1976. ا ع 34 . المرسوم الجمهوري موجود في االضبارة الخاصة رقم الملف 132 / 699 - 185 .والموجودة في كلية االدارة واالقتصاد ، بغداد اا ي 35. وزارة التعليم العالي، جامعة بغداد، كلية االدارة واالقتصاد ، رقم الملف1855. اا ي 35. وزارة التعليم العالي، جامعة بغداد، كلية االدارة واالقتصاد ، رقم الملف1855. 36كتاب مجلس الوزراء ذي العدد16662في27 /7/ 1959والموجود 36. كتاب مجلس الوزراء ذي العدد16662 في27 /7/ 1959 والموجود في االضبارة الخاصة به المرقمة10 ./ ش/م في كلية االدارة واالقتصاد ا ياا م ي 37 . اس ماعيل العارف، اسرار ثورة14 تموز ، بغداد ، ص316 ؛ ثورة 14 ، تموز في عامها السادس ، بغداد1964. ي 38. نوري العاني واخرون، تاريخ الوزارات العراقية في العهد الجمهوري االول، ج1، ط1، المطبعة العربية، بغداد، ص36. جا 39 . الخاتمة وزارة التعليم العالي، جامعة بغداد، كلية االدارة واالقتصاد ، رقم الملف1855. 36. كتاب مجلس الوزراء ذي العدد16662 في27 /7/ 1959 والموجود في االضبارة الخاصة به المرقمة10 ./ ش/م في كلية االدارة واالقتصاد 37 . اس ماعيل العارف، اسرار ثورة14 تموز ، بغداد ، ص316 ؛ ثورة 14 ، تموز في عامها السادس ، بغداد1964. 38. نوري العاني واخرون، تاريخ الوزارات العراقية في العهد الجمهوري االول، ج1، ط1، المطبعة العربية، بغداد، ص36. 39 . ذكر ابراهيم كبة اثناء دفاعه عن نفسه امام المحكمة العسكرية ، ان سبب استقالته التي تعود الى قناعته بتدخل االجهزة القاسمية في اختصاصاتي الوزارية ومحاوالتها الحداث تعديالت في سياسته وقانون االصالح الزراعي ، تحت ضغط االقطاع والبرجوازية ، مما يؤكد عدم اصالح النظام القاسمي بالطرق الدستورية المعروفة، للمزيد ينظر : قرار المرسوم الجمهوري رقم11 والمنشور في جريدة الوقائع العراقية في25 شباط1960 ؛ ابراهيم كبة ، هذا هو طريق14 تموز ، ط1، دار الطليعة ، ،بيروت1969، ص31. 40. امير الحلو،ابراهيم كبة صاحب فكر ثابت ورحيل صامت ، جريدة المدى في13 /2/ 265. 32. ،جريدة الزمان، العدد2691 في17 تموز1958. 33 . تضم مجموعة من االحزاب والتيارات السياسية كحزب االستقالل والحزب الشيوعي وحزب البعث والحزب الوطني الديمقراطي وبعض المستقلين ، اصدرت بيانها االول في9 اذار1957 وتدعو فيه الى اطالق الحريات الديمقراطية والدستورية وتنحية نوري السعيد من منصبه وحل ،المجلس النيابي للمزيد ينظر: هادي رشيد الجاوشلي مشاكل العراق الداخلية ،مع االيام ، بغداد1967، ص7 ؛ علي محمد النوري، جبهة االتحاد الوطني منطلق هام لتطور الحركة الوطنية في العراق ، مقالة منشورة في مجلة الثقافة الجريدة، ع97 ، ، اذار1976. 34 . المرسوم الجمهوري موجود في االضبارة الخاصة رقم الملف 132 / 699 - 185 .والموجودة في كلية االدارة واالقتصاد ، بغداد 35. وزارة التعليم العالي، جامعة بغداد، كلية االدارة واالقتصاد ، رقم الملف1855. 36. كتاب مجلس الوزراء ذي العدد16662 في27 /7/ 1959 والموجود في االضبارة الخاصة به المرقمة10 ./ ش/م في كلية االدارة واالقتصاد 37 . اس ماعيل العارف، اسرار ثورة14 تموز ، بغداد ، ص316 ؛ ثورة 14 ، تموز في عامها السادس ، بغداد1964. 38. نوري العاني واخرون، تاريخ الوزارات العراقية في العهد الجمهوري االول، ج1، ط1، المطبعة العربية، بغداد، ص36. 39 . ذكر ابراهيم كبة اثناء دفاعه عن نفسه امام المحكمة االعسكرية ، ان 32. ،جريدة الزمان، العدد2691 في17 تموز1958. 33 . الخاتمة ذكر ابراهيم كبة اثناء دفاعه عن نفسه امام المحكمة العسكرية ، ان سبب استقالته التي تعود الى قناعته بتدخل االجهزة القاسمية في اختصاصاتي الوزارية ومحاوالتها الحداث تعديالت في سياسته وقانون االصالح الزراعي ، تحت ضغط االقطاع والبرجوازية ، مما يؤكد عدم اصالح النظام القاسمي بالطرق الدستورية المعروفة، للمزيد ينظر : قرار المرسوم الجمهوري رقم11 والمنشور في جريدة الوقائع العراقية في25 شباط1960 ؛ ابراهيم كبة ، هذا هو طريق14 تموز ، ط1، دار الطليعة ، ،بيروت1969، ص31. 40. امير الحلو،ابراهيم كبة صاحب فكر ثابت ورحيل صامت ، جريدة المدى في13 /2/ 265. 376 376 مجلة كلية االداب / العدد76 41 . كتاب وزارة التعليم العالي، جامعة بغداد، ذي العدد3174 في12 /3/ 1963 ، الموجود في االضبارة المرقمة1588 في كلية االدارة . واالقتصاد /3/ 1963 ، الموجود في االضبارة المرقمة1588 في كلية االدارة . واالقتصاد 42. كتاب وزارة الدفاع ، العدد ط ق3/ 13 / 897 في11 /4/ 1963 والموجود في االضبارة الخاصة المرقمة1588 .في كلية االدارة واالقتصاد 43 . بموجب كتاب مقر ال حاكم العسكري العام المؤرخ في29 /2/ 1963 والمبلغ بكتاب من وزارة المالية المرقم س67 والمؤرخ في10 /2/ 1963 رقم الملف41 / 65 في9/ 10 / 1963 والموجود في االضبارة الخاصة المرقمة م1588. 44. ابراهيم كبة ، هذا هو طريق14 .تموز ، المصدر السابق 45 . جامعة بغداد ، الذاتية رقم1849 المؤرخ في3/6/ 1968 ، والموجود في االضبارة / الخاصة المرقمة أ1588 في كلية . االدارة واالقتصاد 46. كتاب جامعة بغداد، مديرية شؤون الهيئة التدريسية رقم الملف 15775 والموجود في االضبارة الخاصة له ، المرقمة10 /ش م في كلية .االدارة واالقتصاد 42. كتاب وزارة الدفاع ، العدد ط ق3/ 13 / 897 في11 /4/ 1963 والموجود في االضبارة الخاصة المرقمة1588 .في كلية االدارة واالقتصاد 43 . بموجب كتاب مقر ال حاكم العسكري العام المؤرخ في29 /2/ 1963 والمبلغ بكتاب من وزارة المالية المرقم س67 والمؤرخ في10 /2/ 1963 رقم الملف41 / 65 في9/ 10 / 1963 والموجود في االضبارة الخاصة المرقمة م1588. 44. ابراهيم كبة ، هذا هو طريق14 .تموز ، المصدر السابق 45 . جامعة بغداد ، الذاتية رقم1849 المؤرخ في3/6/ 1968 ، والموجود في االضبارة / الخاصة المرقمة أ1588 في كلية . االدارة واالقتصاد 46. كتاب جامعة بغداد، مديرية شؤون الهيئة التدريسية رقم الملف 15775 والموجود في االضبارة الخاصة له ، المرقمة10 /ش م في كلية .االدارة واالقتصاد 44. ابراهيم كبة ، هذا هو طريق14 .تموز ، المصدر السابق 45 . الخاتمة ت ، ص20 ، 22 ؛ مجلة العمال ، ع1367 ، 1975. 62. ،محمود حبيب، اقتصاديات العراق، البصرة1969،ص180. 63 . وهي يوغسالفيا وروسيا والمانيا الديمقراطية وجيكوسلوفاكيا ورومانيا وبولندا وهنغاريا وكوريا الش مالية وفيتنام وبلغاريا والبانيا والصين الشعبية ؛ للمزيد ينظر: بينروز ، ج1، المصدر السابق، ص405. 64. ، اداميشين واخرون، السياسة الخارجية لالتحاد السوفيتي ، دار التقدم موسكو، ج2 ، 1975، ص398. 54 . مجلة النفط والعالم، ع45، حزيرا ، ن1977. 55. ، مجلة االقتصاد ، اذار1964. 56 . ضمت اللجنة من رئاسة عبد الكريم قاسم وعضوية العقيد الركن ناجي طالب وزير الشؤون االجتماعية ومحمد حديد وزير المالية ، ومحمد صديق شنشل وزير االرشاد كذلك وزير االقتصاد ابراهيم كبة ، للمزيد ينظر: عبد ، هللا اسماعيل( مفاوضات العراق النفطية952 - 1968 ،)، لندن1989 ؛ مفردات مجلس الوزراء يوم14 اب1958. 57 . احمد ساجر الدليمي، نفط العراق دراسة تاريخية1963 - 1968 ، كلية ،التربية ، بغداد1997، ص19 ؛ ينظر عبد اللطيف الشواف، حول قضية ، النفط في العراق ، المكتبة العصرية ، بيروت1966، ص ر7 - 8. 58. ابراهيم كبة، هذا هو طريق14 تموز، المصدر السابق، ص43 - 44 ؛ للمزيد ينظر: عبد الكريم قاسم ، اهداف الثورة في الخطاب الذي القاه في المؤتمر الصحفي في2 كانون االول1959 ،، وزارة االرشاد ، بغداد 1959، ص45 - 46. ج س وزر يوم ر14 ب1958. 57 . احمد ساجر الدليمي، نفط العراق دراسة تاريخية1963 - 1968 ، كلية ،التربية ، بغداد1997، ص19 ؛ ينظر عبد اللطيف الشواف، حول قضية ، النفط في العراق ، المكتبة العصرية ، بيروت1966، ص ر7 - 8. 58. ابراهيم كبة، هذا هو طريق14 تموز، المصدر السابق، ص43 - 44 ؛ للمزيد ينظر: عبد الكريم قاسم ، اهداف الثورة في الخطاب الذي القاه في المؤتمر الصحفي في2 كانون االول1959 ،، وزارة االرشاد ، بغداد 1959، ص45 - 46. 59 . ليث عبد الحسن، ثورة14 تموز في العرا ،ق، دار الرشيد ، بغداد 1981،ص65؛ ابراهيم كبة ، المصدر السابق، ص44 - 45 ،؛ جيراسيمون ، النفط العراقي، موسكو1969. 59 . ليث عبد الحسن، ثورة14 تموز في العرا ،ق، دار الرشيد ، بغداد 1981،ص65؛ ابراهيم كبة ، المصدر السابق، ص44 - 45 ،؛ جيراسيمون ، النفط العراقي، موسكو1969. 60. محمد كاظم علي، المصدر السابق، ص126 ؛ صباح الدرة، التطور ،العراق، مطبعة النجوم ، بغداد ف الصناع1968،ص327 62. ر ر ق ب ي و بيب 1969ص180. 63 . الخاتمة جامعة بغداد ، الذاتية رقم1849 المؤرخ في3/6/ 1968 ، والموجود في االضبارة / الخاصة المرقمة أ1588 في كلية . االدارة واالقتصاد 46. كتاب جامعة بغداد، مديرية شؤون الهيئة التدريسية رقم الملف اا 47 . وزارة ال مالية ، مديرية التقاعد العامة ، رقم77553 في25 /6/ 1977 الموجود في االضبارة الخاصة له والمرقمة م1588 في كلية االدارة .واالقتصاد ا ا 48. توفي في الساعة التاسعة صباحاً من يوم الثالثاء26 / 10 / 2004 في داره الواقع في الكرادة داخل / الزوية / شارع الوزير محلة57 ز1 د15. 49. مجلة الثقافة الجديدة ، ع81 / ، ايار1976. 50. محمد كاظم علي، الوزارة في عهد عبد الكريم قاسم، دراسة في القوى السياسية والصراع االيديولوجي1958 - 1963 ،، اليقظة العربية، بغداد 1989، ص106. 51 . فاضل عباس مهدي، التنمية االقتصادية والتخطيط في العراق ، ط8 ، دا، ر الطليعة ، بيروت1977،ص85. 52. ينظر نص البيان في موسوعة عبد الوهاب الكيالي، الموسوعة السياسية ، ط1 ، ، المؤسسة العربية ، بيروت1974، ص700. 53. ،منشورات المؤسسة الثقافية العمالية، النفط ، دار الحرية، بغداد 1968، ص30 - 31. 377 مجلة كلية االداب / العدد76 54 . مجلة النفط والعالم، ع45، حزيرا ، ن1977. 55. ، مجلة االقتصاد ، اذار1964. 56 . ضمت اللجنة من رئاسة عبد الكريم قاسم وعضوية العقيد الركن ناجي طالب وزير الشؤون االجتماعية ومحمد حديد وزير المالية ، ومحمد صديق شنشل وزير االرشاد كذلك وزير االقتصاد ابراهيم كبة ، للمزيد ينظر: عبد ، هللا اسماعيل( مفاوضات العراق النفطية952 - 1968 ،)، لندن1989 ؛ مفردات مجلس الوزراء يوم14 اب1958. 57 . احمد ساجر الدليمي، نفط العراق دراسة تاريخية1963 - 1968 ، كلية ،التربية ، بغداد1997، ص19 ؛ ينظر عبد اللطيف الشواف، حول قضية ، النفط في العراق ، المكتبة العصرية ، بيروت1966، ص ر7 - 8. 58. ابراهيم كبة، هذا هو طريق14 تموز، المصدر السابق، ص43 - 44 ؛ للمزيد ينظر: عبد الكريم قاسم ، اهداف الثورة في الخطاب الذي القاه في المؤتمر الصحفي في2 كانون االول1959 ،، وزارة االرشاد ، بغداد 1959، ص45 - 46. 59 . ليث عبد الحسن، ثورة14 تموز في العرا ،ق، دار الرشيد ، بغداد 1981،ص65؛ ابراهيم كبة ، المصدر السابق، 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(عبد المناف شكر، العالقات العراقية السوفيتية1944 - 8 شباط 1963 ،)، بغداد1980،ص88 - 92. 67 . ،الوقائع العراقية4 ايار1959 ، كذلك وزارة االصالح الزراعي ، تقرير حول االصالح الزراعي في اعوامه الثالثة ،بغداد1962. 68. الخاتمة مجيد خدوري ، العراق الجمهوري ، ط1 ، ، الدار المتحدة ، بيروت 1974، ص213 ؛ دورين ورنر، االصالح الزراعي بين المبدا والتطبيق،ط1 ،، دار الطليعة، بيروت1975، ص101. 69. صدر قانون االصالح الزراعي رقم30 في ايلول1958 وكانت استقالته في شباط1960. 70 . ابراهيم كبة، حول ثورة14 تموز1958 ، مجلة المثقف، المصدر .السابق 71. ،حسن الخطيب، االقطاع وقانون االصالح الزراعي، بغداد1959 ، ص4. اا 66. (عبد المناف شكر، العالقات العراقية السوفيتية1944 - 8 شباط 1963 ،)، بغداد1980،ص88 - 92. اا 71. ،حسن الخطيب، االقطاع وقانون االصالح الزراعي، بغداد1959 ، ص4. 72 . ،ابراهيم كبة، مسائل االصالح الزراعي ، جريدة اتحاد الشعب9 شباط1959 ؛ سعد محمد عثمان، االسس النظرية للتطبيق االشتراكي في ال ،عراق، دار الرشيد ، بغداد1981، ص115 - 116. 73. ابراهيم كبة ، هذا طريق14 تموز ، المصدر السابق؛ تقرير وزارة .االعالم، المصدر السابق ماا 74 . بينروز، ج1، المصدر السابق، ص387 ؛ ينظر أ. د. نوري عبد ، الحميد العاني واخرون، تاريخ الوزارات العراقية في العهد الجمهوري ج2 ، المصدر السابق، ص266. 75. ابراهيم كبة، حول ثورة14 تموز1958 ، مجلة المثقف، ع6 ، شباط ،، بغداد1962. 76. صبيح علي غالب، قصة ثورة14 ،تموز والضباط االحرار، الجاحظ ،بغداد1971، ص69. 77 . ابراهيم كبة، هذا طريق14 ،تموز، المصدر السابق؛ الوقائع العراقية ع1 ، 23 ت موز1958. 379 مجلة كلية االداب / العدد76 78. محمد سلمان حسن، دراسات في االقتصاد العراقي، ط1 ، دار الطليعة ،، بيروت1966،ص245 - 246. 79. ، هوشيار معروف، االقتصاد العراقي بين التبعية واالستقالل ، بغداد 1977، ص129. 78. محمد سلمان حسن، دراسات في االقتصاد العراقي، ط1 ، دار الطليعة ،، بيروت1966،ص245 - 246. 79. ، هوشيار معروف، االقتصاد العراقي بين التبعية واالستقالل ، بغداد 1977، ص129. 80. ابراهيم كبة، هذا هو طريق14 تموز، المصدر السابق، ص16. 81 . ليث عبد الحسن ال زبيدي، ثورة14 ،تموز في العراق،المصدر السابق ص265 ؛ ابراهيم كبة ، حول ثورة14 .تموز، المصدر السابق 82. ابراهيم كبة، محمد سلمان حسن، مصطفى علي وعبد الوهاب ، محمود؛ للمزيد ينظر: مجلة دراسات عربية، اكتوبر1966 ،؛ جريدة البلد ع3 ، نيسان1967. 83 . مركز دراسات وا بحاث الماركسية واليسار، اراء ابراهيم كبة حول : الفكر الرجعي في العراقsota l iraq. Com .iraqi press. Com. Htm . 78. محمد سلمان حسن، دراسات في االقتصاد العراقي، ط1 ، دار الطليعة ،، بيروت1966،ص245 - 246. 79. ، هوشيار معروف، االقتصاد العراقي بين التبعية واالستقالل ، بغداد 1977، ص129. 80. ابراهيم كبة، هذا هو طريق14 تموز، المصدر السابق، ص16. 81 . الخاتمة ليث عبد الحسن ال زبيدي، ثورة14 ،تموز في العراق،المصدر السابق ص265 ؛ ابراهيم كبة ، حول ثورة14 .تموز، المصدر السابق 82. ابراهيم كبة، محمد سلمان حسن، مصطفى علي وعبد الوهاب ، محمود؛ للمزيد ينظر: مجلة دراسات عربية، اكتوبر1966 ،؛ جريدة البلد ع3 ، نيسان1967. 83 . مركز دراسات وا بحاث الماركسية واليسار، اراء ابراهيم كبة حول : الفكر الرجعي في العراقsota l iraq. Com .iraqi press. Com. 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https://openalex.org/W2790574923	https://europepmc.org/articles/pmc6414998?pdf=render	English	null	Poly(butylene 2,5-thiophenedicarboxylate): An Added Value to the Class of High Gas Barrier Biopolyesters	Polymers	2,018	cc-by	11,067	Giulia Guidotti 1, Matteo Gigli 2,* ID , Michelina Soccio 1, Nadia Lotti 1,* ID , Massimo Gazzano 3, Valentina Siracusa 4 ID and Andrea Munari 1 1 Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy; giulia.guidotti9@unibo.it (G.G.); m.soccio@unibo.it (M.S.); andrea.munari@unibo.it (A.M.) 1 Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy; giulia.guidotti9@unibo.it (G.G.); m.soccio@unibo.it (M.S.); andrea.munari@unibo.it (A.M.) 2 Department of Chemical Science and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientiﬁca 1, 00133 Roma, Italy 3 Organic Synthesis and Photoreactivity Institute, CNR, Via Gobetti 101, 40129 Bologna, Italy; massimo.gazzano@cnr.it 4 Department of Chemical Science, University of Catania, Viale A. Doria 6, 95125 Catania, Italy; vsiracus@dmfci.unict.it * Correspondence: matteo.gigli@uniroma2.it (M.G.); nadia.lotti@unibo.it (N.L.); Tel.: +39-067-259-4488 (M.G.); +39-051-209-0354 (N.L.) * Correspondence: matteo.gigli@uniroma2.it (M.G.); nadia.lotti@unibo.it (N.L.); Tel.: +39-067-259-4488 (M.G.); +39-051-209-0354 (N.L.) Received: 15 January 2018; Accepted: 6 February 2018; Published: 9 February 2018 Received: 15 January 2018; Accepted: 6 February 2018; Published: 9 February 2018 Abstract: Many efforts are currently devoted to the design and development of high performance bioplastics to replace traditional fossil-based polymers. In response, this contribution presents a new biobased aromatic polyester, i.e., poly(butylene 2,5-thiophenedicarboxylate) (PBTF). Here, PBTF is characterized from the molecular, thermo-mechanical and structural point of view. Gas permeability is evaluated at different temperatures, in the range below and above glass transition, providing a full insight into the performances of this material under different operating conditions, and demonstrating the superior gas barrier behavior of PBTF with respect to other polyesters, such as PEF and PET. The combination of calorimetric and diffractometric studies allows for a deep understanding of the structure of PBTF, revealing the presence of a not-induced 2D-ordered phase (meso-phase), responsible for its outstanding gas permeability behavior. The simple synthetic strategy adopted, the exceptional barrier properties, combined with the interesting mechanical characteristics of PBTF open up new scenarios in the world of green and sustainable packaging materials. Keywords: poly(butylene 2,5-thiophenedicarboxylate); biopolymers; meso-phase; mechanical properties; gas barrier properties 1. Introduction In recent years, great attention has been paid to the exploitation of renewable resources in response to fossil fuel depletion and growing environmental pollution. Plastic materials, which nowadays are employed in nearly all aspects of our daily life, are not an exception. Indeed, global plastic production has constantly increased, reaching 322 Mtons in 2015 [1]. This generates huge amounts of plastic trash (about 30 Mtons/year only in Europe) that end up continuously in the waste stream, causing well-known terrestrial and aquatic contamination problems. In this respect, packaging probably represents the most pressing issue for various reasons: it accounts for ca. 40% of global plastic demand [1], it is a disposable item and, in case of food contact, its recycling is not very desirable [2]. Polymers 2018, 10, 167; doi:10.3390/polym10020167 www.mdpi.com/journal/polymers www.mdpi.com/journal/polymers Polymers 2018, 10, 167 Polymers 2018, 10, 167 2 of 14 Therefore, the use of bioplastics, i.e., plastics obtained from renewable resources and/or biodegradable, may represent a solution to these urgent needs. As reported by the European Bioplastics, their actual market is 4.1 Mtons/year and the growth expectations are about 50% in 5 years, thus at a much higher rate with respect to traditional plastics [3]. Many natural polymers such as starch, chitin, lignin, cellulose and vegetable oils have been extensively studied to promote the shift toward a greener economy. More recently, the scientiﬁc community has devoted its efforts to the synthesis of bio-based monomers for the production of highly performing biopolymers. In this framework, aliphatic polyesters (APs) are probably the most successful example, since various products based on poly(lactic acid), polyhydroxyalcanoates, poly(alkylene succinate)s and poly(alkylene adipate)s have been brought to the market. Although all commercially available, none of the above-mentioned APs displays gas barrier properties high enough to be considered a real alternative to poly(ethylene terephthalate) (PET). In the last years, another class of biopolymers received considerable attention because of the excellent permeability characteristics, far better than those of PET, i.e., furan-based polyesters, and in particular poly(ethylene furanoate) (PEF). Although known since the ﬁfties, the great success of 2,5-furandicarboxylic acid (FDCA) is mainly due to the discovery of a green route for its production [4,5]. Today, furan-based monomers can be obtained from sugar dehydration in large amounts and high purity. 1. Introduction The interest in these compounds is so high that the US Department of Energy included FDCA, 5-hydroxymethyl-furfural (HMF) and furfural in the top “10 + 4” list of bio-based chemicals [6]. The industry’s interest in these building blocks is high as well. Consequently, in fall 2016. Avantium and BASF announced the establishment of a joint venture called Synvina to build a plant with a capacity of 50,000 tons/year for the production of FDCA, the raw material for the synthesis of furanoate-based polymers [7]. The exceptional gas permeability behavior of PEF as compared to PET is linked to the different molecular structure and chain mobility. Indeed, it has been reported that the substitution of apolar benzene ring of PET with polar furan ring, together with nonlinear axis of ring rotation in PEF hinders the furan ring-ﬂipping, thus preventing permeant diffusion [8]. Furthermore, recent studies have demonstrated the enzymatic degradability of PEF and PET, opening up new possibilities in eco-friendly industrial depolymerization processes for recycling purposes [9–11]. In particular, a recent work demonstrated the degradability of PEF by cutinase from Humicola insolens, leading to complete polymer solubilisation in 72 h of incubation [11]. In this contribution, we propose a novel homopolymer with high gas barrier properties as an alter ego of FDCA-based polyesters, i.e., poly(butylene 2,5-thiophenedicarboxylate) (PBTF). Both starting materials, 2,5-thiophenedicarboxylic acid (TFDCA) and 1,4-butanediol (BD), can be derived from renewable resources, thus obtaining a fully biobased polymer. TFDCA is industrially produced from the reaction of adipic acid with thionyl chloride [12,13]. In turn, adipic acid can be obtained from glucaric or muconic acid [14]. Lastly, bio-based BD is prepared by hydrogenation of succinic acid [15]. Molecular, thermal, structural, mechanical and barrier properties of PBTF have been deeply investigated and correlated with the chemical structure, in order to establish structure-property relationships. 2.3. Molecular, Thermal and Structural Characterization 2.3. Molecular, Thermal and Structural Characterization Polymer structure was checked by 1H-NMR spectroscopy at RT. A Varian Inova (Palo Alto, CA, USA) 400-MHz was used for the measurements. Molecular weight was determined by gel-permeation chromatography (GPC) at 30 ◦C with an Agilent 1100 HPLC system (Santa Clara, CA, USA) equipped with PLgel 5-µm MiniMIX-C column. A UV-detector was employed. A Hexaﬂuoro-2-propanol/chloroform mixture (5:95 v/v) was used as eluent with a 0.3 mL/min ﬂow. A molecular weight calibration curve was obtained with polystyrene standards in the molecular weight range 800–100,000 g/mol. TGA was carried out under nitrogen atmosphere by means of a Perkin Elmer TGA7 apparatus (Waltham, MA, USA). Gas ﬂow of 30 mL/min and heating scan of 10 ◦C/min were used for the analysis. A Perkin Elmer DSC6 (Waltham, MA, USA) was used for the calorimetric measurements. Weighed samples were encapsulated in aluminum pans and heated to about 40 ◦C above fusion temperature at a rate of 20 ◦C/min (first scan), held there for 3 min, and then quenched to −40 ◦C. Finally, they were reheated from −10 ◦C to a temperature well above the melting at a heating rate of 20 ◦C/min (second scan). A heating rate of 60 ◦C/min was also used. Thermal annealing was performed at 65, 100, 130 and 145 ◦C and consisted of holding each sample for 10 min at the indicated temperature, followed by quenching and a scan collection (20 ◦C/min). Other specific thermal treatments are described in the text. X-ray diffraction patterns of polymeric ﬁlms were performed in the wide angle region by means of a PANalytical X’PertPro diffractometer equipped with a fast X’Celerator detector (Almelo, the Netherlands). The radiation was supplied by a copper target (λ = 0.1548 nm), and 567 points at interval 0.1◦(2θ) were scanned for 100 s each. In situ XRD analysis was performed by using an Anton Paar TTK-450 (Graz, Austria) sample stage. Temperature was increased at 20 ◦C/min, and data collection was carried out as reported in the ﬁgures, by scanning from 10 to 35◦2θ degrees counting 40 s each 0.1◦step (with the fast X’Celerator detector an XRD scan was collected in 40 s). The 1st temperature scan was performed from 23 ◦C up to 145 ◦C. After holding for 3 min at this temperature, the samples were quenched in liquid nitrogen. 2.1. Materials 2.1. Materials TFDCA (97%) was purchased from TCI (Tokyo, Japan). Hexaﬂuoro-2-propanol, chloroform, methanol, BD (99%), titanium tetrabutoxide (TBT, 97%) and titanium isopropoxide (TTIP, 97%) were obtained from Sigma Aldrich (Saint Louis, MO, USA). TBT and TTIP were both distilled before use, while other products and used as received. 3 of 14 Polymers 2018, 10, 167 2.2. Polyester Synthesis PBTF was prepared by two-step melt polycondensation by reacting TFDCA (15.2 g, 0.883 mol) with BD (19.9 g, 0.22 mol) in the presence of 200 ppm/gpolymer of TBT and 200 ppm/gpolymer of TTIP. A 200 mL glass reactor was placed in a silicon oil bath, and the reaction mixture was stirred at 100 rpm by a two-bladed centrifugal stirrer connected to a overhead motor (IKA-Werke GmbH & Co., Staufen, Germany). Nitrogen ﬂow was applied and the temperature was set to 170 ◦C. When more than 90% of the water produced during esteriﬁcation was distilled off (about 3 h), pressure and temperature were gradually reduced to 0.1 mbar and increased to 200 ◦C. Polymerization was stopped when constant torque was measured (4 additional hours). As-synthesized polymer was puriﬁed through dissolution in a mixture hexaﬂuoro-2-propanol/ chloroform and precipitation in methanol. The puriﬁed polymer, in the form of white ﬂoccules, was dried at 30 ◦C under vacuum to constant weight. Thin ﬁlms of about 150 µm thickness were obtained by compression molding using a Carver press. The puriﬁed polymer was melted at 180 ◦C and kept for 2 min at a pressure of 5 tons/m2. Lastly, the ﬁlm was cooled to 23 ◦C in press by tap water. Film thickness was determined by a Sample Thickness Tester DM-G (Brugger Feinmechanik GmbH, Munich, Germany). The reported value represents the mean thickness of three experimental tests, each run on 10 different points of the polymer ﬁlm surface at RT. 2.5. Color Evaluation The color of ﬁlm samples was measured using a HunterLab ColorFlex EZ 45/0◦color spectro-photometer (Reston, VA, USA), with D65 illuminant, 10◦observer (according to ASTM E308). Measurements were made using CIE Lab scale. The instrument was calibrated with a black and white tile before the measurements. Results are expressed as L* (lightness), a* (red/green) and b* (yellow/blue) parameters. The total color difference (∆E) was calculated using the following equation: ∆E = [(∆L)2 + (∆a)2 + (∆b)2]0.5 (1) (1) where ∆L, ∆a and ∆b are the differentials between a sample color parameter (L, a, b) and the color parameter of a standard white plate used as the ﬁlm background (L′ = 66.39, a′ = −0.74, b′ = 1.25). Chromaticity C = [(a)2 + (b)2]0.5 and hue angle hab = tan−1 (b/a) were determined. Measurements were carried out in triplicate at random positions over the ﬁlm surface. 2.6. Gas Permeability Permeability tests were performed by the manometric method using a Permeance Testing Device, type GDP-C (Brugger Feinmechanik GmbH, München, Germany), in accordance with ASTM 1434-82, DIN 53 536 in compliance with ISO/DIS 15 105-1 and to Gas Permeability Testing Manual (Brugger Feinmechanik GmbH). After a preliminary high vacuum desorption of the system, the upper chamber was ﬁlled with the gas test at ambient pressure. A pressure transducer, set in the chamber below the ﬁlm, recorded the increasing of gas pressure as a function of time. The gas transmission rate (GTR, expressed as cm3·cm·m−2·d−1·bar−1) was determined considering the pressure increase in relation to time and volume of the device. All the measurements were carried out by using a gas stream of 100 cm3/min, 0% of gas RH. Food grade O2, CO2 and N2 were used. Permeability was determined at 5, 10, 15, 23, 35, 40, 45 and 50 ◦C. Experiments were performed at least in triplicate, and results are presented as the average ± standard deviation. Method A was used for the analysis, as reported in the literature [16,17]. Sample temperature was set by an external thermostat HAAKE-Circulator DC10-K15 type (Thermo Fisher Scientiﬁc, Waltham, MA, USA). 2.4. Mechanical Properties Tensile measurements were carried out on rectangular ﬁlms (5 mm wide and 0.2 mm thick) with a crosshead speed of 10 mm/min by using a Instron 4465 tensile testing machine (Norwood, MA, USA), equipped with a rubber grip and a 100 N load cell. A preload of 1 MPa was applied to each Polymers 2018, 10, 167 4 of 14 specimen prior to testing. At least ﬁve replicates were run, and the results are presented as the average ± standard deviation. 3. Results and Discussion 3.1. Molecular, Thermal and Structural Characterization 3.1. Molecular, Thermal and Structural Characterization Degradation followed a one-step path (Figure S2) and no residual mass was detected at 750 °C. The first DSC scan of the PBTF film provided evidence of the semicrystalline nature of this polyester (Figure 1A). The glass transition temperature is not clearly detectable, while an endothermic peak at about 50 °C (I) and a more pronounced one at higher temperature (II) have been highlighted (Table 1). The second DSC scan, recorded after melt quenching of the sample, is typical of an amorphous material that crystallizes during heating (Figure 1A). It presents the classic endothermic step ascribable to the glass transition located at 25 °C, an exothermic peak centered at d d h l d The amorphous nature of the material after melt quenching is conﬁrmed as the exothermic (∆Hcc) and endothermic (∆Hm) heats are both equal to 28 J/g (Table 1). 89 °C and an endothermic one located at 150 °C. The amorphous nature of the material after melt quenching is confirmed as the exothermic (ΔHcc) and endothermic (ΔHm) heats are both equal to 28 J/g (Table 1). The amorphous nature of the material after melt quenching is conﬁrmed as the exothermic (∆Hcc) and endothermic (∆Hm) heats are both equal to 28 J/g (Table 1). 89 °C and an endothermic one located at 150 °C. The amorphous nature of the material after melt quenching is confirmed as the exothermic (ΔHcc) and endothermic (ΔHm) heats are both equal to 28 J/g (Table 1). ( ) q J g ( ) Figure 1. DSC curves of PBTF film: (A) I and II scan (20 °C/min); (B) untreated and annealed (10 min at the indicated temperatures) film; (C) different heating rates (20 and 60 °C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 °C. Figure 1. DSC curves of PBTF ﬁlm: (A) I and II scan (20 ◦C/min); (B) untreated and annealed (10 min at the indicated temperatures) ﬁlm; (C) different heating rates (20 and 60 ◦C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 ◦C. Figure 1. DSC curves of PBTF film: (A) I and II scan (20 °C/min); (B) untreated and annealed (10 min at the indicated temperatures) film; (C) different heating rates (20 and 60 °C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 °C. Figure 1. 3.1. Molecular, Thermal and Structural Characterization As synthesized, the polymer appeared as a yellowish hard solid material, while the puriﬁed one was obtained as white ﬂoccules. 1H-NMR analysis conﬁrmed the expected structure (Figure S1) and no impurities were detected in the spectrum. High molecular weight (Mn = 26,500 g/mol) and fairly low polydispersity (PDI = 2.5) were measured by GPC. This result conﬁrms that optimized reaction conditions were achieved, especially when comparing PBTF molecular weight to that reported in the literature for PEF and PET synthesized by two-step melt polycondensation (Mn = 11,200 and 13,700 g/mol, respectivley) [18]. Therefore, melt polycondensation was revealed as a winning strategy for the preparation of highly pure PBTF. It is worth highlighting that the reagents were used as received and that the adopted protocol is very close to industrial procedures for the preparation of polyesters. In addition, by starting from dicarboxylic acid, the esteriﬁcation of TFDCA and the consequent formation of methanol during polymerization were avoided. On the contrary, to obtain high molecular weight furanoate-based polymers, various authors reported the need for methyl-esteriﬁcation of FDCA before polymerization [19–21]. 5 of 14 Polymers 2018, 10, 167 PBTF displayed good thermal stability, higher than that of poly(butylene furanoate) (PBF) [22], up to 391 ◦C. At this temperature the degradation process started and reached its maximum rate at 411 ◦C. Degradation followed a one-step path (Figure S2) and no residual mass was detected at 750 ◦C. Polymers 2018, 10, x FOR PEER REVIEW 5 of 14 PBTF displayed good thermal stability, higher than that of poly(butylene furanoate) (PBF) [22], up to 391 °C At this temperature the degradation process started and reached its maximum rate at 411 °C The ﬁrst DSC scan of the PBTF ﬁlm provided evidence of the semicrystalline nature of this polyester (Figure 1A). The glass transition temperature is not clearly detectable, while an endothermic peak at about 50 ◦C (I) and a more pronounced one at higher temperature (II) have been highlighted (Table 1). The second DSC scan, recorded after melt quenching of the sample, is typical of an amorphous material that crystallizes during heating (Figure 1A). It presents the classic endothermic step ascribable to the glass transition located at 25 ◦C, an exothermic peak centered at 89 ◦C and an endothermic one located at 150 ◦C. to 391 C. At this temperature the degradation process started and reached its maximum rate at 411 C. 3.1. Molecular, Thermal and Structural Characterization DSC curves of PBTF ﬁlm: (A) I and II scan (20 ◦C/min); (B) untreated and annealed (10 min at the indicated temperatures) ﬁlm; (C) different heating rates (20 and 60 ◦C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 ◦C. 6 of 14 Polymers 2018, 10, 167 Table 1. I and II calorimetric scan data of PBTF ﬁlm and stretched ﬁlm. Table 1. I and II calorimetric scan data of PBTF ﬁlm and stretched ﬁlm. I Scan II Scan Sample Tm,I (◦C) ∆Hm,I (J/g) Tm,II (◦C) ∆Hm,II (J/g) Tg (◦C) ∆Cp (J/g◦C) Tcc (◦C) ∆Hcc (J/g) Tm (◦C) ∆Hm (J/g) ﬁlm 51 4 150 30 25 0.287 89 28 150 28 stretched ﬁlm 51 4 149 45 25 0.283 87 25 150 25 To shed light on the nature of the low temperature endothermic peak, annealing treatments were performed, and the calorimetric traces of the so-treated ﬁlm samples are reported in Figure 1B. After annealing, peak I shifted to higher temperature, while the position of peak II was not affected by the thermal treatment. The higher the annealing temperature, the sharper the peak I and the more consistent its temperature shift: from 51 to 78 ◦C, 109 and 140 ◦C, respectively, suggesting an improvement of its associated phase. Moreover, an intense baseline deviation due to the glass transition phenomenon became fully visible in the DSC scans of the annealed samples (Figure 1B), thus allowing for the detection of Tg (at 30 ◦C ca.). In the case of the higher annealing temperature (145 ◦C), the calorimetric trace is characterized by the presence of only one endothermic phenomenon located at a higher temperature with respect to the most intense peak of the other annealed samples (peak II). This result could be explained as being due to: (i) an improvement of the phase corresponding to the endothermic peak located at 150 ◦C or to (ii) the development of a new ordered phase. The effect of the heating rate on the two endothermic peaks was also evaluated. The corresponding calorimetric traces are reported in Figure 1C. A different behavior for the two endotherms can be highlighted: the position of peak I is dependent on the scanning rate, whereas that of peak II is not inﬂuenced. As is well known, the melting phenomenon is a ﬁrst-order transition and, consequently, it does not depend on the heating rate. 3.1. Molecular, Thermal and Structural Characterization On the other hand, the dependence of a transition on the scanning rate is indicative of its second-order nature. Typical second-order transitions are the glass to rubber transitions and the melting of two-dimension ordered phases, like meso-phase. The presence of meso-phase has been already observed in other polyesters, such as PET [23,24] and polylactide (PLA) [25,26], although in these cases it mainly develops upon straining and/or thermal treatment. To better understand the nature of the low temperature endothermic peak present in the DSC trace of PBTF ﬁlm, the reversibility of this transition has been also veriﬁed. In this view, a PBTF ﬁlm sample was subjected to the following thermal treatment (20 ◦C/min): heating step to 65, 100, and 130 ◦C, each followed by quenching to −40 ◦C and heating to the subsequent temperature of 100, 130, and 180 ◦C (Figure 1D). According to the intrinsic low crystallization rate of PBTF homopolymer (as clearly shown in Table 1 and Figure 1A, it can be frozen in its amorphous state by melt quenching), after the adopted thermal treatment, peak I should not appear if associated with a ﬁrst-order transition. On the contrary, it is still evident in the subsequent DSC curve (Figure 1D). In particular, after heating to 100 ◦C (2nd cycle), peak I is visible, yet at a higher temperature. The same behavior was observed after the 3rd cycle (130 ◦C) and the 4th cycle (180 ◦C), with a continuous shift of peak I towards peak II. The observed behavior conﬁrms the reversible character of the transition associated with peak I. X-ray diffraction (XRD) was carried out to clarify the nature of the ordered phases of PBTF. The XRD scanning at RT of PBTF film (Figure 2A-b) shows a low defined pattern: few, broad peaks of low intensity overlap on a bell-shaped intense background. The two most intense peaks are located at 2θ 22.9 and 24.7◦(d = 3.88, 3.60 Å) and possible other peaks are located at 8.7, 15.1, 17.1◦(d = 10.1, 5.8, 5.1 Å). With the aim of improving the diffractometric pattern, PBTF ﬁlm was subjected to tensile tests (this sample is hereafter deﬁned as stretched ﬁlm). Stretching is indeed an efﬁcient tool to induce sample crystallization and, consequently, to improve the quality of the crystalline phase. 3.1. Molecular, Thermal and Structural Characterization It is therefore possible to hypothesize that the reversible and second order low-temperature endothermic peak, characteristic of both PBTF film and stretched film, is related to the meso-phase id d b XRD l i T if th b ti d t t t l d PBTF fil d The presence of meso-PBTF in the ﬁlm is also supported by the calorimetric measurements carried out on the stretched ﬁlm subjected to the same thermal treatments of the ﬁlm. PBTF stretched ﬁlm presented an analogous thermal behavior with respect to the ﬁlm (Figure 3). Figure 2. XRD patterns of PBTF: (A) from bottom to top, stretched film (a), film (b), well crystallized sample (c) obtained by slow cooling after melting; XRD scans performed at RT on untreated and annealed (10 min at the indicated temperatures) film (B) and stretched film (C). The presence of meso PBTF in the film is also supported by the calorimetric measurements evidenced by XRD analysis. To verify the above-mentioned statement, annealed PBTF films and stretched films were subjected to X-ray diffraction analysis. The corresponding XRD patterns are reported in Figure 2B,C, respectively. XRD profiles of annealed film showed a progressive improvement of the ordered phase. In particular, the higher the annealing temperature, the higher the sharpness, intensity and definition of the peaks located at 23.1 d 26 9° (d 3 84 d 3 31 Å) Thi ff i l i d b h i f l ddi i l It is therefore possible to hypothesize that the reversible and second order low-temperature endothermic peak, characteristic of both PBTF ﬁlm and stretched ﬁlm, is related to the meso-phase evidenced by XRD analysis. To verify the above-mentioned statement, annealed PBTF ﬁlms and stretched ﬁlms were subjected to X-ray diffraction analysis. The presence of meso-PBTF in the film is also supported by the calorimetric measurements carried out on the stretched film subjected to the same thermal treatments of the film. PBTF stretched film presented an analogous thermal behavior with respect to the film (Figure 3). It is therefore possible to hypothesize that the reversible and second order low-temperature endothermic peak, characteristic of both PBTF film and stretched film, is related to the meso-phase and 26.9° (d = 3.84 and 3.31 Å). This effect is also accompanied by the increase of several additional reflections at 8.4, 18.2, 20.2, 22.2 and 24.8° (d = 10.5, 4.87, 4.39, 3.99, 3.60 Å) (Figure 2B). 3.1. Molecular, Thermal and Structural Characterization After this treatment, XRD pattern of PBTF stretched ﬁlm (Figure 2A-a) did not look like that of a tridimensional crystalline phase, as it displayed two partially overlapped very broad reﬂections, positioned at 16.7 and 24.2◦(d = 5.3, 3.7 Å). On the contrary, such a proﬁle is typical of a roughly 2D-ordered phase, 7 of 14 Polymers 2018, 10, 167 hereinafter referred to as meso-PBTF. The comparison of ﬁlm and stretched ﬁlm diffraction proﬁles (Figure 2A) suggests the simultaneous presence of a crystalline phase and of a meso-phase in the ﬁlm, this latter being the majority. Moreover, the elongation test performed on the ﬁlm seems to favor the formation of the meso-PBTF phase over the crystalline one. Polymers 2018, 10, x FOR PEER REVIEW 7 of 14 hereinafter referred to as meso-PBTF. The comparison of film and stretched film diffraction profiles (Figure 2A) suggests the simultaneous presence of a crystalline phase and of a meso-phase in the film, this latter being the majority. Moreover, the elongation test performed on the film seems to favor the formation of the meso-PBTF phase over the crystalline one. Polymers 2018, 10, x FOR PEER REVIEW 7 of 14 h f f d h f f l d h d f l d ff f l Figure 2. XRD patterns of PBTF: (A) from bottom to top, stretched film (a), film (b), well crystallized sample (c) obtained by slow cooling after melting; XRD scans performed at RT on untreated and annealed (10 min at the indicated temperatures) film (B) and stretched film (C). The presence of meso PBTF in the film is also supported by the calorimetric measurements Figure 2. XRD patterns of PBTF: (A) from bottom to top, stretched ﬁlm (a), ﬁlm (b), well crystallized sample (c) obtained by slow cooling after melting; XRD scans performed at RT on untreated and annealed (10 min at the indicated temperatures) ﬁlm (B) and stretched ﬁlm (C). p p (Figure 2A) suggests the simultaneous presence of a crystalline phase and of a meso-phase in the film, this latter being the majority. Moreover, the elongation test performed on the film seems to favor the formation of the meso-PBTF phase over the crystalline one. p ase and of a meso-phase in the fil med on the film seems to favor t p us presence of a crystalline ph ver, the elongation test perfor ver the crystalline one. 3.1. Molecular, Thermal and Structural Characterization ure 2A) suggests the simultane latter being the majority. More mation of the meso-PBTF phase Figure 2. XRD patterns of PBTF: (A) from bottom to top, stretched film (a), film (b), well crystallized sample (c) obtained by slow cooling after melting; XRD scans performed at RT on untreated and annealed (10 min at the indicated temperatures) film (B) and stretched film (C). Th f PBTF i th fil i l t d b th l i t i t Figure 2. XRD patterns of PBTF: (A) from bottom to top, stretched ﬁlm (a), ﬁlm (b), well crystallized sample (c) obtained by slow cooling after melting; XRD scans performed at RT on untreated and annealed (10 min at the indicated temperatures) ﬁlm (B) and stretched ﬁlm (C). carried out on the stretched film subjected to the same thermal treatments of the film. PBTF stretched film presented an analogous thermal behavior with respect to the film (Figure 3). It is therefore possible to hypothesize that the reversible and second order low-temperature endothermic peak, characteristic of both PBTF film and stretched film, is related to the meso-phase id d b XRD l i T if th b ti d t t t l d PBTF fil d The presence of meso-PBTF in the ﬁlm is also supported by the calorimetric measurements carried out on the stretched ﬁlm subjected to the same thermal treatments of the ﬁlm. PBTF stretched ﬁlm presented an analogous thermal behavior with respect to the ﬁlm (Figure 3). Figure 2. XRD patterns of PBTF: (A) from bottom to top, stretched film (a), film (b), well crystallized sample (c) obtained by slow cooling after melting; XRD scans performed at RT on untreated and annealed (10 min at the indicated temperatures) film (B) and stretched film (C). The presence of meso PBTF in the film is also supported by the calorimetric measurements carried out on the stretched film subjected to the same thermal treatments of the film. PBTF stretched film presented an analogous thermal behavior with respect to the film (Figure 3). 3.1. Molecular, Thermal and Structural Characterization The same trend characterizes the XRD profiles of annealed PBTF stretched films (Figure 2C). In this case, it is worth noting that the meso-phase underwent a continuous perfection as the temperature of the annealing treatment increased, the two peaks at 2θ 16.7 and 24.2° becoming sharper and more intense. Such improvement of the meso-phase occurred up to 100 °C. The corresponding XRD patterns are reported in Figure 2B,C, respectively. XRD proﬁles of annealed ﬁlm showed a progressive improvement of the ordered phase. In particular, the higher the annealing temperature, the higher the sharpness, intensity and deﬁnition of the peaks located at 23.1 and 26.9◦(d = 3.84 and 3.31 Å). This effect is also accompanied by the increase of several additional reﬂections at 8.4, 18.2, 20.2, 22.2 and 24.8◦(d = 10.5, 4.87, 4.39, 3.99, 3.60 Å) (Figure 2B). evidenced by XRD analysis. To verify the above-mentioned statement, annealed PBTF films and stretched films were subjected to X-ray diffraction analysis. The corresponding XRD patterns are reported in Figure 2B,C, respectively. XRD profiles of annealed film showed a progressive improvement of the ordered phase. In particular, the higher the annealing temperature, the higher the sharpness, intensity and definition of the peaks located at 23.1 and 26.9° (d = 3.84 and 3.31 Å). This effect is also accompanied by the increase of several additional The same trend characterizes the XRD proﬁles of annealed PBTF stretched ﬁlms (Figure 2C). In this case, it is worth noting that the meso-phase underwent a continuous perfection as the temperature of the annealing treatment increased, the two peaks at 2θ 16.7 and 24.2◦becoming sharper and more intense. Such improvement of the meso-phase occurred up to 100 ◦C. p y reflections at 8.4, 18.2, 20.2, 22.2 and 24.8° (d = 10.5, 4.87, 4.39, 3.99, 3.60 Å) (Figure 2B). The same trend characterizes the XRD profiles of annealed PBTF stretched films (Figure 2C). In this case, it is worth noting that the meso-phase underwent a continuous perfection as the temperature of the annealing treatment increased, the two peaks at 2θ 16.7 and 24.2° becoming sharper and more intense. Such improvement of the meso-phase occurred up to 100 °C. Figure 3. Cont. Figure 3. Cont. 8 of 14 Polymers 2018, 10, 167 Figure 3. 3.1. Molecular, Thermal and Structural Characterization DSC curves of PBTF stretched film: (A) I and II scan (20 °C/min); (B) untreated and annealed (10 min at the indicated temperatures) stretched film; (C) different heating rates (20 and 60 °C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 °C. On the other hand a decrease of the signal at 16 7° associated with the meso phase together Figure 3. DSC curves of PBTF stretched ﬁlm: (A) I and II scan (20 ◦C/min); (B) untreated and annealed (10 min at the indicated temperatures) stretched ﬁlm; (C) different heating rates (20 and 60 ◦C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 ◦C. Polymers 2018, 10, x FOR PEER REVIEW 8 of 14 Figure 3. DSC curves of PBTF stretched film: (A) I and II scan (20 °C/min); (B) untreated and annealed (10 min at the indicated temperatures) stretched film; (C) different heating rates (20 and 60 °C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65 100 130 and 180 °C EER REVIEW Figure 3. DSC curves of PBTF stretched film: (A) I and II scan (20 °C/min); (B) untreated and annealed (10 min at the indicated temperatures) stretched film; (C) different heating rates (20 and 60 °C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 °C. Figure 3. DSC curves of PBTF stretched ﬁlm: (A) I and II scan (20 ◦C/min); (B) untreated and annealed (10 min at the indicated temperatures) stretched ﬁlm; (C) different heating rates (20 and 60 ◦C/min, dotted lines represent eye guides); (D) consecutive heating steps to 65, 100, 130 and 180 ◦C. Figure 3. DSC curves of PBTF stretched film: (A) I and II scan (20 °C/min); (B) untreated and annealed (10 min at the indicated temperatures) stretched film; (C) different heating rates (20 and 60 °C/min, d d li id ) (D) i h i 65 100 130 d 180 °C with the appearance of a shoulder at 26.6°, related to the crystal phase, was observed when an annealing temperature of 130 °C was used. Finally, stretched film annealed at 145° showed a profile typical of a semicrystalline polymer with the two main sharp peaks at 23.1 and 26.7°. 3.1. Molecular, Thermal and Structural Characterization These two peaks are also present in the XRD pattern of a well crystallized sample of PBTF, obtained by slow cooling after melting (Figure 2A-c), which is characterized by two major peaks at 23.1° and 26.8° (d = 3.84 and 3.32 Å) and several additional reflections at 8.4, 18.2, 20.2, 22.2 and 24.8° (d = 10.5, 4.87, 4.39, 3.99, 3.60 Å). Hereinafter, this phase is named α-PBTF. The stability of the crystalline phase of the film was investigated by in situ XRD temperature scans (Figure 4A). On the other hand, a decrease of the signal at 16.7◦, associated with the meso-phase, together with the appearance of a shoulder at 26.6◦, related to the crystal phase, was observed when an annealing temperature of 130 ◦C was used. Finally, stretched ﬁlm annealed at 145◦showed a proﬁle typical of a semicrystalline polymer with the two main sharp peaks at 23.1 and 26.7◦. These two peaks are also present in the XRD pattern of a well crystallized sample of PBTF, obtained by slow cooling after melting (Figure 2A-c), which is characterized by two major peaks at 23.1 and 26.8◦(d = 3.84 and 3.32 Å) and several additional reﬂections at 8.4, 18.2, 20.2, 22.2 and 24.8◦(d = 10.5, 4.87, 4.39, 3.99, 3.60 Å). Hereinafter, this phase is named α-PBTF. On the other hand, a decrease of the signal at 16.7°, associated with the meso-phase, together with the appearance of a shoulder at 26.6°, related to the crystal phase, was observed when an annealing temperature of 130 °C was used. Finally, stretched film annealed at 145° showed a profile typical of a semicrystalline polymer with the two main sharp peaks at 23.1 and 26.7°. These two peaks are also present in the XRD pattern of a well crystallized sample of PBTF, obtained by slow cooling after melting (Figure 2A-c), which is characterized by two major peaks at 23.1° and 26.8° (d = 3.84 and 3.32 Å) and several additional reflections at 8.4, 18.2, 20.2, 22.2 and 24.8° (d = 10.5, 4.87, 4.39, 3.99, 3.60 Å). Hereinafter, this phase is named α-PBTF. The stability of the crystalline phase of the film was investigated by in situ XRD temperature The stability of the crystalline phase of the ﬁlm was investigated by in situ XRD temperature scans (Figure 4A). The stability of the crystalline phase of the film was investigated by in situ XRD temperature scans (Figure 4A). 3.1. Molecular, Thermal and Structural Characterization Figure 4. In situ XRD patterns collected at different temperatures (I scan and II scan after quenching from the melt) for PBTF film (A) and stretched film (B). In the first scan, a significant enhancement of peak intensities occurred as temperature increased, up to melting. Since the patterns show an evolution towards the full profile characteristic of the α crystal phase, it can be stated that the low intensity and broad reflections detected in the untreated film belong to this phase. After melt quenching, during the 2nd thermal scan, the sample, initially Figure 4. In situ XRD patterns collected at different temperatures (I scan and II scan after quenching from the melt) for PBTF film (A) and stretched film (B). Figure 4. In situ XRD patterns collected at different temperatures (I scan and II scan after quenching from the melt) for PBTF ﬁlm (A) and stretched ﬁlm (B). 4. In situ XRD patterns collected at differen e melt) for PBTF film (A) and stretched film f h f emperatures (I scan and II scan after qu B). k d In the first scan, a significant enhancement of peak intensities occurred as temperature increased, up to melting. Since the patterns show an evolution towards the full profile characteristic of the α crystal phase, it can be stated that the low intensity and broad reflections detected in the untreated film belong to this phase After melt quenching during the 2nd thermal scan the sample initially Figure 4. In situ XRD patterns collected at different temperatures (I scan and II scan after quenching from the melt) for PBTF film (A) and stretched film (B). Figure 4. In situ XRD patterns collected at different temperatures (I scan and II scan after quenching from the melt) for PBTF ﬁlm (A) and stretched ﬁlm (B). amorphous, crystallized arranging in the α-PBTF lattice. In situ XRD temperature scans were also In the first scan, a significant enhancement of peak intensities occurred as temperature increased, up to melting. Since the patterns show an evolution towards the full profile characteristic of the α crystal phase, it can be stated that the low intensity and broad reflections detected in the untreated film belong to this phase. After melt quenching, during the 2nd thermal scan, the sample, initially amorphous, crystallized arranging in the α-PBTF lattice. 3.2. Mechanical Characterization and Color Evaluation 3.2. Mechanical Characterization and Color Evaluation Film transparency and color is very important especially if food packaging application is Such diverse properties for polymers with very similar chemical structure can be mainly ascribed to the different Tgs and to the peculiar structural arrangement of PBTF polymer chains. Since tensile tests were carried out at room temperature, therefore below Tg for PBF (i.e., 35–40 ◦C) [30] and PEF (i.e., 77 ◦C) [19] and around Tg for PBTF, the polymers are respectively in the glassy (PBF and PEF) and in the rubbery state (PBTF). Moreover, as mentioned above, PBTF is characterized by the presence of the meso-phase that constitutes the majority of the ordered domains, thus lowering the overall degree of crystallinity. Film transparency and color is very important, especially if food packaging application is envisioned. Given the high amount of pigments present in food, its color has been always considered one of the key factors used to evaluate quality and taste by the final consumer. Therefore, packaging should interfere as little as possible with the color of the product, not to cause a decrease in attractiveness. The results of the PBTF film surface color determination is reported in Table S1 as compared to a white standard. An explanation of the significance of the various parameters is also provided in SI. PBTF shows an L* close to white, while a* and b* indicate a faint tendency toward a yellowish color (hab over 90°), as due to the presence of sulphur atoms along the polymer backbone. A very low C* has been recorded, meaning low color saturation and thus a good transparency of the film. Film transparency and color is very important, especially if food packaging application is envisioned. Given the high amount of pigments present in food, its color has been always considered one of the key factors used to evaluate quality and taste by the ﬁnal consumer. Therefore, packaging should interfere as little as possible with the color of the product, not to cause a decrease in attractiveness. The results of the PBTF ﬁlm surface color determination is reported in Table S1 as compared to a white standard. An explanation of the signiﬁcance of the various parameters is also provided in SI. PBTF shows an L* close to white, while a* and b* indicate a faint tendency toward a yellowish color (hab over 90◦), as due to the presence of sulphur atoms along the polymer backbone. 3.2. Mechanical Characterization and Color Evaluation 3.2. Mechanical Characterization and Color Evaluation The mechanical behavior of PBTF, evaluated by tensile measurements on thin ﬁlms, is presented in Figure 5. As can be seen, PBTF ﬁlm displays high elongation (εb = 555% ± 50%) and stress at break (σb = 24.5 ± 0.5 MPa). In addition, no necking was recorded at the yield point, and the elastic modulus is equal to 89 ± 7 MPa. Thus, mechanical characteristics of PBTF are quite peculiar, especially as compared to that of its furanoate-based counterparts, i.e., PEF and PBF. PBTF exhibits a hard and tough behavior making it very interesting, e.g., for ﬂexible packaging applications. On the contrary, PBF shows yield and high elastic modulus, above 800 MPa [22,28–30]. Similar characteristics, i.e., very high elastic modulus and brittle fracture, have been highlighted for PEF [19,31]. The mechanical behavior of PBTF, evaluated by tensile measurements on thin films, is presented in Figure 5. As can be seen, PBTF film displays high elongation (εb = 555% ± 50%) and stress at break (σb = 24.5 ± 0.5 MPa). In addition, no necking was recorded at the yield point, and the elastic modulus is equal to 89 ± 7 MPa. Thus, mechanical characteristics of PBTF are quite peculiar, especially as compared to that of its furanoate-based counterparts, i.e., PEF and PBF. PBTF exhibits a hard and tough behavior making it very interesting, e.g., for flexible packaging applications. On the contrary, PBF shows yield and high elastic modulus, above 800 MPa [22,28–30]. Similar characteristics, i.e., very high elastic modulus and brittle fracture, have been highlighted for PEF [19,31]. Figure 5. Representative stress-strain curve. Figure 5. Representative stress-strain curve. Figure 5. Representative stress-strain curve. Figure 5. Representative stress-strain curve. Such diverse properties for polymers with very similar chemical structure can be mainly ascribed to the different Tgs and to the peculiar structural arrangement of PBTF polymer chains. Since tensile tests were carried out at room temperature, therefore below Tg for PBF (i.e., 35–40 °C) [30] and PEF (i.e., 77 °C) [19] and around Tg for PBTF, the polymers are respectively in the glassy (PBF and PEF) and in the rubbery state (PBTF). Moreover, as mentioned above, PBTF is characterized by the presence of the meso-phase that constitutes the majority of the ordered domains, thus lowering the overall degree of crystallinity. 3.1. Molecular, Thermal and Structural Characterization In situ XRD temperature scans were also In the ﬁrst scan, a signiﬁcant enhancement of peak intensities occurred as temperature increased, up to melting. Since the patterns show an evolution towards the full proﬁle characteristic of the α crystal phase, it can be stated that the low intensity and broad reﬂections detected in the untreated ﬁlm belong to this phase. After melt quenching, during the 2nd thermal scan, the sample, initially amorphous, crystallized arranging in the α-PBTF lattice. In situ XRD temperature scans were also carried out on PBTF stretched ﬁlm. The results (Figure 4B) show that meso-phase peaks became sharper and more intense as the temperature increased, up to 100 ◦C, then at 140 ◦C a small worsening occurred. Polymers 2018, 10, 167 carried out on PB sharper and more 9 of 14 ecame sening At 145 ◦C, a temperature very close to the melting point, signiﬁcant changes of the in situ XRD pattern occurred, with a shift toward a proﬁle reminiscent of a low crystalline α-PBTF. p y g p g g XRD pattern occurred, with a shift toward a profile reminiscent of a low crystalline α-PBTF. The results of both the annealing treatment (Figure 2B) and of the in situ XRD temperature The results of both the annealing treatment (Figure 2B) and of the in situ XRD temperature measurements (Figure 4A) of the ﬁlm samples conﬁrm that the meso-phase covers the largest fraction of the ordered phase. By proper thermal treatments, the fraction of the α-crystal phase can be increased, through the conversion of meso-phase into α-crystal phase, as already observed for poly(L-lactide) [27]. measurements (Figure 4A) of the film samples confirm that the meso-phase covers the largest fraction of the ordered phase. By proper thermal treatments, the fraction of the α-crystal phase can be increased, through the conversion of meso-phase into α-crystal phase, as already observed for poly(L- lactide) [27]. 3.3. Gas Permeability In particular, given the near-ambient temperature Tg of PBTF (i.e., 25 °C), two different behaviors can be outlined: below Tg and above Tg. In addition, although very close to Tg, GTR was also measured at 23 °C since it is a very common temperature used for barrier properties evaluation. A sudden increase in permeability was recorded at this temperature, while a lower dependence A sudden increase in permeability was recorded at this temperature, while a lower depe on T can be observed both in the range 5–15 ◦C and 35–45 ◦C. behaviors can be outlined: below Tg and above Tg. In addition, although very close to Tg, GTR was a measured at 23 °C since it is a very common temperature used for barrier properties evaluation. A sudden increase in permeability was recorded at this temperature, while a lower dependen Below Tg, the chain segments have little mobility; therefore, gas molecules must follow a more tortuous path to diffuse through the polymer matrix, also because of the reduction in free volume. Thus, GTR in the range 5–15 ◦C is lower than at 23 ◦C or above. p y p p on T can be observed both in the range 5–15 °C and 35–45 °C. Below Tg, the chain segments have little mobility; therefore, gas molecules must follow a more tortuous path to diffuse through the polymer matrix, also because of the reduction in free volume. Th GTR i h 5 15 °C i l h 23 °C b It is worth noticing that the barrier performances of PBTF remain very high in all the explored ranges of temperature, both below and above Tg, thus broadening its range of possible operating conditions. This result can be ascribed to the presence, together with the crystalline domains, of the meso-phase that acts as an additional obstacle for the diffusion of the gas molecules, because of the dense packing of the macromolecular chains in this 2D-ordered phase. Thus, GTR in the range 5–15 °C is lower than at 23 °C or above. It is worth noticing that the barrier performances of PBTF remain very high in all the explored ranges of temperature, both below and above Tg, thus broadening its range of possible operating conditions. 3.3. Gas Permeability As reported in the literature [32], several factors affect ﬁlm gas permeability, such as molecular structure, chain stiffness, molecular symmetry, degree of order, crystallinity and orientation, presence of cross-linking, glass transition temperature. It is well known that gas transmission through a polymeric material is linked to the tortuosity induced by impermeable domains, like the crystalline phase, within the polymer matrix. Polymers 2018, 10, x FOR PEER REVIEW 10 of 14 3.3. Gas Permeability As reported in the literature [32], several factors affect film gas permeability, such as molecular structure, chain stiffness, molecular symmetry, degree of order, crystallinity and orientation, presence of cross-linking, glass transition temperature. It is well known that gas transmission through To evaluate the diffusion of small molecules through PBTF ﬁlm, barrier properties have been investigated using N2, O2 and CO2 gas, due to their different van der Waals molar volume and inertness towards organic polymers. The collected data are reported in Figure 6A and Table S2 as gas transmission rate (GTR). p ese ce o c oss i i g, g ass a si io e pe a u e I is e o a gas a s issio oug a polymeric material is linked to the tortuosity induced by impermeable domains, like the crystalline phase, within the polymer matrix. To evaluate the diffusion of small molecules through PBTF film, barrier properties have been investigated using N2, O2 and CO2 gas, due to their different van der Waals molar volume and d l Th ll d d d F 6A d T bl S As expected, GTR depends on the temperature and, more speciﬁcally, it increases by enhancing the temperature. In particular, given the near-ambient temperature Tg of PBTF (i.e., 25 ◦C), two different behaviors can be outlined: below Tg and above Tg. In addition, although very close to Tg, GTR was also measured at 23 ◦C since it is a very common temperature used for barrier properties evaluation. A sudden increase in permeability was recorded at this temperature, while a lower dependence on T can be observed both in the range 5–15 ◦C and 35–45 ◦C. inertness towards organic polymers. The collected data are reported in Figure 6A and Table S2 as gas transmission rate (GTR). As expected, GTR depends on the temperature and, more specifically, it increases by enhancing the temperature. 3.2. Mechanical Characterization and Color Evaluation 3.2. Mechanical Characterization and Color Evaluation A very low C* has been recorded, meaning low color saturation and thus a good transparency of the ﬁlm. 10 of 14 Polymers 2018, 10, 167 10 of 14 3.3. Gas Permeability This result can be ascribed to the presence, together with the crystalline domains, of the meso-phase that acts as an additional obstacle for the diffusion of the gas molecules, because of the dense packing of the macromolecular chains in this 2D-ordered phase. Figure 6. Gas transmission rate (A) and Arrhenius plot of GTR (B) in the range 5–45 °C for CO2, O2, and N2. Figure 6. Gas transmission rate (A) and Arrhenius plot of GTR (B) in the range 5–45 ◦C for CO2, O2, and N2. Figure 6. Gas transmission rate (A) and Arrhenius plot of GTR (B) in the range 5–45 °C for CO2, O2, and N2. Figure 6. Gas transmission rate (A) and Arrhenius plot of GTR (B) in the range 5–45 ◦C for CO2, O2, and N2. Polymers 2018, 10, 167 Polymers 2018, 10, 167 11 of 14 Perm-selectivity ratios (Table S2) are not constant throughout all the temperature range and rather show a certain dependence on the measuring temperature, conﬁrming that this parameter is correlated not only with the chemical structure of the material, but also with T [33]. When compared to the barrier properties of other polyesters (Table S3), the values found for PBTF acquire much more value. Indeed, O2 and CO2 permeabilities are well below those of PLA, PEF and amorphous PET and comparable to those already excellent of PPF [34–37]. For temperatures where no transitions in polymers (e.g., glass transition) and in permeants (e.g., boiling point) are detected, the permeation dependence on the temperature can be described through the Arrhenius model [34]. A linear correlation between the logarithm of a transport parameter and the reciprocal of the absolute temperature exists: P = P0exp (−Ep/RT) (2) (2) where P is the gas permeability (GTR), P0 is a pre-exponential factor of permeation, Ep is the activation energy for permeation and R is the gas constant. As mentioned above, gas permeability changes across polymer Tg. Ep can therefore be greater or smaller above Tg than below Tg [38]. Ep was then calculated in the two following ranges of temperature: 5–15 ◦C (below Tg) and 35–45 ◦C (above Tg). Figure 6B reports the GTR dependence of the studied gases on the temperature according to Equation (2). From the linear ﬁtting of the experimental data (dashed lines) the activation energies have been calculated and reported in Table 2. 3.3. Gas Permeability Experimental data well ﬁt the theoretical behavior, thus indicating a good correlation between permeability and temperature for all gases. Table 2. Activation energy of the gas transmission rate process for O2, N2 and CO2. In brackets: R2 coefﬁcient. Table 2. Activation energy of the gas transmission rate process for O2, N2 and CO2. In brackets: R2 coefﬁcient. Temperature range (◦C) Ep (kJ/mol) O2 N2 CO2 5–15 8.91 (0.98) 10.65 (0.98) 3.73 (0.99) 35–45 2.68 (0.89) 4.21 (0.93) 2.25 (0.99) Table 2. Activation energy of the gas transmission rate process for O2, N2 and CO2. In brackets: R2 coefﬁcient. Temperature range (◦C) Ep (kJ/mol) O2 N2 CO2 5–15 8.91 (0.98) 10.65 (0.98) 3.73 (0.99) 35–45 2.68 (0.89) 4.21 (0.93) 2.25 (0.99) Ep values vary for all gases by changing the temperature range from below to above Tg. However, different trends can be highlighted. Ep for O2 and N2 follows a similar path: a higher Ep is observed below Tg, while in the range 35–45 ◦C a lower dependence of GTR on the temperature has been detected (lower values of Ep). On the contrary, for CO2, Ep remains similar both in the range above Tg and below Tg. g However, in both temperature ranges, a clear dependence on the permeant size can be observed, as Ep decreases with the increase of the permeant size in the following order: CO2 < O2 < N2, as already observed in the literature [39]. Furthermore, above Tg, the activation energies of the permeation process for the three gases are less inﬂuenced by the permeant size, probably because of the enhanced mobility of the macromolecular chains that allows for an easier crossing of the gas molecules. Although further investigations are necessary for a comprehensive characterization of the gas permeability behavior, the results presented provide meaningful evidence for the potential of PBTF as a high gas barrier polymeric ﬁlm. 4. Conclusions Poly(butylene 2,5-thiophene dicarboxylate), an aromatic polyester derived from renewable resources, has been successfully synthesized through melt polycondensation. PBTF shows good thermal properties, i.e., stability above 390 ◦C, glass transition at around room temperature (25 ◦C) and melting point of 150 ◦C, coupled with an intriguing mechanical behavior. The absence of yielding Polymers 2018, 10, 167 12 of 14 point, elongation at break above 500% and stress at break of 25 MPa, together with an elastic modulus of about 90 MPa, have been indeed demonstrated by tensile testing. point, elongation at break above 500% and stress at break of 25 MPa, together with an elastic modulus of about 90 MPa, have been indeed demonstrated by tensile testing. In addition, with respect to PEF, PBTF displays a reduction of permeability to O2 and CO2 of 2.0× and 4.75×, respectively [34,35]. These exceptional barrier properties are maintained both above and below Tg, owing to the peculiar structural arrangement that allows for the formation of a not-induced 2D-ordered phase, i.e., meso-phase, and of 3D-crystalline domains, named α-PBTF. The former, together with the effect provided by the crystalline regions, causes a dense packing of PBTF macromolecules, thus hampering the gas permeation. g p Calorimetric and diffractometric studies evidenced that the meso-phase is highly stable, up to 140 ◦C, although by annealing it is possible to increase the amount of α-phase, by inducing a conversion of the meso-phase into α-PBTF. Furthermore, the α-crystal phase is the most thermodynamically stable since, after melt quenching, it is the only one that forms. Thanks to the combination of all the above-mentioned characteristics, PBTF establish itself as a very important member of the biobased polyester family, opening up new possibilities in sustainable packaging, particularly for ﬂexible ﬁlms purposes. Supplementary Materials: The following are available online at www.mdpi.com/2073-4360/10/2/167/s1, Figure S1: 1H-NMR spectrum of PBTF with resonance assignments, Figure S2. Thermogravimetric curve (solid line) under nitrogen ﬂow of PBTF and its derivative (dashed line), Table S1: L, a, b, total color difference (∆E), C and hab of PBFT ﬁlm (ﬁlm color determination data), Table S2: Gas transmission rate (GTR) of N2, O2 and CO2 in the range 5–45 ◦C and perm-selectivity ratios for PBTF ﬁlm, Table S3: Gas transmission rate (GTR) of PBTF with respect to other polyesters. 4. Conclusions Author Contributions: Matteo Gigli and Nadia Lotti conceived and designed the experiments; Giulia Guidotti, Michelina Soccio, Valentina Siracusa and Massimo Gazzano performed the experiments; all the authors analyzed the data; Matteo Gigli and Nadia Lotti wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. PlasticsEurope—Plastics the Fact, Brussels, Belgium. 2016. Available online: www.plasticseurope.org (accessed on 15 July 2017). 1. 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https://openalex.org/W4396659381	https://www.frontiersin.org/journals/psychology/articles/10.3389/fpsyg.2024.1418051/pdf	English	null	Editorial: Multidisciplinary approach in health: new strategies from the perspective of education, management, culture and gender	Frontiers in psychology	2,024	cc-by	1,779	TYPE Editorial PUBLISHED 06 May 2024 DOI 10.3389/fpsyg.2024.1418051 TYPE Editorial PUBLISHED 06 May 2024 DOI 10.3389/fpsyg.2024.1418051 KEYWORDS COPYRIGHT © 2024 Gomez-Cantarino, Ugarte-Gurrutxaga, Solano-Ruiz and Oliveira Xavier. 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. education, management, culture, health, gender, sexuality, health habits frontiersin.org CITATION Gomez-Cantarino S, Ugarte-Gurrutxaga MI, Solano-Ruiz C and Oliveira Xavier Bd (2024) Editorial: Multidisciplinary approach in health: new strategies from the perspective of education, management, culture and gender. Front. Psychol. 15:1418051. d i 10 3389/f 2024 1418051 1Department of Nursing, Physiotherapy and Occupational Therapy, Faculty of Physiotherapy and Nursing, University of Castilla-La Mancha, Toledo, Spain, 2Department of Nursing, Faculty of Nursing, University of Alicante, Alicante, Spain, 3Nursing School of Coimbra, Coimbra, Portugal OPEN ACCESS EDITED AND REVIEWED BY Kath Woodward, The Open University, United Kingdom CORRESPONDENCE M. Idoia Ugarte-Gurrutxaga maria.ugarte@uclm.es RECEIVED 15 April 2024 ACCEPTED 22 April 2024 06 M 2024 EDITED AND REVIEWED BY Kath Woodward, The Open University, United Kingdom CORRESPONDENCE M. Idoia Ugarte-Gurrutxaga maria.ugarte@uclm.es RECEIVED 15 April 2024 ACCEPTED 22 April 2024 PUBLISHED 06 May 2024 CITATION Gomez-Cantarino S, Ugarte-Gurrutxaga MI, Solano-Ruiz C and Oliveira Xavier Bd (2024) Editorial: Multidisciplinary approach in health: new strategies from the perspective of education, management, culture and gender. Front. Psychol. 15:1418051. doi: 10.3389/fpsyg.2024.1418051 EDITED AND REVIEWED BY Kath Woodward, The Open University, United Kingdom CORRESPONDENCE M. Idoia Ugarte-Gurrutxaga maria.ugarte@uclm.es RECEIVED 15 April 2024 ACCEPTED 22 April 2024 PUBLISHED 06 May 2024 CITATION Gomez-Cantarino S, Ugarte-Gurrutxaga MI, Solano-Ruiz C and Oliveira Xavier Bd (2024) Editorial: Multidisciplinary approach in health: new strategies from the perspective of education, management, culture and gender. Front. Psychol. 15:1418051. doi: 10.3389/fpsyg.2024.1418051 Sagrario Gomez-Cantarino1, M. Idoia Ugarte-Gurrutxaga1, Carmen Solano-Ruiz2 and Beatriz de Oliveira Xavier3 Frontiers in Psychology sexual en el aula de clases. Sinergias educativas 5, 391–406. doi: 10.37954/se. v5i2.149 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. Funding The author(s) declare that no ﬁnancial support was received for the research, authorship, and/or publication of this article. Editorial on the Research Topic It is concluded that sexuality constitutes a crucial educational issue requiring further exploration to overcome barriers to its addressing, with the aim of improving the preparation and competence of future healthcare professionals. The discrimination, stigma, and lack of access to culturally competent health services, including mental health, can exacerbate the challenges faced by LGBTQ+ individuals in terms of emotional and psychological wellbeing. Thus, to address these disparities, both students and healthcare professionals must recognize and address the speciﬁc mental health needs of LGBTQ+ individuals, creating safe and prejudice-free environments where they can express their concerns and receive support. This situation is addressed in: Gender and sexuality in mental health: perspectives on the rights and mental health of lesbians, gays, bisexuals, and transgender people (LGBT) in the ASEAN region (Alibudbud). Training in cultural competence and gender sensitivity is emphasized to ensure inclusive and respectful care. It is also important to promote policies that protect LGBTQ+ rights, such as laws that promote anti-discrimination and equal access to healthcare (Ugarte-Gurrutxaga, 2020). Alzain et al. recognize the value of volunteering in personal and professional development. Thus, it is evaluated how this experience fosters competencies such as intercultural communication, teamwork, and problem-solving, as well as speciﬁc technical skills in healthcare specialties. The authors Santiago et al. present a multicenter study investigating nursing students’ knowledge about sexuality, sex, and gender diversity. It is imperative that nursing curricula integrate a deep understanding of sexuality and its diversity. This will ensure adequate preparation to comprehensively and sensitively address sexual health needs. Acosta-Leal, D., Ponce-Martínez, E., and González-Martínez, C. (2020). La educación superior como escenario para la inclusión de la diversidad Author contributions SG-C: Conceptualization, Formal analysis, Investigation, Project administration, Validation, Visualization, Writing – original draft. MU-G: Conceptualization, Formal analysis, Investigation, Project administration, Visualization, Writing – review & editing. CS-R: Data curation, Methodology, Supervision, Writing – original draft. BO: Resources, Visualization, Writing – review & editing. It is important to foster critical thinking, ethical reﬂection, and a commitment to continuous improvement in higher education within the health sciences. Indeed, self-esteem and professional identity are crucial elements for healthcare professionals and students. In the literature, a study reveals that perceived prejudice and psychological distress can inﬂuence these aspects (Wu et al.). Stereotypes and perceived discrimination in the workplace that can undermine self-esteem and professional conﬁdence, negatively impacting the quality of care provided, are shown. It is essential to address these challenges to promote an inclusive and healthy work environment where healthcare professionals develop a strong identity and maintain optimal mental health to provide quality care. It is also worth noting that historically, as documented by Espina-Jerez et al., early female predecessors of modern nursing faced socio-cultural diﬃculties solely because of their gender due to their scientiﬁc activities such as herbalism and healing, among others, being condemned and even imprisoned for these practices. Another issue related to training in health sciences is observed in the study presented by Shiningayamwe, which addresses the implementation of educational policies in Namibia and student pregnancies in rural schools. In fact, these authors propose essential ideas to reduce pregnancies and school dropout rates, reinforcing formal education. It is recognized that sex education leads to changes in behaviors and norms, as expressed by Gradellini et al., where perspectives such as students’ educational level, prior knowledge, and possible reactions to sexual topics are analyzed, without forgetting religious and cultural inﬂuences. This issue is raised and continued in the literature with a question: Purity 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. Editorial on the Research Topic Multidisciplinary approach in health: new strategies from the perspective of education, management, culture and gender The training within the European Higher Education Area must promote equality and inclusion in both academic and social spheres, fostering respect toward cultural diversity and sexual orientation, and ensuring non-discriminatory access to education, while integrating a gender perspective across all university areas. From this standpoint, within the ﬁeld of Health Sciences, there is a need for training in both knowledge and skills that facilitate the development of cultural competence (Berenguel Chacón et al., 2023) and education in aﬀective-sexual matters (Cantarino et al., 2016). However, do educational programs include comprehensive sexual education? Scientiﬁc literature paints a rather discouraging picture in this regard, revealing signiﬁcant deﬁciencies that need to be addressed (Cunha-Oliveira et al., 2021). There is also evidence of inadequate training in providing healthcare in culturally diverse contexts (Shariﬁet al., 2019). It is necessary to enhance new educational approaches diﬀerent from conventional ones, where the focus is not only on conceptual competencies related to the discipline but also on the development of attitudes free from biases and prejudices, and behaviors that do not discriminate against people based on their cultural background or sexual orientation (Acosta-Leal et al., 2020). Undoubtedly, the educational project will be enriched through interdisciplinary collaboration among healthcare professionals (social workers, psychologists, educators, and public health experts). In this regard, there is a proposal that examines the interaction of sex and gender psychological roles on symptoms of stress, scatter, and anxiety: Sex and gender role diﬀerences in stress, depression, and anxiety symptoms in response to the COVID-19 pandemic over time (Arcand et al.), which states that sexual and psychological gender diﬀerences contribute to heterogeneous patterns of stress and anxiety symptoms over time in response to the COVID-19 pandemic. However, training in health sciences Frontiers in Psychology 01 frontiersin.org 10.3389/fpsyg.2024.1418051 Gomez-Cantarino et al. a should include experiential learning opportunities in diverse rotations and ﬁeldwork, allowing students to apply theoretical knowledge in real-life situations and develop empathy and teamwork skills. Therefore, mental health and healthcare are fundamental aspects that must be addressed comprehensively in the context of gender and healthcare. or perversion? From taboo to fact: reﬂections of kindergarten teachers on age-appropriate sexuality (Lehn et al.). Gomez-Cantarino et al. 10.3389/fpsyg.2024.1418051 Cunha-Oliveira, A., Camarneiro, A. P., Gómez-Cantarino, S., Cipriano-Crespo, C., Queirós, P. J. P., Cardoso, D., et al. (2021). The integration of gender perspective Berenguel Chacón, P., Plaza del Pino, F. J., Molina-Gallego, B., and Ugarte- Gurrutxaga, M. I. (2023). The perception of nurses about migrants after the COVID-19 pandemic: close contact improves the relationship. Int. J. Environ. Res. Public Health 20:1200. doi: 10.3390/ijerph20021200 References Frontiers in Psychology Frontiers in Psychology 02 frontiersin.org frontiersin.org Gomez-Cantarino et al. 10.3389/fpsyg.2024.1418051 into young people’s sexuality education in Spain and Portugal: legislation and educational models. Int. J. Environ. Res. Public Health 18:11921. doi: 10.3390/ijerph182 211921 Berenguel Chacón, P., Plaza del Pino, F. J., Molina-Gallego, B., and Ugarte- Gurrutxaga, M. I. (2023). The perception of nurses about migrants after the COVID-19 pandemic: close contact improves the relationship. Int. J. Environ. Res. Public Health 20:1200. doi: 10.3390/ijerph20021200 Berenguel Chacón, P., Plaza del Pino, F. J., Molina-Gallego, B., and Ugarte- Gurrutxaga, M. I. (2023). The perception of nurses about migrants after the COVID-19 pandemic: close contact improves the relationship. Int. J. Environ. Res. Public Health 20:1200. doi: 10.3390/ijerph20021200 into young people’s sexuality education in Spain and Portugal: legislation and educational models. Int. J. Environ. Res. Public Health 18:11921. doi: 10.3390/ijerph182 211921 Shariﬁ, N., Adib-Hajbaghery, M., and Najaﬁ, M. (2019). Cultural competence in nursing: a concept analysis. Int. J. Nurs. Stud. 99:103386. doi: 10.1016/j.ijnurstu.2019.103386 Cantarino, S. G., Pinto, J. M., Fabião, J. A., García, A. M., Abellán, M. V., and Rodrigues, M. A. (2016). The importance of religiosity/spirituality in the sexuality of pregnant and postpartum women. PLoS ONE 11:e0156809. doi: 10.1371/journal.pone.0156809 Cantarino, S. G., Pinto, J. M., Fabião, J. A., García, A. M., Abellán, M. V., and Rodrigues, M. A. (2016). The importance of religiosity/spirituality in the sexuality of pregnant and postpartum women. PLoS ONE 11:e0156809. doi: 10.1371/journal.pone.0156809 Ugarte-Gurrutxaga, M. I. (2020). La salud reproductiva de las mujeres inmigrantes: el “plus” de la desigualdad. Atlánticas. Revista Internacional De Estudios Feministas 4, 179–196. doi: 10.17979/arief.2019.4. 1.3705 Cunha-Oliveira, A., Camarneiro, A. P., Gómez-Cantarino, S., Cipriano-Crespo, C., Queirós, P. J. P., Cardoso, D., et al. (2021). The integration of gender perspective 03 03 Frontiers in Psychology Frontiers in Psychology frontiersin.org
https://openalex.org/W2762355270	https://discovery.ucl.ac.uk/1574440/1/Plumb_Rapid%20volumetric%20photoacoustic%20tomographic%20imaging%20with%20a%20Fabry-Perot%20ultrasound%20sensor_VoR.pdf	English	null	Rapid volumetric photoacoustic tomographic imaging with a Fabry-Perot ultrasound sensor depicts peripheral arteries and microvascular vasomotor responses to thermal stimuli	European radiology	2,017	cc-by	6,128	Eur Radiol (2018) 28:1037–1045 DOI 10.1007/s00330-017-5080-9 Eur Radiol (2018) 28:1037–1045 DOI 10.1007/s00330-017-5080-9 EXPERIMENTAL Rapid volumetric photoacoustic tomographic imaging with a Fabry-Perot ultrasound sensor depicts peripheral arteries and microvascular vasomotor responses to thermal stimuli Andrew A. Plumb1 & Nam Trung Huynh2 & Jamie Guggenheim2 & Edward Zhang2 & Andrew A. Plumb1 & Nam Trung Huynh2 & Jamie Guggenheim2 & Edward Zhang2 & Paul Beard2 Received: 16 May 2017 /Revised: 3 September 2017 /Accepted: 13 September 2017 /Published online: 10 October 2017 # The Author(s) 2017. This article is an open access publication beds with 100% accuracy (95% CI 77.2-100.0%, p < 0.001). The number of voxels exhibiting vascular signal was significantly smaller after cold water immersion (cold: 5263 voxels; warm: 363,470 voxels, p < 0.001). The DP artery was visible in 7/8 participants (87.5%). Abstract Purpose To determine if a new photoacoustic imaging (PAI) system successfully depicts (1) peripheral arteries and (2) microvascular circulatory changes in response to thermal stimuli. Methods Following ethical permission, 8 consenting subjects underwent PAI of the dorsalis pedis (DP) artery, and 13 completed PAI of the index fingertip. Finger im- ages were obtained after immersion in warm (30-35 °C) or cold (10-15 °C) water to promote vasodilation or va- soconstriction. The PAI instrument used a Fabry-Perot interferometeric ultrasound sensor and a 30-Hz 750-nm pulsed excitation laser. Volumetric images were acquired through a 14 × 14 × 14-mm volume over 90 s. Images were evaluated subjectively and quantitatively to deter- mine if PAI could depict cold-induced vasoconstriction. The full width at half maximum (FWHM) of resolvable vessels was measured. Conclusion PAI achieves rapid, volumetric, high-resolution imaging of peripheral limb vessels and the microvasculature and is responsive to vasomotor changes induced by thermal stimuli. Key points • Fabry-Perot interferometer-based photoacoustic imaging (PAI) generates volumetric, high-resolution images of the peripheral vasculature. • The system reliably detects thermally induced peripheral va- soconstriction (100% correct identification rate, p < 0.001). • Vessels measuring less than 100 μm in diameter can be depicted in vivo. Results Fingertip vessels were visible in all participants, with mean FWHM of 125 μm. Two radiologists used PAI to correctly identify vasoconstricted fingertip capillary Keywords Photoacoustic techniques . Interferometry . Ultrasound . Peripheral vascular diseases . Vasoconstriction Keywords Photoacoustic techniques . Interferometry . Ultrasound . Peripheral vascular diseases . Vasoconstriction Abbreviations DP Dorsalis pedis FP Fabry-Perot FWHM Full width at hal LV Large vessel PA Photoacoustic PAD Peripheral arteri PAI Photoacoustic im PAT Photoacoustic to SV Small vessel SVD Small vessel dis Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00330-017-5080-9) contains supplementary material, which is available to authorized users. * Andrew A. Plumb andrew.plumb@nhs.net; yangcan.gong@nhs.net 1 Centre for Medical Imaging, Division of Medicine, University College London, Podium Level 2, 235 Euston Road, London NW1 2BU, UK 2 Photoacoustic Imaging Group, Department of Medical Physics and Biomedical Engineering, University College London, London, UK Abbreviations DP Dorsalis pedis FP Fabry-Perot FWHM Full width at half maximum LV Large vessel PA Photoacoustic PAD Peripheral arterial disease PAI Photoacoustic imaging PAT Photoacoustic tomography SV Small vessel SVD Small vessel disease Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00330-017-5080-9) contains supplementary material, which is available to authorized users. * Andrew A. Abstract Plumb andrew.plumb@nhs.net; yangcan.gong@nhs.net 1 Centre for Medical Imaging, Division of Medicine, University College London, Podium Level 2, 235 Euston Road, London NW1 2BU, UK 2 Photoacoustic Imaging Group, Department of Medical Physics and Biomedical Engineering, University College London, London, UK Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00330-017-5080-9) contains supplementary material, which is available to authorized users. * Andrew A. Plumb andrew.plumb@nhs.net; yangcan.gong@nhs.net 1 Centre for Medical Imaging, Division of Medicine, University College London, Podium Level 2, 235 Euston Road, London NW1 2BU, UK 2 Photoacoustic Imaging Group, Department of Medical Physics and Biomedical Engineering, University College London, London, UK Eur Radiol (2018) 28:1037–1045 1038 detector array [15]. Further ex vivo animal model work has shown that volumetric PAI can successfully depict subcutane- ous veins during endovenous laser therapy [16]. However, PAI has not yet been shown capable of detecting vascular changes induced by either normal physiology or disease in humans, nor has it been subject to appropriately powered, prospective clin- ical studies with interpreter blinding and pre-specified end- points aimed at clinical validation, as befits development of a new imaging technology [17]. Since SVs are readily able to vasoconstrict and vasodilate in response to cold and heat re- spectively [18], they are an excellent experimental test bed for evaluation of PAI-based vascular imaging, serving as a safe, laboratory-controlled paradigm for the constriction of the SVs seen in atherosclerotic disease. We therefore wished to deter- mine if our FP sensor-based PAI system can successfully depict (1) peripheral leg arteries and (2) microvascular circulatory changes in response to thermal stimuli as a reliable, controllable means of safely inducing SV vasoconstriction and vasodilation. Introduction Peripheral arterial disease (PAD) is a common, important con- dition, affecting approximately 27 million individuals across the USA and Europe [1]. Most patients with PAD have generalised atherosclerosis throughout the cardiovascular system, including significant coronary artery disease in one-third, meaning that cardiovascular deaths are increased six-fold [2]. Furthermore, severe PAD itself causes significant local morbidity via tissue necrosis and ultimately amputation or even death. Healthcare costs are substantial (>$4.5 billion annually in the USA alone), similar to cerebrovascular disease and cardiac failure [3]. Therefore, prompt identification and treatment of PAD are im- portant to patients, healthcare services, and wider society [4]. PAD involves both large vessels (LVs) and small vessels (SVs) beyond named arterial branches. Whereas LVs are well depicted by existing techniques, primarily ultrasonography (US) and computed tomographic or magnetic resonance angi- ography (CTA/MRA), limited spatial resolution precludes meaningful assessment of SV. This is problematic, because SV-PAD is closely associated with diabetes mellitus [5], which is increasing in incidence globally [6]. Accordingly, there is clear need to develop newer techniques to accurately assess SV-PAD non-invasively. Participants We recruited healthy volunteers, aged ≥18 years and taking no medication, via internal advertisement. Exclusion criteria were inability to provide informed written consent, vascular/ cardiorespiratory disease, and any skin disorder preventing safe imaging probe placement. Ultimately, we recruited 15 volunteers (13 male); 13 participated in the thermal stimulus study and 8 in the limb artery imaging study (5 completed both studies). Materials and methods Ethical permission was granted for this prospective single- centre study by the University College London Research Ethics Committee (Project ID: 1133/001). One possible solution is photoacoustic (optoacoustic) im- aging (PAI), which exploits the photoacoustic (PA) effect, whereby laser illumination of tissue provokes broadband ul- trasound emission. These ultrasound waves can be recon- structed into high-resolution images, based on the optical ab- sorption properties of the tissue [7]. PAI is particularly suitable for vascular imaging because haemoglobin has strong optical absorption, maximising ultrasound emission and image con- trast. However, imaging at clinically relevant depths can be problematic because normal tissues have high optical and acoustic attenuation, mandating highly sensitive ultrasound detectors. Furthermore, it is challenging to simultaneously il- luminate tissue and detect ultrasound without the ultrasound detectors obscuring the laser light. We have developed a PA system that uses a Fabry-Perot (FP) interferometer as the ul- trasound sensor [8]. This is optically transparent to the excita- tion laser, avoiding such obscuration. Furthermore, the FP sensor outperforms conventionally used piezoelectric ultra- sound sensors in sensitivity, bandwidth, and element size, yielding improving 3D image resolution and contrast [8] for sub-centimetre scale vascular imaging. Eur Radiol (2018) 28:1037–1045 Fig. 1. Fabry-Perot photoacoustic imaging system. A schematic of the imaging setup is shown in (a), with a photograph of the system being used for image acquisition (in this case, of the index fingertip) in (b), with detail in the inset Fig. 1. Fabry-Perot photoacoustic imaging system. A schematic of the imaging setup is shown in (a), with a photograph of the system being used for image acquisition (in this case, of the index fingertip) in (b), with detail in the inset Fig. 1. Fabry-Perot photoacoustic imaging system. A schematic of the imaging setup is shown in (a), with a photograph of the system being used for image acquisition (in this case, of the index fingertip) in (b), with detail in the inset °C). After 3-min immersion, their contralateral (non-immersed) index fingertip was imaged to investigate for reflex vasocon- striction (Breflex stimulus^). At 5 min, the immersed hand’s index finger was removed from the water and immediately imaged (Bdirect stimulus^). Five minutes was chosen as the length of immersion because in normal physiology a more prolonged cold stimulus than this often induces vascular dila- tation in order to protect the peripheries from tissue damage due to cold [18]. Images were obtained using a 20-mm-diameter excitation beam and incident fluence at the skin surface of 6.5 mJ, four fold below maximum regulatory exposure limits (BS- EN-60825-1). The sensor was scanned over a 14 × 14-mm area in 106-μm steps, thus acquiring 17,443 PA waveforms; acqui- sition time (for the entire imaged volume) was 90 s. Participants then transferred their non-dominant hand to warm water (30-35 °C) and the experiment was repeated for both the reflex stimu- lus (contralateral hand) and direct stimulus (immersed hand). scanning the sensor with a second Binterrogation^ laser, thus synthesising a 2D array of tens of thousands of highly sensi- tive individual ultrasound detectors. We used a parallelised Santec TSL-550 1550-nm laser to interrogate the sensor and InGaAs photo-detectors (Hamamatsu G9801-22) to permit measurement of the optical power changes induced by the photoacoustic waves at a sampling rate of 60 MHz. The scan- ner provides a spatial resolution in the range 75-125 μm de- pending on the imaged depth and has an ultrasound bandwidth (-3dB) of 30 MHz. Eur Radiol (2018) 28:1037–1045 The field of view of the instrument is dependent on the area of the FP sensor that is scanned by the interrogation laser, typically 1-2 cm in each of the x and y dimensions. Depth of view (the z dimension) is governed by penetration of laser light into the imaged volume. PAI reconstruction methods for an earlier scanner prototype have been described previously [8, 19, 20]. The algorithm uses a k-space fast Fourier transform method to reconstruct the pho- toacoustic image from the spatio-temporal distribution of the PA-induced ultrasound measured by the FP sensor. For imme- diate feedback at the time of scanning, rapid reconstruction was performed using non-upsampled data, generating images with- in 2-4 s. However, since the sensor captures higher frequency components in the time domain (60 MHz sampling rate) than in the spatial domain (scanning steps of 106 μm), it is possible to reconstruct higher-quality images by upsampling spatially (in this case, two fold) so that the high frequency information from the time domain is used in the reconstruction [21]. This in- creases the computation time to 10-20 s. Images were interpo- lated (×2) after reconstruction to generate voxels of approxi- mately 25 μm for radiologist manipulation. DICOM images were exported for radiologist manipulation and interpretation (Osirix, Pixmeo Sarl, Switzerland). At a second visit, the dorsalis pedis (DP) artery was im- aged. An experienced radiologist (A.A.P.) used the 40-MHz linear probe of a Viewsonics SonixMDP scanner (Analogic Ultrasound, Richmond, Canada) to obtain longitudinal and axial vessel images. Immediately thereafter, location- matched PAI images were obtained by positioning the PA scan head at the same position that had been used for sonography. To determine if PAI could detect SV changes induced by thermal stimuli, we assessed the images (1) subjectively and (2) quantitatively. For the former, paired images for each par- ticipant in each imaging condition (i.e. cold and warm) were viewed independently in random order by two radiologists. Reflex and direct stimulus images were viewed separately. Each radiologist judged whether SVs were more readily visi- ble after cold or warm water immersion. Subsequently, quan- titative measures were obtained by using the DICOM viewer to count the number of voxels returning vascular signal. An elliptical region of interest (ROI) was drawn within the largest imaged vessel, and the vascular signal was defined as the mean signal intensity within this ROI, ± 2 standard deviations. Imaging platform The PAI platform is based on a previously described prototype [19]. The system (Fig. 1) comprises a fibre-coupled Bexcitation^ laser that illuminates the target tissue, provoking photoacoustic ultrasound emission for subsequent detection by the FP sensor. Here, we used a fibre-coupled, 30 Hz, optical parametric oscillator (OPO) excitation laser system (SpitLight- 600, InnoLas Laser GmbH, Krailling, Germany) at a nominal wavelength of 750 nm. This wavelength balances high absorp- tion by haemoglobin and good tissue depth penetration. Several previous PAI studies have depicted human vascula- ture successfully, although most only imaged superficial skin vessels [9–11], or while investigating dermatological disease [12], although clear proof-of-concept of deeper vessel imaging has been reported, of both larger named arterial vessels [13] and within organs such as the breast [14]. A further recent article described high-resolution two-dimensional peripheral foot ves- sel imaging using a handheld probe and a concave ultrasound The FP interferometer comprises a polymer spacer sandwiched between two mirrors. Incident ultrasound waves modulate the spacer thickness, producing a corresponding change in optical reflectivity. This reflectivity change can be mapped very precisely in two dimensions by rapidly raster 1039 Eur Radiol (2018) 28:1037–1045 Fig. 1. Fabry-Perot photoacoustic imaging system. A schematic of the imaging setup is shown in (a), with a photograph of the system being used for image acquisition (in this case, of the index fingertip) in (b), with detail in the inset Subjective assessments Subjective assessments SVs were depicted successfully in all 13 volunteers. Both readers judged there to be fewer visible vessels after cold water immersion vs. warm in all cases for the direct stimulus (correct identification rate = 100%, 95% CI 77.2 to 100.0%, p < 0.001, Fig. 2 and Supplemental Material 1 and 2). For the reflex stimulus, both readers judged there to be fewer visible SVs after cold water immersion in all cases except one (in which the two conditions were judged equivalent; correct identification rate = 92.3%, 95%CI 66.7 to 98.6%, p = 0.006). DP artery conspicuity was judged subjectively on a four- point scale (0, imperceptible; 1, barely perceptible; 2, percep- tible, but with some artefacts; 3, clearly visible, minimal/no artefacts) by viewing maximum-intensity projection (MIP) and multiplanar reformats (MPRs). For each artery, the depth from the skin surface to its superficial and deep margins was measured using MPR images. Quantitative measurements The primary power was based on readers’ ability to determine which of a given image pair had been obtained after cold water immersion (representing a vasoconstricted state) using the di- rect stimulus. We assumed that a correct identification rate of ≥90% (vs. the 50% expected by chance) would imply future clinical value. Therefore, at a power of 80% and significance level of 5%, we required 13 participants (G*Power version 3.1.9.2 for Mac). The mean number of voxels exhibiting vascular signal was significantly lower after cold water immersion than warm wa- ter immersion for both the direct stimulus and the reflex stim- ulus (direct: cold = 5263 voxels; warm = 363,470 voxels, p < 0.001; reflex: cold = 50,388 voxels, warm = 365,037 voxels, p = 0.007). Each individual volunteer showed less PAI signal after cold vs. warm water immersion after the direct stimulus (Fig. 3a), mirroring the 100% discrimination recorded subjec- tively. For the reflex stimulus, only a single subject had more PAI signal after cold vs. warm water (Fig. 3b). The smallest vessels depicted by PAI had mean FWHM dimensions of 125 μm (range: 75–150 μm). These tiny vessels were completely undetectable by 40-MHz B-mode and Doppler ultrasound (not shown). Statistical analysis The primary outcome was the proportion of individuals in whom cold (vs. warm) water immersion was correctly identi- fied by the radiologists for the direct stimulus. This was cal- culated separately for each reader and compared to chance (50% rate) using a one-sample test of proportions. Secondary outcomes were the results of the qualitative radiol- ogist assessment for the reflex stimulus and the number of voxels demonstrating vascular signal (for the direct and the reflex stimulus). The latter were compared for cold vs. warm images using the Wilcoxon signed-rank test. Descriptive sta- tistics were calculated for other outcomes. We used R version 3.2.0 for Mac software, taking probability values of < 0.05 as significant. Peripheral limb arteries Images were reconstructed to depths of 14 mm. The dorsalis pedis (DP) artery was visible (score of ≥2) for all eight par- ticipants except one (examples in Fig. 4 and Supplementary material 3). This participant had dark skin (Fitzpatrick scale = 6) and deeper vessels were obscured by strong laser absorp- tion by melanin at the wavelength used. A subsequent attempt at re-imaging this individual at a different excitation wave- length (900 nm) was successful. Mean distance from the skin to the DP was 2.9 mm to its superficial border (range: 1.8 to 4.5 mm) and 4.6 mm to its deep border (range: 3.5 to 6.9 mm). Several accompanying veins demonstrated internal fold-like structures, taken to be normal venous valves (Fig. 5). Results There were no symptomatic side effects from PAI for any participant. No skin damage, irritation, or discomfort was re- ported either immediately following image acquisition or in the days thereafter. High-quality, three-dimensional spatially resolved data sets were successfully acquired in all cases. Average acquisition time was 90 s; the SV experiment was completed in approximately 15 min (including the 10 min of water immersion time, half for each hand) and the LV exper- iment was completed in approximately 5 min. Imaging protocol Images were acquired over two visits. On the first, participants submerged their non-dominant hand in a cold water bath (10-15 1040 Eur Radiol (2018) 28:1037–1045 Fingertip SVs The number of voxels within each imaged volume meeting these conditions was then measured. We also measured the dimension of PAI-depicted SVs by drawing a linear ROI across their short axis and calculating the full width at half maximum (FWHM). The number of voxels within each imaged volume meeting these conditions was then measured. We also measured the dimension of PAI-depicted SVs by drawing a linear ROI across their short axis and calculating the full width at half maximum (FWHM). Discussion We have shown that PAI generates high-resolution, three- dimensional images of both SV and LV in vivo and depicts thermally induced peripheral vasoconstriction, both directly and via reflex action. Importantly, this was using a robust 1041 Eur Radiol (2018) 28:1037–1045 Fig. 2. Maximum intensity projection (MIP) fingertip PA images after cold (left hand panels) and warm (right hand panels) water immersion using the direct stimulus, in three different subjects, color-coded for depth. Arrows show the same vessels in each imaging condition Fig. 2. Maximum intensity projection (MIP) fingertip PA images after cold (left hand panels) and warm (right hand panels) water immersion using the direct stimulus, in three different subjects, color-coded for depth. Arrows show the same vessels in each imaging condition to multi- vs. single-detector row CT). Theoretically, such parallelisation is limited solely by the technical complexity and cost, although in practical terms, scanning more than 100 channels will be challenging. As well as these hardware improvements, it is possible to interrogate the FP sensor using compressed sensing techniques, which speeds acquisition by reducing the amount of data that needs to be collected without significantly compromising image quality [22]. As well as these approaches on the Bsensor read-out^ side of the device, the excitation step can also be hastened by using excitation lasers with a higher pulse repetition frequency. Lasers similar to that used in the current study but operating at 200 Hz (vs. 30 Hz) are commercially available and would reduce the acqui- sition time to <30 s per imaged volume. Even faster rates are possible using alternative technology such as fibre lasers [23]. However, ultimately this will be limited by more rapid energy deposition in the imaged volume, potentially contravening safety limits. We anticipate that up to 1 kHz excitation lasers experimental design with pre-specified outcomes, adequate sample size to achieve statistical power, and radiologist blinding during image interpretation. The system has a conve- nient, easily manipulated probe and acquires volumetric im- ages rapidly (< 90 s to acquire over 17,000 PAwaveforms and image at 14 × 14 × 14-mm volume). The findings suggest that PAI may be a powerful technique for assessing arterial disease. Our PA system uses a different method of ultrasound de- tection from most previously described devices, namely, a Fabry-Perot interferometer (vs. a piezoelectric array). Fig. 2. Maximum intensity projection (MIP) fingertip PA images after cold (left hand panels) and warm (right hand panels) water immersion using the direct stimulus, in three different subjects, color-coded for depth. Arrows show the same vessels in each imaging condition Discussion This has several major advantages; first, it can be placed directly on tissues of interest without obscuring the excitation laser, simplifying imaging geometry. Second, FP sensors are highly sensitive to ultrasound. Third, it is possible to parallelise sen- sor interrogation by using a multi-beam laser, thereby accel- erating image acquisition considerably. Here, we accelerated eight fold; future upgrades to 16- and 24-beam systems are planned and will reduce acquisition times further (analogous 1042 Eur Radiol (2018) 28:1037–1045 Fig. 3. Boxplots and strip charts showing the number of voxels exhibiting vascular signal (y-axis, logarithmic scale) for all subjects in each imaging condition (x-axis). Dashed grey lines show the change for each individual subject. Panel (a) shows results for the direct thermal stimulus, in which all subjects showed an increase in the number of vascular signal voxels after immersion in warm vs. cold water. Panel (b) shows results for the reflex stimulus: All subjects except one showed an increase after warm water immersion of the contralateral hand 1042 Eur Radiol (2018) 28:1037–1045 stimulus, in which all subjects showed an increase in the number of vascular signal voxels after immersion in warm vs. cold water. Panel (b) shows results for the reflex stimulus: All subjects except one showed an increase after warm water immersion of the contralateral hand Fig. 3. Boxplots and strip charts showing the number of voxels exhibiting vascular signal (y-axis, logarithmic scale) for all subjects in each imaging condition (x-axis). Dashed grey lines show the change for each individual subject. Panel (a) shows results for the direct thermal stimulus, in which all subjects showed an increase in the number of vascular signal voxels after immersion in warm vs. cold water. Panel (b) shows results for the reflex stimulus: All subjects except one showed an increase after warm water immersion of the contralateral hand reach the imaging target) and, to a lesser degree, because of sonographic attenuation (i.e. inability of the laser-generated ultrasound to reach the detector). Despite these concerns, the data reported here show that we were able to routinely depict the DP artery at depths of several millimetres from the skin surface, meaning larger vessels are well within reach of the instrument. Accordingly, PAI as employed here is highly com- plementary to conventional Duplex US, which images larger and deeper vessels effectively. Discussion Presently, PAI is unlikely to compete directly with US for macrovascular imaging, but in- stead permits high-resolution, volumetric imaging of the SVs that Duplex US cannot detect. Nonetheless, we anticipate that PAI imaging depths will increase further. For example, it will be possible to increase the power of the incident excitation could, in theory, be deployed successfully and safely (using energies of a few mJ). The combination of all these elements means that video frame-rate three-dimensional acquisition is ultimately likely to be achievable. Fourth, the system is ideally suited to refinement by adding conventional ultrasound (for detection by the same FP sensor), which will permit fused, perfectly co-registered US/PAI images from a single device. Finally, the sensor can be constructed in a variety of geome- tries, permitting adaptation to specific clinical tasks (e.g. laparoscope- or endoscope-mounted sensors for intra- operative or endocavitary imaging [24]). A concern for the clinical application of PAI is its relatively limited penetration depth [25]. This problem arises primarily because of optical attenuation (i.e. inability of the laser light to Eur Radiol (2018) 28:1037–1045 1043 w the maximum ultrasound detec- improve imaging d 1 cm (using the ave also success- ectric ultrasound array to depict both LVs and SVs with similar resolution to that reported here [15]. These highly complementary data to our own strongly imply that the underlying PAI technology will translate successfully to routine clinical practice. There are several limitations to our study. First, we recruit- ed healthy volunteers and induced vasomotor changes using Fig. 4. Example short axis 2D slices taken from the volumetric PA imaging series (left upper panels) with contemporaneous, location-matched ultrasound (right upper panels) for two separate volunteers (a and b respectively). The corresponding PAI volumetric data sets, presented as coronal and sagittal maximum intensity projection (MIP) images, are shown in the lower panels. MIP images are color-coded for depth (see scale). The dashed line indicates the plane through which the axial image has been reconstructed. The DP artery, with its accompanying venae comitantes, is arrowed, with a superficial vein (large arrowhead) and the skin surface (small arrowheads) also shown in a. The axes are as follows; y, proximal-distal; x, medial-lateral; z, dorsal-plantar laser since we imaged at powers well below the maximum tolerable limit. Discussion Additionally, more sensitive ultrasound detec- tors are being developed [26] and will likely improve imaging depths from the current maximum of around 1 cm (using the present system) to over 2 cm. Other groups have also success- fully used PAI based on a concave piezoelectric ultrasound array to depict both LVs and SVs with similar resolution to that reported here [15]. These highly complementary data to our own strongly imply that the underlying PAI technology will translate successfully to routine clinical practice. There are several limitations to our study. First, we recruit- ed healthy volunteers and induced vasomotor changes using e dashed line d cates t e plane through which the axial image has been reconstructed. The DP artery, with its accompanying venae comitantes, is arrowed, with a superficial vein (large arrowhead) and the skin surface (small arrowheads) also shown in a. The axes are as follows; y, proximal-distal; x, medial-lateral; z, dorsal-plantar laser since we imaged at powers well below the maximum tolerable limit. Additionally, more sensitive ultrasound detec- tors are being developed [26] and will likely improve imaging depths from the current maximum of around 1 cm (using the present system) to over 2 cm. Other groups have also success- fully used PAI based on a concave piezoelectric ultrasound array to depict both LVs and SVs with similar resolution to that reported here [15]. These highly complementary data to our own strongly imply that the underlying PAI technology will translate successfully to routine clinical practice. There are several limitations to our study. First, we recruit- ed healthy volunteers and induced vasomotor changes using 1044 Eur Radiol (2018) 28:1037–1045 Fig. 5. Example coronal and sagittal maximum intensity projection (MIP) images through a superficial foot vein of a healthy volunteer, depicting a pair of angled, linear webs of reduced signal (arrows) within the vessel at a site of slight venous dilatation. These were not visible on ultrasound, but from their morphology and location they are presumed to represent a venous valve Funding This study has received funding by the University College London and University College London Hospitals National Institute for Health Research Biomedical Research Centre scheme. Compliance with ethical standards Guarantor The scientific guarantor of this publication is Andrew Plumb. Conflict of interest The authors of this manuscript declare no relation- ships with any companies, whose products or services may be related to the subject matter of the article. Statistics and biometry No complex statistical methods were neces- sary for this paper. Fig. 5. Example coronal and sagittal maximum intensity projection (MIP) images through a superficial foot vein of a healthy volunteer, depicting a pair of angled, linear webs of reduced signal (arrows) within the vessel at a site of slight venous dilatation. These were not visible on ultrasound, but from their morphology and location they are presumed to represent a venous valve Informed consent Written informed consent was obtained from all individuals in this study. Ethical approval Institutional Review Board approval was obtained from the University College London Ethics Committee. thermal stimuli, which may not be representative of changes in disease. It is possible that the degree of vasoconstriction induced by cold water immersion is greater than that caused by atherosclerosis of typical severity. These data should there- fore be taken as proof of the concept that PAI can depict changes to vascular beds in general rather than atherosclerosis specifically. Second, we imaged only at a single wavelength; since haemoglobin and deoxyhaemoglobin have different ab- sorption spectra, it is theoretically possible to estimate oxygen saturation by multi-wavelength imaging [27], an important future avenue for development. Third, we imaged volumetri- cally in 3D mode, sacrificing temporal resolution and real- time device navigation for a larger field of view. The device is entirely capable of real-time 2D imaging (at frame rates exceeding 10 frames/second) and in the future we plan to permit user-controlled switching between the two, 2D for de- vice navigation and positioning and 3D for detailed assess- ment. Finally, for reasons of convenience and practicality we imaged volunteers’ fingertips rather than their toes, which is the more common location for peripheral arterial disease—it is possible (although unlikely) that vasomotor changes differ between these sites. thermal stimuli, which may not be representative of changes in disease. It is possible that the degree of vasoconstriction induced by cold water immersion is greater than that caused by atherosclerosis of typical severity. These data should there- fore be taken as proof of the concept that PAI can depict changes to vascular beds in general rather than atherosclerosis specifically. Discussion The work was also supported by the Engineering and Physical Sciences Research Council (EPSRC), European Union project FAMOS (FP7 ICT, Contract 317744), and University College London Comprehensive Cancer Imaging Centre, Cancer Research UK & Engineering and Physical Sciences Research Council, in association with the Medical Research Council and Department of Health, UK. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR, the EPSRC, the MRC, or the UK Department of Health. Methodology • prospective • experimental • experimental • performed at one institution • performed at one institution 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. Compliance with ethical standards Second, we imaged only at a single wavelength; since haemoglobin and deoxyhaemoglobin have different ab- sorption spectra, it is theoretically possible to estimate oxygen saturation by multi-wavelength imaging [27], an important future avenue for development. Third, we imaged volumetri- cally in 3D mode, sacrificing temporal resolution and real- time device navigation for a larger field of view. 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https://openalex.org/W2886940279	https://www.nature.com/articles/s41598-018-29613-1.pdf	English	null	Subjective time expansion with increased stimulation of intrinsically photosensitive retinal ganglion cells	Scientific reports	2,018	cc-by	7,797	Subjective time expansion with increased stimulation of intrinsically photosensitive retinal ganglion cells Received: 30 August 2017 Accepted: 12 July 2018 Published: xx xx xxxx Pei-Ling Yang1, Sei-ichi Tsujimura2, Akiko Matsumoto2, Wakayo Yamashita2 & Su-LingYeh1,3,4,5 Intrinsically photosensitive retinal ganglion cells (ipRGCs) contain photoreceptors that are especially sensitive to blue light. Nevertheless, how blue light and ipRGCs affect time perception remains unsolved. We used the oddball paradigm and manipulated the background light to examine whether and how blue light and ipRGCs affect perceived duration. In the oddball paradigm, participants were asked to judge the duration of the target (oddball), compared to that of the standard, with a two alternative-forced-choice procedure. When the background light was controlled to be either blue or red in Experiment 1, results showed that blue light led to longer subjective duration compared to red light. Experiment 2 further clarified the contribution of the ipRGCs. A set of multi-primary projector system that could manipulate the ipRGC stimulation were used, while the color and luminance of the background lights were kept constant throughout. Results showed that increased stimulation of ipRGCs under metameric background expanded subjective time. These results suggest that ipRGC stimulation increases arousal/attention so as to expand subjective duration. In this study, we examined the role of a population of intrinsically photosensitive retinal ganglion cells (ipRGCs) on observers’ duration judgment. The ipRGCs, a newly discovered third type of photoreceptors1,2, are easily stim- ulated by blue light emitted from various commonly used electrical devices (e.g., cellphone, pad, computer, room light, etc.) and affect circadian rhythm3, one kind of timing mechanisms in our body that governs metabolic func- tion and sleep-awake cycle. The ipRGCs have been identified in rats1,4, monkeys, and human beings5. These cells are sensitive to light peaking at 481~493 nm (blue light) and are responsible for the effect of blue light on circadian rhythm. Especially, ipRGCs project to suprachiasmatic nuclei (SCN), which is the endogenous biological clock that allows external signal (i.e., ipRGC signal) to mediate the circadian rhythm6,7. Further, SCN is also associated with percived duration8,9, the kind of time perception we are interested in the current study. y However, little is known about the relationship between ipRGCs and perceived duration, since most previous studies focus on how blue light (compared to other color lights) affects perceived duration. Also, the studies that investigated time perception under blue light have rendered inconsistent results. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Subjective time expansion with increased stimulation of intrinsically photosensitive retinal ganglion cells Time production task asks participants to produce a specific time interval. Time estimation task demands participants to evaluate length of durations and response on scales (i.e., 1 = “slow” and 9 = “fast”). Time comparison task requires participants to categorize target durations into short or long. The various time perception paradigms have their own pros and cons. For example, time production task is more straightforward for evaluating par- ticipants’ subjective duration, but its variance is greater than the other paradigms. Time comparison task also requests participants to remember the standard time interval for categorization and thus also involves working memory. As to the different time scales used, they ranged from 0.4 s to 180 s across sub-second and supra-second scales, and these two scales have been shown to involve different mechanisms8,9. Specifically, the supra-second level is correlated with high cognitive functions, while the sub-second level is correlated with sensation or auto- matic processing14–16.ff Most importantly, comparing time perception under different color lights necessarily involves different ipRGC stimulation and yet the role of ipRGCs on time perception has not been systematically examined in past stud- ies. To examine the role of ipRGCs in the effects of blue light on perceived duration, especially bearing in mind that the sub-second level is more sensation involved, in this study we used the oddball paradigm to investigate an effect of attention/arousal process that is related to ipRGC stimulations17,18 on time perception. The oddball paradigm used in Tse, Intriligator, Rivest, and Cavanagh19 was adopted. In the oddball paradigm, participants are required to judge the duration of a low-probability but salient stimulus (oddball) and compare it to a string of high-probability standard stimuli with a fixed duration. The performance of the oddball paradigm could be influenced by attention level19 or arousal level20, which is suitable for examing the relationship between ipRGCs and time perception. The oddball paradigm has advantages over the production task, including that information of parameters in psychometric function could be collected and confounding factors from motor responses and participants’ memory ability can be eliminated21. y y By adopting the oddball paradigm, we were able to obtain the psychometric function and estimated the point of subjective equality (PSE) by comparing the judged duration of the oddball with that of the standards under different background conditions. In Experiment 1, the stimuli (standards and oddball) were presented in blue or red background (Fig. 1). Subjective time expansion with increased stimulation of intrinsically photosensitive retinal ganglion cells These inconsistent results may result from different amount of ipRGC stimulation (i.e., in different color lights used), tasks (i.e., time produc- tion), or target time intervals (i.e., sub- or supra-second). For example, Caldwell and Jones10 used a production task for 30 and 40 seconds, and found no difference in time perception between red, white, and blue lights. Gorn11 used a supra-second estimation and found that time perception was shortened under blue light compared to red or yellow light. Katsuura, Yasuda, Shimomura, and Iwanaga12 used a production task to measure supra-second time interval, manipulating the background light as blue or red. Their results showed no effect between the two background conditions, except that time perception was lengthened at 180 s duration under blue light compared to that under red light. Shibasaki and Masataka13 used a time comparison task under blue and red light, with the 1Department of Psychology, National Taiwan University, Taipei, Taiwan. 2Faculty of Science and Engineering, Kagoshima University, Kagoshima, Japan. 3Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan. 4Neurobiology and Cognitive Neuroscience Center, National Taiwan University, Taipei, Taiwan. 5Center for Artificial Intelligence and Advanced Robotics, National Taiwan University, Taipei, Taiwan. Correspondence and requests for materials should be addressed to S.-L.Y. (email: suling@ntu.edu.tw) SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 1 www.nature.com/scientificreports/ Study Method Results: time perception under blue light compared to other lights Task Time interval Peak wavelength Caldwell et al. (1985) Time production 30 s, 40 s Not Available No effect Gorn et al. (2004) Time estimation 17.5 s Not Available Shorter than yellow/red Katsuura et al. (2007) Time production 90 s, 180 s Blue: 436 nm Red: 612 nm Longer than red (180 s) Shibasaki et al. (2014) Time comparison 0.4 s~1.6 s Not Available Shorter than red (male) Table 1. Summaries of studies on blue light effect on time perception. Table 1. Summaries of studies on blue light effect on time perception. Table 1. Summaries of studies on blue light effect on time perception. sub-second to supra-second time intervals, and found shortened time perception under blue light, but only for sub-second to supra-second time intervals, and found shortened time perception under blue light, but only for men.h Table 1 summarizes the methods and results of these studies. This table shows that relationships between blue light and time perception have been examined by applying various tasks and with different time scales. Subjective time expansion with increased stimulation of intrinsically photosensitive retinal ganglion cells As the explanations to the effect of blue light on perceived duration in past studies failed to consider the influence of ipRGCs, merely paying attention to the effect of background color, we examined the role of ipRGCs on perceived duration in Experiment 2. It is predicted that higher stimulation of ipRGCs would lead to greater stimulation of SCN and higher arousal level, then causing time perception to be lengthened. Experiment 1 p In the oddball paradigm, we chose black circles as the standard stimuli and black square as the target, same as in Tsai and Yeh21. Methods. Participants. Eight healthy, male naïve volunteers (mean age = 23.25 years old) took part in Experiment 1. In this study, all participants had normal or corrected-to-normal vision, and if they wore glasses it was confirmed that their glasses did not contain blue light filters. Only male participants were recruited because time perception was only distorted with the male participants between blue and red13. They all gave informed consent before their participation. All experiments were approved by the Research Ethics Committee at National Taiwan University (NTU REC: 201505HS071) and conducted in accordance with applicable research subject guidelines. Stimuli and Apparatus. The visual stimuli were controlled by E-Prime 1.0 and presented on a 19′′ CRT screen with 60 Hz refresh rate. The visual standards were black circles, and the oddball was a black square. The radius of the standards and the side length of the oddball were 1.7° visual angle, and they were presented on either blue light or red light background at the center of the screen. The duration of the standards was 1050 ms, and the duration of the oddball was randomly selected from one of the following nine durations with equal probability: 750, 833, 900, 983, 1050, 1133, 1200, 1283, and 1350 ms. The inter-stimulus intervals were randomly assigned as 900, 1050, or 1200 ms. Using a Spectroradiometer (PR650, Photo Research) to estimate the composition of blue light and red light background, the peak wavelength of the blue light was 452 nm (CIE xy color coordinate (0.14, 0.10)) and the peak of the red light was 628 nm (CIE coordinate (0.62, 0.34)). The luminance values in CIE 200622 for blue light and red light was 9.51 cd/m2 and 5.99 cd/m2, respectively. We calculated the stimulation of ipRGCs based on the sensitivity curve of ipRGCs23,24 that has a peak wavelength of 493 nm, and stimulation of cones based SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 2 www.nature.com/scientificreports/ Figure 1. Procedure and stimuli used in this study. The duration of the standard stimuli (the circles) was fixed at 1050 ms, while that of the oddball (the square) varied and was chosen from one of the nine durations: 750, 833, 900, 983, 1050, 1133, 1200, 1283 and 1350 ms. Experiment 1 Seven to 12 standards were presented randomly between two oddballs in the series. The inter-stimulus intervals were randomly chosen from one of the three durations: 900, 1050, and 1200 ms. The task was to judge whether the duration of the oddball was longer or shorter than that of the standards. In Experiment 1, the background could either be blue or red, while in Experiments 2 the background was gray. The displays are not plotted to scale. Figure 1. Procedure and stimuli used in this study. The duration of the standard stimuli (the circles) was fixed at 1050 ms, while that of the oddball (the square) varied and was chosen from one of the nine durations: 750, 833, 900, 983, 1050, 1133, 1200, 1283 and 1350 ms. Seven to 12 standards were presented randomly between two oddballs in the series. The inter-stimulus intervals were randomly chosen from one of the three durations: 900, 1050, and 1200 ms. The task was to judge whether the duration of the oddball was longer or shorter than that of the standards. In Experiment 1, the background could either be blue or red, while in Experiments 2 the background was gray. The displays are not plotted to scale. on cone fundamentals at peripheral visual field in human25,26. The amount of ipRGC stimulation in the blue light condition was 42.16 times greater than in the red light condition. See Supplementary Materials 1 and 2 for cone and ipRGC stimulations and spectra for each condition. n cone fundamentals at peripheral visual field in human25,26. The amount of ipRGC stimulation in the blue ligh ondition was 42.16 times greater than in the red light condition. See Supplementary Materials 1 and 2 for cone nd ipRGC stimulations and spectra for each condition. Design. In Experiment 1, we adopted a 2 (background color: blue, red) × 9 (durations: 750, 833, 900, 983, 1050, 1133, 1200, 1283, and 1350 ms) within-subject factorial design. Background colors were counterbalanced between participants and each condition lasted for about one hour. In order to control the influence of circadian rhythm, participants performed the task at the same time of the day across the all the sessions. Each condition contained 378 oddballs in total, and each duration of oddball appeared 42 times in each condition. Procedure. After dark adapted for five minutes, participants started the oddball task. Experiment 1 Before the main task, par- ticipants needed to perform a practice session. The practice session had the same procedure with the main task. Seven oddball durations (750, 833, 900, 1050, 1200, 1283, and 1350 ms) randomly appeared once. In the main task, after 7~12 standard stimuli (randomly assigned between trials), an oddball would appear with one of the nine randomly assigned durations. In the task, participants were asked to judge whether the duration of the target stimulus (square) was longer or shorter than the standard stimuli (circles). They needed to respond immediately after the square disappeared, followed by a blank screen waiting for the response. No feedback was given as to the correctness of the response. Results. All analyses were executed using R27 and package ‟modelfree”28. We analyzed the three parameters: Point of Subjective Equality (PSE, the 50% chance that the duration was perceived as longer), Threshold, and Slope of psychometric functions for each of the participants and the group data (Table 2 and Fig. 2). All psycho- metric functions were fitted by the Weibull function. All the data can be found in the Data Availability Session below. All parameters except PSE of the red light condition have passed the Shapiro-Wilk normality test. To deal with the non-normality issue of PSE data in the red light condition, a bootstrapping paired t-test was conducted for comparing the mean PSE from both conditions, using “wBoot” R package29 for 100000 iterations. The mean difference of PSE of both conditions was significant (PSE: bootstrapped mean = −25.79, 95% CI of the mean difference = [−51.88, −4.204], p = 0.0169). For comparing the threshold and slope of the two conditions, two paired t-test was conducted, respectively (Threshold: t(7) = −1.478, p = 0.183, 95% CI = [−21.663, 4.996]; Slope: t(7) = 1.284, p = 0.240, 95% CI = [−0.00017, 0.00058]). See Fig. 2C for the PSEs under the two (blue light vs. red light) conditions. SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 3 3 www.nature.com/scientificreports/ Mean (SE) PSE in ms Threshold in ms Slope Exp1 Blue 1056.76 (16.65) 101.43 (11.33) 0.00244 (0.00035) Red 1082.51 (19.26) 109.76 (12.31) 0.00223 (0.00029) Exp2 ipRGC High 1042.72 (15.89) 115.02 (9.78) 0.00196 (0.00015) Lightflux High 1063.66 (16.54) 102.85 (12.16) 0.00233 (0.00029) Control 1064.87 (18.91) 107.88 (11.57) 0.00216 (0.00021) Table 2. Results of Experiment 1 and 2. Table 2. Results of Experiment 1 and 2. Table 2. Experiment 2 p In Experiment 2, we used a multi-primary stimulator (Fig. 3) that can independently manipulate stimulation of ipRGCs and the three types of cones24,30,31. This system allows us to further examine the influence of increased stimulation of ipRGCs and cones under the same color and luminance (i.e., metameric) backgrounds. By isolating each of these factors, it is expected to clarify the influences of each factors on time perception. Methods. Participants. Eight healthy, naïve volunteers (all males, mean age = 22.4 years old) took part in Experiment 2. Same participant inclusion criteria were used as in Experiment 1. They all gave informed con- sent before their participation. The experiment was approved by the Research Ethics Committee at Kagoshima University and all methods were performed in accordance with applicable research subject guidelines. Stimuli and Apparatus. Spatial arrangement and timing of the presentation of the test stimuli were the same as in Exp.1, except that the background color was always gray (CIE coordinate (0.40, 0. 38)) in all conditions. The observers were seated 81.0 cm in front of the display, which subtended 20.4° × 16.8° in visual angle. A multi-primary stimulator consists of three projectors and interference filters that enables independent stimu- lation of each photoreceptor class. The peak wavelengths of the four primaries were 455 nm, 530 nm, 580 nm, and 595 nm. We used this system to create three conditions, ipRGC High, Lightflux High and Control condition (Fig. 4). The luminance in the Control condition and the ipRGC High condition was 110 cd/m2 while the lumi- nance in the Lightflux High condition was 228 cd/m2. The same calculation of cone stimulations was used as in Experiment 1. The display in the ipRGC High condition and that in the Control condition had the same lumi- nance and color, indicating a metameric pair. The stimulation of ipRGCs in the ipRGC High condition was 2.1 times higher than that in the Control condition. By comparing the three conditions, we were able to tease apart the contribution of luminance, cone stimulation, and ipRGC stimulation to the duration judgment. Design and Procedure. In Experiment 2, we adopted a 3 (condition: ipRGC High, Lightflux High, and Control) × 9 (duration: 750, 833, 900, 983, 1050, 1133, 1200, 1283, and 1350 ms) within-subject design. Each condition repeated three times. www.nature.com/scientificreports/ Figure 3. A set of multi-primary stimulation used in Experiment 2. (A) A multi-primary stimulator is a customized illumination system consisting of three projectors and interference filters which exploits a four- primary illumination system that enables independent stimulation of ipRGCs and the three types of cones. Three projectors on the left created a metameric background with different light components and projected to the screen (the light square on the right) in front of the participant, who sat at the right side that is out of the range of this photo. Black curtain covering the whole system was used in the experiment and it was removed for demonstration purpose here. (B) A schematic illustration of the experimental setting as described above. Figure 3. A set of multi-primary stimulation used in Experiment 2. (A) A multi-primary stimulator is a customized illumination system consisting of three projectors and interference filters which exploits a four- primary illumination system that enables independent stimulation of ipRGCs and the three types of cones. Three projectors on the left created a metameric background with different light components and projected to the screen (the light square on the right) in front of the participant, who sat at the right side that is out of the range of this photo. Black curtain covering the whole system was used in the experiment and it was removed for demonstration purpose here. (B) A schematic illustration of the experimental setting as described above. Discussion. Our analyses in Experiment 1 yielded that perceived duration was lengthened under blue light compared to that under red light. Difference in color, cone, luminance, and ipRGC stimulation were possible factors contributing to this result. Thus, to clarify the role of ipRGCs in affecting perceived duration, in the next experiment, we created a pair of lights with the same color and luminance (i.e., the metameric pair) and manipu- lated different ipRGC stimulations to tease apart the effect of color and luminance30,31. Discussion. Our analyses in Experiment 1 yielded that perceived duration was lengthened under blue light compared to that under red light. Difference in color, cone, luminance, and ipRGC stimulation were possible factors contributing to this result. www.nature.com/scientificreports/ Thus, to clarify the role of ipRGCs in affecting perceived duration, in the next experiment, we created a pair of lights with the same color and luminance (i.e., the metameric pair) and manipu- lated different ipRGC stimulations to tease apart the effect of color and luminance30,31. Experiment 1 Results of Experiment 1 and 2. Figure 2. Results of Experiment 1. (A) The fitted PSE for blue and red light conditions in Experiment 2. The white bar in the middle indicates the bootstrapped 95% confidence intervals of each condition and the black horizontal line in the middle is the group mean. Each dot represents one participant’s fitted PSE value. The gray dashed line in the middle shows the duration of standard stimuli (1050 ms). (B) The group-averaged psychometric functions. (C) The PSE in the blue vs. red light conditions with bootstrapped 95% confidence intervals. The PSE under blue light was significantly smaller than that under red light, indicating a longer perceived duration under blue light than red light. Figure 2. Results of Experiment 1. (A) The fitted PSE for blue and red light conditions in Experiment 2. The white bar in the middle indicates the bootstrapped 95% confidence intervals of each condition and the black horizontal line in the middle is the group mean. Each dot represents one participant’s fitted PSE value. The gray dashed line in the middle shows the duration of standard stimuli (1050 ms). (B) The group-averaged psychometric functions. (C) The PSE in the blue vs. red light conditions with bootstrapped 95% confidence intervals. The PSE under blue light was significantly smaller than that under red light, indicating a longer perceived duration under blue light than red light. SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 4 www.nature.com/scientificreports/ Experiment 2 We conducted the one-way repeated measure ANOVA for the three parameters, including PSE, threshold, and slope. Except for the signifi- cant main effect of PSE (PSE: F(2,14) = 4.929, p = 0.024, ηp 2 = 0.413) (Fig. 5C), both main effects of threshold and slope were not significant (Threshold: F(2,14) = 1.416, p = 0.275, ηp 2 = 0.168; Slope: F(2,14) = 1.640, p = 0.229, ηp 2 = 0.190). Then, the pairwise comparison showed that the significance of PSE came from the difference between ipRGC High condition and Control condition (Bonferroni corrected p = 0.021, 95% CI = [−40.515, −3.779]). Data Availability Statement. The R commands of psychometric function fitting and the raw data in this study are available from the link: http://epa.psy.ntu.edu.tw/data_repository/Time_perception_Data. Discussion. We used a set of multi-primary stimulator to increase stimulation of ipRGCs while color and luminance were kept constant and showed that increasing the stimulation of ipRGCs would affect perceived dura- tion: Increased stimulation of ipRGC led to lengthened perceived duration compared to the Control condition. No difference between the other paired comparison of conditions indicates that cone stimulation might have an opposite effect to that of the ipRGCs. In the Lightflux High condition, since the cone stimulation increased in comparison with that in the ipRGC High condition, cones may inhibit the effects of ipRGCs’ lengthening perceived duration, resulting in no difference between the ipRGC High and Lightflux High conditions. That is, higher ipRGC stimulation would lengthen the perceived duration, while higher cone stimulation (i.e., luminance) might have the opposite effect of the ipRGCs. Experiment 2 The other details were the same as in Experiment 1, except that the seven oddball durations in the practice session were randomly chosen from the nine durations (durations: 750, 833, 900, 983, 1050, 1133, 1200, 1283, and 1350 ms). All participants had been through five minutes light adaptation to a back- ground in each condition. Results. We analyzed the psychometric functions of eight participants (Fig. 5A), and the corresponding parameters. The data were fitted by Weibull model. The group-averaged data (Fig. 5B) of three conditions in SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 5 www.nature.com/scientificreports/ Figure 4. Three conditions in Experiment 2. Three conditions were manipulated, including ipRGC High condition, Control condition, and Lightflux High condition, and every two of the three conditions could be compared to examine the influence of ipRGC as well as cone stimulations (i.e., luminance). For the x-axis, “L”, “M”, and “S” refer to stimulations of the three kinds of cones: Long-wavelength, Middle-wavelength, and Short- wavelength cones. Figure 4. Three conditions in Experiment 2. Three conditions were manipulated, including ipRGC High condition, Control condition, and Lightflux High condition, and every two of the three conditions could be compared to examine the influence of ipRGC as well as cone stimulations (i.e., luminance). For the x-axis, “L”, “M”, and “S” refer to stimulations of the three kinds of cones: Long-wavelength, Middle-wavelength, and Short- wavelength cones. Experiment 2 was summarized in Table 2. First, all the data for three parameters have passed the Shapiro-Wilk test for normality. In addition, we could do the further inference analysis with this data. We conducted the one-way repeated measure ANOVA for the three parameters, including PSE, threshold, and slope. Except for the signifi- cant main effect of PSE (PSE: F(2,14) = 4.929, p = 0.024, ηp 2 = 0.413) (Fig. 5C), both main effects of threshold and slope were not significant (Threshold: F(2,14) = 1.416, p = 0.275, ηp 2 = 0.168; Slope: F(2,14) = 1.640, p = 0.229, ηp 2 = 0.190). Then, the pairwise comparison showed that the significance of PSE came from the difference between ipRGC High condition and Control condition (Bonferroni corrected p = 0.021, 95% CI = [−40.515, −3.779]). Experiment 2 was summarized in Table 2. First, all the data for three parameters have passed the Shapiro-Wilk test for normality. In addition, we could do the further inference analysis with this data. General Discussion To reveal the role of ipRGCs on perceived duration and to resolve the controversy about the effect of blue light on time perception, we used an oddball paradigm as the task for time perception and manipulated different background conditions when judging the duration of the oddball. In Experiment 1, longer time perception was found under blue light compared to red light. In Experiment 2, we attempted to solely control the stimulation of ipRGCs with no change in luminance and color, using a multi-primary stimulator to create metameric conditions to understand the role of increased stimulation of ipRGCs and cones. Results yielded that perceived duration was lengthened for higher stimulation of ipRGCs, while the stimulation of cone may have opposite effect on perceived duration than the ipRGCs. Together, this study provided evidence to dissociate the effect of cones and ipRGCs in contributing the influence of blue light on time perception. gl g p p In past researches, the explanations of blue light on time perception mainly focused on the contribution of color properties, without clarifying the role of ipRGCs. Caldwell and Jones10 found no difference between blue light and other lights, using supra-second time production task, and indicated that their results were influenced by different SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 6 www.nature.com/scientificreports/ com/scientificreports/ Figure 5. The results of Experiment 2. (A) The fitted PSE for all three conditions in Experiment 2. The white bar in the middle indicates the 95% confidence intervals of each condition and the black line is the group mea Each dot represents one participant’s fitted PSE value. The gray dashed line in the middle shows the duration o standard stimuli. (B) The group-averaged psychometric function in the three conditions. (C) The PSE for each condition. The PSE in the ipRGC High condition was significantly lower than that in the Control condition, while no other differences were found between other pairs of conditions. Figure 5. The results of Experiment 2. (A) The fitted PSE for all three conditions in Experiment 2. The white bar in the middle indicates the 95% confidence intervals of each condition and the black line is the group mean. Each dot represents one participant’s fitted PSE value. The gray dashed line in the middle shows the duration of standard stimuli. (B) The group-averaged psychometric function in the three conditions. (C) The PSE for each condition. General Discussion The PSE in the ipRGC High condition was significantly lower than that in the Control condition, while no other differences were found between other pairs of conditions. characteristics of light, such as cold and warm. Gorn11 found time shrank under blue light, through conducting a supra-second time estimation task, and claimed that their results could be explained by different relaxing states induced by different colors. Katsuura et al.12 performed a supra-second time production task, and found time expanded under blue light only at 180 s duration. They listed various possible reasons, such as age, sex, arousal level, and ipRGC influences, but no further test was provided. Shibasaki and Masataka13 used a sub-second time comparison task, and found time shrank under blue light for men. They suggested that this was due to different implicit social meanings for blue than for red. Notably, previous researches did not clarify whether the changed time perception resulted from increased stimulation of ipRGC, or color perception, or both. In this study, we cannot distinguish the contribution of which components in Experiment 1 (cones, luminance, or ipRGCs) lengthened the perceived duration. However, the results from Experiment 2 strongly suggest that higher stimulation of ipRGC was responsible for lengthened perceived duration with the contribution from color excluded and luminance controlled. Although it seems that a larger PSE difference was found between the blue light condition and the red light condi- tion (Experiment 1, mean = −25.75 ms, SE = 12.10 ms) than between the ipRGC High condition and the Control condition (Experiment 2, mean = −22.15 ms, SE = 5.49 ms), the difference between the two experiments was not statistically significant (t(9.7697) = −0.25, p = 0.805, 95% CI = [−35.35, 28.14]). characteristics of light, such as cold and warm. Gorn11 found time shrank under blue light, through conducting a supra-second time estimation task, and claimed that their results could be explained by different relaxing states induced by different colors. Katsuura et al.12 performed a supra-second time production task, and found time expanded under blue light only at 180 s duration. They listed various possible reasons, such as age, sex, arousal level, and ipRGC influences, but no further test was provided. Shibasaki and Masataka13 used a sub-second time comparison task, and found time shrank under blue light for men. They suggested that this was due to different implicit social meanings for blue than for red. General Discussion Notably, previous researches did not clarify whether the changed time perception resulted from increased stimulation of ipRGC, or color perception, or both. In this study, we cannot distinguish the contribution of which components in Experiment 1 (cones, luminance, or ipRGCs) lengthened the perceived duration. However, the results from Experiment 2 strongly suggest that higher stimulation of ipRGC was responsible for lengthened perceived duration with the contribution from color excluded and luminance controlled. Although it seems that a larger PSE difference was found between the blue light condition and the red light condi- tion (Experiment 1, mean = −25.75 ms, SE = 12.10 ms) than between the ipRGC High condition and the Control condition (Experiment 2, mean = −22.15 ms, SE = 5.49 ms), the difference between the two experiments was not statistically significant (t(9.7697) = −0.25, p = 0.805, 95% CI = [−35.35, 28.14]). characteristics of light, such as cold and warm. Gorn11 found time shrank under blue light, through conducting a supra-second time estimation task, and claimed that their results could be explained by different relaxing states induced by different colors. Katsuura et al.12 performed a supra-second time production task, and found time expanded under blue light only at 180 s duration. They listed various possible reasons, such as age, sex, arousal level, and ipRGC influences, but no further test was provided. Shibasaki and Masataka13 used a sub-second time comparison task, and found time shrank under blue light for men. They suggested that this was due to different implicit social meanings for blue than for red. Notably, previous researches did not clarify whether the changed time perception resulted from increased stimulation of ipRGC, or color perception, or both. In this study, we cannot distinguish the contribution of which components in Experiment 1 (cones, luminance, or ipRGCs) lengthened the perceived duration. However, the results from Experiment 2 strongly suggest that higher stimulation of ipRGC was responsible for lengthened perceived duration with the contribution from color excluded and luminance controlled. General Discussion Although it seems that a larger PSE difference was found between the blue light condition and the red light condi- tion (Experiment 1, mean = −25.75 ms, SE = 12.10 ms) than between the ipRGC High condition and the Control condition (Experiment 2, mean = −22.15 ms, SE = 5.49 ms), the difference between the two experiments was not statistically significant (t(9.7697) = −0.25, p = 0.805, 95% CI = [−35.35, 28.14]). 7 SCIENTIFIC REPOrTS \| (2018) 8:11693 \| DOI:10.1038/s41598-018-29613-1 7 www.nature.com/scientificreports/ One might suspect that the subjective time expansion we found here might have been caused by rods rather than ipRGCs, since the peak wavelengths for spectral sensitivity curves of rods and ipRGCs are close to each other23,31–33. To minimize rod contribution in our setup, we used bright stimuli with luminance values between 110 cd/m2 and 228 cd/m2 in Experiment 2. The retinal illuminance was between 738 and 1533 scotopic tro- lands with a pupil size of 3.0 mm. According to Fuortes, Gunkel, and Rushton34, the incremental threshold for rod-based detection increased sharply at a light level above 100 scotopic trolands, suggesting that rods would sat- urate at the retinal illuminance we used in Experiment 2. Although we cannot completely rule out the possibility of rod intrusion, the involvement of rods in our Experiment 2 should be small and negligible34–37.h p g g There are two possible explanations for time expansion with higher ipRGC stimulation: attention and arousal. First, duration judgment of the oddball could be affected by attention19. Lockley et al.38 found that participants showed higher sustained attention under blue light compared to green light. Also, the ipRGCs project to SCN, which controls circadian rhythm6 and is associated with attention39. In addition, blue light might increase the ipRGC stimulation and affect sustained attention, causing lengthened perceived duration. Second, it might be affected by arousal. We adopted the Scalar Timing Theory (STT) of time perception40,41 to explain the results we obtained. The variation of the PSEs obtained from the oddball paradigm has been shown to be contributed by the pacemaker com- ponent in the STT model20, and pacemaker can be accelerated by higher arousal state42. Moreover, higher ipRGC stimulation was correlated with greater arousal state, because the ipRGC neurons would transmit positive signals to SCN38. In addition, ipRGC neurons could modulate the SCN stimulation level43, further lengthening the perceived duration. General Discussion Some behavioral experiments also showed the connection between circadian rhythm (controlled by SCN) and duration judgment; participants responded longer at night and morning than in the middle of the day in repro- duction task44, a common time perception task asking participants to reproduce durations. Regardless of the exact underlying mechanism, both explanations (attention and arousal) need to base on the role of SCN and circadian rhythm. Through influencing the SCN, higher ipRGC stimulation would lengthen the duration judgment. yh gl g g p g j g Notably, the perceived duration did not expand when the cones and ipRGC stimulation increased simul- taneously, compared to the Control condition in Experiment 2. One possible explanation is that the influence on SCN between cones and ipRGCs could be opposite. According to Pilorz et al.45, the effect of delayed sleep onset, controlled by SCN, induced by blue light (470 nm) was established in melanopsin-normal mice but not in melanopsin-deficient mice. Moreover, Allen et al.46 showed that light adaptation ability was different between melanopsin-normal and -deficient mice, using receptor silent substitution. In ipRGCs, melanopsin serves a role as transducing light information to brain regions1,4. Hence, both studies support that some opposite interaction exists between cones and ipRGCs. In addition, here, higher ipRGC stimulation might lengthen perceived dura- tion through increased SCN activation, while higher cone stimulation might shorten perceived duration and cancel out the lengthening effect caused by ipRGCs.hi g gf y p This is the first study using the oddball paradigm and manipulating the background light components to clarify the contributions of ipRGCs and color on time perception. This finding reveals how blue light and ipRGCs affect sub-second interval judgment. Future studies may further investigate the contribution of cones and ipRGCs to the attention and arousal process and conduct experiments on ipRGC-gene knockout mice to confirm the neu- ral mechanism. The multi-primary stimulation used in Experiment 2, on the other hand, can easily manipulate the stimulation of ipRGCs and luminance without changes in color perception, and thus is suitable for conducting experiments in lab settings. References 1. Berson, D. M., Dunn, F. A. & Takao, M. Phototransduction by retinal ganglion cells that set the circadian clock. Science 295 1070–1073 (2002). 2. Do, M. T. H. & Yau, K.-W. Intrinsically photosensitive retinal ganglion cells. Physiological reviews 90, 1547–1581 (2010). 3. Chang, A. M., Aeschbach, D., Duffy, J. F. & Czeisler, C. A. Evening use of light-emitting eReaders negatively affects sleep, circadian timing, and next-morning alertness. 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PLoS biology 14, e1002482 (2016). 46 Allen A E et al Melanopsin driven light adaptation in mouse vision Curr Biol 24 2481 2490 (2014) Acknowledgementsh g This research was supported by Grants from Taiwan’s Ministry of Science and Technology (MOST104-2410-H- 002-061-MY3) to S.-L.Y. and the Ministry of Education, Science, Sports and Culture of Japan, Grants-in-Aid for Scientific Research (B) 26280103 and (B) 17H01808 to S.T. y Scientific Research (B) 26280103 and (B) 17H01808 to S.T. Author Contributions S.-L.Y. and P.-L.Y. developed and designed the study. P.-L.Y. performed Experiment 1 and data analyses for all experiments. A.M. and W.Y. performed Experiment 2; S.T. designed the multi-primary stimulator system in Experiment 2 and analyzed all the spectrums of cones and ipRGCs. P.-L.Y., S.-L.Y. and S.T. wrote the manuscript. S.-L.Y. supervised the whole work. References 39, 2901–2927 (1999). 6. Stockman, A. & Sharpe, L. T. 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Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29613-1.h Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29613-1. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W4238521969	https://www.qeios.com/read/5LFXJB/pdf	English	null	AKAP4 wt Allele	Definitions	2,020	cc-by	88	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios AKAP4 wt Allele National Cancer Institute National Cancer Institute Qeios ID: 5LFXJB · https://doi.org/10.32388/5LFXJB Source National Cancer Institute. AKAP4 wt Allele. NCI Thesaurus. Code C142995. Human AKAP4 wild-type allele is located in the vicinity of Xp11.22 and is approximately 10 kb in length. This allele, which encodes A-kinase anchor protein 4, plays a role in both protein kinase A localization and sperm motility. Aberrant expression may be associated with neoplastic disease. Qeios ID: 5LFXJB · https://doi.org/10.32388/5LFXJB 1/1
https://openalex.org/W3203921994	https://backend.orbit.dtu.dk/ws/files/257768894/1_s2.0_S0956053X21005079_main.pdf	English	null	A life cycle assessment framework for large-scale changes in material circularity	Waste management	2,021	cc-by	13,699	Citation (APA): Andreasi Bassi, S., Tonini, D., Ekvall, T., & Astrup, T. F. (2021). A life cycle assessment framework for large- scale changes in material circularity. Waste Management, 135, 360-371. https://doi.org/10.1016/j.wasman.2021.09.018 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal 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. A life cycle assessment framework for large-scale changes in material circularity ndreasi Bassi, Susanna; Tonini, Davide; Ekvall, Tomas; Astrup, Thomas F. Andreasi Bassi, Susanna; Tonini, Davide; Ekvall, Tomas; Astrup, Thomas F. Published in: Waste Management Link to article, DOI: 10.1016/j.wasman.2021.09.018 Document Version Publisher's PDF, also known as Version of record Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research Citation (APA): Andreasi Bassi, S., Tonini, D., Ekvall, T., & Astrup, T. F. (2021). A life cycle assessment framework for large- scale changes in material circularity. Waste Management, 135, 360-371. https://doi.org/10.1016/j.wasman.2021.09.018 A life cycle assessment framework for large-scale changes in material circularity Susanna Andreasi Bassi a,b,, Davide Tonini b, Tomas Ekvall c, Thomas F. Astrup a Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet, Bygning 115, DK-2800 Kgs. Lyngby, Denmark b European Commission, Joint Research Centre, Edificio Expo, Calle Inca Garcilaso 3, 41092 Seville, Spain c Division of Environmental Systems Analysis, Chalmers University of Technology, 412 96 G¨oteborg, Sweden A R T I C L E I N F O Even if no univocal definition of CE exists (Geisendorf and Pietrulla, 2018) and the focus of CE legislation varies significantly in different geographical areas (McDowall et al., 2017), the majority of CE policies and CE literature focused on waste recovery and recycling to close the materials loops (Ghisellini et al., 2016; Merli et al., 2018; Morseletto, 2020). In Europe, material circularity has been implemented in particular through policy targets focusing on recycling (Morseletto, 2020), where recycling in dicates “any recovery operation by which waste materials are reproc essed into products, materials or substances whether for the original or other purposes” (EC, 2008). A R T I C L E I N F O Keywords: Circular economy Environmental assessment PET trays Policy targets Recycling Increasing material circularity is high on the agenda of the European Union in order to decouple environmental impacts and economic growth. While life cycle assessment (LCA) is useful for quantifying the associated envi ronmental impacts, consistent LCA modeling of the large-scale changes arising from policy targets addressing material circularity (i.e., recycled content and recycling rate) is challenging. In response to this, we propose an assessment framework addressing key steps in LCA, namely, goal definition, functional unit, baseline versus alternative scenario definition, and modeling of system responses. Regulatory and economic aspects (e.g., trends in consumption patterns, market responses, market saturation, and legislative side-policies affecting waste management) are emphasized as critical for the identification of potential system responses and for supporting regulatory interventions required to reach the intended environmental benefits. The framework is recommended for LCA studies focusing on system-wide consequences where allocation between product life cycles is not relevant; however, the framework can be adapted to include allocation. The application of the framework was illustrated by an example of implementing a policy target for 2025 of 70% recycled content in PET trays in EU27+1. It was demonstrated that neglecting large-scale market responses and saturation lead to an over estimation of the environmental benefits from the policy target and that supplementary initiatives are required to achieve the full benefits at system level. models (Bao et al., 2019; Rashid et al., 2013). Even if no univocal definition of CE exists (Geisendorf and Pietrulla, 2018) and the focus of CE legislation varies significantly in different geographical areas (McDowall et al., 2017), the majority of CE policies and CE literature focused on waste recovery and recycling to close the materials loops (Ghisellini et al., 2016; Merli et al., 2018; Morseletto, 2020). In Europe, material circularity has been implemented in particular through policy targets focusing on recycling (Morseletto, 2020), where recycling in dicates “any recovery operation by which waste materials are reproc essed into products, materials or substances whether for the original or other purposes” (EC, 2008). models (Bao et al., 2019; Rashid et al., 2013). Link back to DTU Orbit Link back to DTU Orbit Citation (APA): Andreasi Bassi, S., Tonini, D., Ekvall, T., & Astrup, T. F. (2021). A life cycle assessment framework for large- scale changes in material circularity. Waste Management, 135, 360-371. https://doi.org/10.1016/j.wasman.2021.09.018 this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim. Waste Management 135 (2021) 360–371 Available online 30 September 2021 0956-053X/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.wasman.2021.09.018 Received 28 March 2021; Received in revised form 15 September 2021; Accepted 19 September 2021 Abbreviations: CE, Circular economy; LCA, Life cycle assessment; PEF, Product Environmental Footprint; CAGR, Compound annual growth rate. Corresponding author at: Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet, Bygning 115, DK-2800 Kgs. Lyngby, Denmark. E-mail address: suan@env.dtu.dk (S. Andreasi Bassi). Available online 30 September 2021 0956-053X/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Received 28 March 2021; Received in revised form 15 September 2021; Accepted 19 September 2021 p 0956-053X/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2.1. Framework overview This section describes the assessment framework (Fig. 1) and focuses on those parts of the LCA we believe are most important to address, without aiming to provide comprehensive guidance on how to perform an LCA. The application of the framework is illustrated in Section 3, but several other examples of different goals, functional units, and system boundaries are provided in the Supplementary Material (SM) to support the reader. Since material circularity can be increased by leveraging both recycled content and recycling rates, the framework especially focuses on how to model changes in the recycled content and the recy cling rate. y g g Second, increased material recycling has been assumed to be completely absorbed by the market and to always substitute primary material (as in Andreoni et al., 2015; Gibbs et al., 2014; Hestin et al., 2015; Tallentire and Steubing, 2020), without considering potential market saturation or the scale of the impact (i.e., small-scale versus large-scale changes relative to unsatisfied demand), because all modeled systems have been assumed to be linear without accounting for the volume of recycled material. The majority of studies on non-linearity focus on the problem of modeling the upscaling of emerging and scal able technologies (Arvidsson et al., 2018; Pizzol et al., 2021). However, far less attention has been focused on the different kinds of non-linearity occurring when individual material sources or markets are limited. Ex ceptions to this include Andreasi Bassi et al. (2020) who quantified the risk of recycling market saturation and environmental dispersion via export, Binnemans et al. (2013) who determined whether the market could absorb a co-product of rare earth material mining, and S¨oderman et al. (2016) and Ekvall et al. (2016) that combined macro-economic models with LCAs. g From this point forward, primary materials are materials that have been extracted from or produced from nature (i.e., fossil fuels, metal ores, forests, and plantations), while secondary materials are materials produced from recycling. Furthermore, this framework introduces the concept of side policies that is similar to the economic side policies found in Domenech and Bahn-Walkowiak (2019). In fact, since the legislation on CE is a complex constellation of policy frameworks, economic in centives, and economic side policies (Domenech and Bahn-Walkowiak, 2019), it is often not possible to quantify and isolate the impacts of introducing a single policy target independently from other legislative tools. 1. Introduction Growing awareness of the environmental impacts caused by anthropogenic activities has encouraged decision-makers to factor in environmental implications along with socio-economic aspects when deciding new policies. In recent years, circular economy (CE) has gained traction both at governmental and business levels as a solution to sup port economic growth while reducing environmental footprint (EC, 2015a). CE is a restorative industrial economy concept that is based on three principles: to design out waste and pollution, to keep products and materials in use, and to regenerate natural systems (Ellen MacArthur Foundation, 2017). CE aims at increasing material circularity by reducing the need for resource extraction (i.e., materials, nutrients, etc.), encouraging reuse, repair, and recycling instead of the linear “extract-use-discard” consumption, and supporting innovative business To support the development of policy targets leading to the intended effects, assessment of the environmental consequences associated with full-scale implementation of these policies should reflect appropriate system and framework conditions (Cantzler et al., 2020). Policy targets Abbreviations: CE, Circular economy; LCA, Life cycle assessment; PEF, Product Environmental Footprint; CAGR, Compound annual growth rate. * Corresponding author at: Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet, Bygning 115, DK-2800 Kgs. Lyngby, Denmark. E-mail address: suan@env.dtu.dk (S. Andreasi Bassi). Abbreviations: CE, Circular economy; LCA, Life cycle assessment; PEF, Product Environmental Footprint; CAGR, Compoun * Corresponding author at: Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet, Denmark. Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. for recycling are inherently intended to cause large-scale changes in society, industry, and markets, and thereby affect material and product production capacity. A large-scale change is defined as a consequence that cannot be considered “marginal” nor likely to affect only parts of a market, and thereby warrant assumptions of linearity in market re sponses and material supply (EC, 2010). While the environmental con sequences from implementing such policy targets can be quantified with life cycle assessment (LCA) (EC, 2015b), little methodological guidance has been provided for the application of LCA to large-scale changes with market implications at meso/macro levels. and scope to model changes in recycled content and recycling rates, ii) accounts for jointly assessing the supply and use of recycled materials, and iii) integrates future developments of market conditions and back ground systems, including associated effects on market saturation. 1. Introduction The specific objectives are to: i) describe and document the individual steps and assumptions associated with the framework, ii) illustrate the implementation of the framework for a hypothetical EU-wide recycling target, and iii) put the framework in perspective by comparing it with existing alternatives and discuss its overall limitations of the framework. The framework departs from the authors’ experiences with consequen tial LCAs (i.e., which impacts are due to a change in the system). Attributional modeling (i.e., “process-based modeling intended to pro vide a static representation of average conditions” (EC, 2013)) is not considered, since we agree with many researchers that the consequential approach is more appropriate for modeling system-wide changes and supporting policy decisions (Frischknecht et al., 2017; Sala et al., 2016; Zamagni et al., 2012). We found two main methodological challenges in LCAs aiming at evaluating CE policies and targets in relation to waste management and recycling. First, the majority of methods modeling recycling in LCA (Allacker et al., 2014; EC, 2018a; JRC-EC, 2020; Schrijvers et al., 2016) focus on individual products and divide the environmental burdens/ savings associated with recycling between the product being recycled after use and the product being produced from the recycled material. The way recycling should be modeled in LCA has been debated exten sively in the scientific literature (e.g., Allacker et al., 2014; Schrijvers et al., 2016), and several LCA standards (e.g., PAS 2050, ISO/TS 14067, BPX 30-323-0) offer contradictory guidance. Already in 1993, SETAC (1993) observed that recycling (and, more generally, any linked system) should be assessed by jointly modeling all involved life cycles since concerted actions between actors in different life cycles are required to reach the desired goal (i.e., recycling) within the system. This is particularly important when assessing environmental consequences associated with policy targets intended to have large-scale implications across value and supply chains, as, for example, the changes required in the European material circularity required to comply with current recycling targets. 2.1. Framework overview Side policies are defined as other legislative tools (e.g., targets, bans, monitoring tools, economic tools) that address the same functional unit of the study. For example, the consequences of increasing recycling can vary depending on the implementation of landfilling and incinera tion bans, exporting or importing embargos, minimum recycled content, design requirements, incentives in sorting and recycling plants, etc. Without addressing the effects on value-chains over several life cy cles, the full consequences of a society-level policy such as recycling targets cannot be encompassed by the LCA. Simply applying individual product LCAs as support for legislative decisions on recycling and, thereby, ignoring scale effects and potential market saturation, may lead to false expectations of savings and burdens associated with new recy cling initiatives. In the case of large-scale policy initiatives, this may lead to the rollout of unsubstantiated regulation, without the intended effects on environmental impacts and resource efficiency. Based on existing literature, further guidance is needed for transparent and consistent LCAs addressing the societal transition and the policies supporting ma terial circularity. 2.3. Functional unit and reference flow 2.3. Functional unit and reference flow 2.3. Functional unit and reference flow life cycle generating the secondary material to the system in focus and the products absorbing the secondary material generated by the system in focus (Fig. 2 and Fig. 3). Expanding the system will also show the intrinsic mutual interaction between legislation covering the secondary material market and legislation covering the waste management market. The functional unit defines the service assessed in the LCA (Fig. 1, b) and the reference flow that fulfills the functional unit. Due to the scope of the framework, the functional unit could address a material flow such as represented by an anthropogenic consumption (e.g., “cardboard consumed in the USA in 2025) or a specific waste generated (e.g., “plastic packaging waste generated in the EU27 between 2020 and 2030”). l For instance, an LCA could have the goal “To quantify the environ mental impact of increasing the recycled content of PET bottles in the EU27+1 from the expected 11% to 25% by 2025, combined with the needed changes in the waste management system to reach such recycled content.” or “To quantify the environmental impact of increasing the recycling rate of PET bottles in the EU27+1 from the expected 50% to 70% by 2025, combined with induced reactions of the market absorbing the generated secondary material”. Here, the PET bottles are the system in focus, and the phrases in italic reveal the system expansion and the avoidance of the allocation to capture all possible effects. On the other hand, the goal “To quantify the environmental impacts of increasing the recycled content of PET bottles in the EU27+1 from the expected 11% to 25% by 2025” would indeed require allocation, because this goal does not include the reaction of the waste management system providing the secondary material (e.g., increasing in source-separation, increasing of recycling, simple diversion from other products that were before using the secondary material). Depending on the study, the composition of the reference flow can be subdivided between different stakeholders (e.g., waste generated from households, industry), sectors (e.g., packaging, textile), plastic polymers (e.g., PET, HDPE), colors, products (e.g., PET bottles and PET trays), or quality (e.g., low- and high-quality waste paper). Often, the composition of multi-material fractions (e.g., scrap vehicles and mixed waste) needs to be estimated or based on other data. 2.2. Goal: Assessing the impact of waste policy targets The goal of an LCA (Fig. 1, a) summarizes the reasons for carrying out a study and its intended application. The goal specifies the material circularity change under investigation, which for waste policy often translates into a target rate (e.g., “collection rate”, “recycling rate”, “landfilling rate”, “incineration rate”). The goal also specifies the geographical and temporal scope of the LCA. i The goal is also the phase where it is clarified whether or not the impacts will be allocated between product life cycles. To assess the impacts of changes in the material circularity in a society, we recom mend avoiding allocation. This is justified by the system-level approach that legislators usually take, whereby the goal is to quantify the overall consequences of a policy and not to subdivide burdens and savings of recycling between different products or stakeholders. Allocation can be avoided through the expansion of the system. In this case, the system is expanded to include the consequences in the waste management of the The goal of this study is to develop a guiding framework supporting a consistent goal and scope definition for consequential LCAs of large- scale changes in material circularity. The focus is on changes induced by implementing policy targets on recycled content and post-consumer recycling rate of products and materials. i The framework i) describes how to consistently define LCAs’ goal 361 Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. Effect of policy targets: • Quantitative target • Geographic scope • Temporal scope, if any • Allocation between different life cycles • Composition of the functional unit • Size (current generation rate) • Expected growth rate Scale? Small Large - Long-term marginal - Dynamic Which other markets are affected? Goal (a) Functional unit and reference flow (b) Modeling and calculation of the system response (f) Baseline scenarios (c) • The handling of the service without implementation of any strategies/policies • Tested initiatives/strategies • System responses • Consumption trends • Technological advancements Alternative scenarios (d) Overlap of baseline and alternative scenarios result in the flows affected by the change Product system and system boundaries (e) Background processes Foreground processes Due to increased recycled content Due to increased recycling rate - Choice of the effectiveness factors - Identification of the competitive markets Fig. 1. Proposed framework to define the goal and scope of consequential LCAs aiming at quantifying the impacts of increasing/ decreasing material circularity. 2.2. Goal: Assessing the impact of waste policy targets The blue ar rows indicate the connections between the different steps. Letters (a) to (f) indicate the steps described in more detail in Sections 2.2 to 2.6. All steps are affected in the case of temporally dynamic modeling (see Section 2.6.9). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 1. Proposed framework to define the goal and scope of consequential LCAs aiming at quantifying the impacts of increasing/ decreasing material circularity. The blue ar rows indicate the connections between the different steps. Letters (a) to (f) indicate the steps described in more detail in Sections 2.2 Fig. 1. Proposed framework to define the goal and scope of consequential LCAs aiming at quantifying the impacts of increasing/ decreasing material circularity. The blue ar rows indicate the connections between the different steps. Letters (a) to (f) indicate the steps described in more detail in Sections 2.2 to 2.6. All steps are affected in the case of temporally dynamic modeling (see Section 2.6.9). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) to 2.6. All steps are affected in the case of temporally dynamic modeling (see Section 2.6.9). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Overlap of baseline and alternative scenarios result in the flows affected by the change Due to increased recycled content Due to increased recycling rate Scale? - Choice of the effectiveness factors - Identification of the competitive markets - Long-term marginal - Dynamic Which other markets are affected? 2.3. Functional unit and reference flow Since this framework focuses on material circularity, we suggest collecting information on how the re cyclables and secondary materials market are subdivided and then base the composition on such understanding. Since policy targets always refer to future years (e.g., by 2035 only 10% of the municipal solid waste should be landfilled in the European Union (EC, 2018b)), the future consumption or waste generation (e.g., municipal solid waste generated in 2035 or between today and 2035) 362 Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. S. Andreasi Bassi et al. Consumption Waste Increased recycling and avoided primary material Competition with a competitive market Increased recycling and avoided primary material + Decrease competitive recycling and induced competitive primary material Waste providing the secondary material for the recycled content Products absorbing the secondary material from the recycling Increased recycling and avoided primary material Competition with a competitive market Increased recycling and avoided primary material + Decrease competitive recycling and induced competitive primary material Fig. 2. System boundaries of increasing/decreasing the recycled content (RIN[kg/kg or %]) and the recycling rate (ROUT [kg/kg or %]) in case the goal of the study avoids (red dotted lines) or includes (gray dotted lines) allocation between different life cycles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Waste providing the secondary material for the recycled content Competition with a competitive market Increased recycling and avoided primary material + Decrease competitive recycling and induced competitive primary material Increased recycling and avoided primary material + Decrease competitive recycling and induced competitive primary material Consumption Waste Competition with a competitive market Increased recycling and avoided primary material + Decrease competitive recycling and induced competitive primary material Increased recycling and avoided primary material Decrease competitive recycling and induced competitive primary material Products absorbing the secondary material from the recycling Fig. 2. System boundaries of increasing/decreasing the recycled content (RIN[kg/kg or %]) and the recycling rate (ROUT [kg/kg or %]) in case the goal of the study avoids (red dotted lines) or includes (gray dotted lines) allocation between different life cycles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) material circularity. 2.4. Baseline and alternative scenarios We propose to calculate the impacts as the difference between the baseline scenario (no change, “frozen policy” situation) and the alter native scenarios, following best practice in impact assessment of policies (EC, 2015b). 2.3. Functional unit and reference flow While the range of alternative scenarios should reflect the range and complexity of potential effects from the imple mentation of the policy target, it is advised that this is limited within an individual study to ensure transparency in interpretation and commu nication towards decision-makers. has to be quantified. Assumptions on future consumption can be based on the expected annual growth rate or compound annual growth rate (CAGR), economic growth, technological innovation, material effi ciencies, etc. The system can be tested for different market trends (e.g., if the consumption increases more than expected, or remains stable) that fulfill the same service to be assessed (i.e., the functional unit), and this can help evaluating the impact of changing the demand (i.e., avoiding consumption) or comparing waste prevention against end-of-pipe solu tions (described in Section 2.6.3). has to be quantified. Assumptions on future consumption can be based on the expected annual growth rate or compound annual growth rate (CAGR), economic growth, technological innovation, material effi ciencies, etc. The system can be tested for different market trends (e.g., if the consumption increases more than expected, or remains stable) that fulfill the same service to be assessed (i.e., the functional unit), and this can help evaluating the impact of changing the demand (i.e., avoiding consumption) or comparing waste prevention against end-of-pipe solu tions (described in Section 2.6.3). Analysis of the economic and legislative context is thus fundamental to derive scenarios and to avoid unrealistic or impossible scenarios. Modeling efforts could benefit from collecting information on the regulation of contaminants in the waste, potential and current markets providing the secondary material (e.g., which markets could supply the secondary materials used in the functional unit?), potential and current end-markets (e.g., which markets could absorb the secondary material produced by recycling the waste), etc. 2.5. System boundaries In the system boundaries (Fig. 1, e), all processes associated with the functional unit are identified. Fig. 2 illustrates the processes and flows that should be included when the alternative scenario has a different recycled content (ΔRIN [kg/kg or %]) or recycling rate (ΔROUT [kg/kg or %]) compared with the baseline scenario. l The baseline (Fig. 1, c) has many synonyms in the literature (“reference”, “business-as-usual”, “current scenario”, “counterfactual”, “conventional scenario”, “status quo“), reflecting “what would happen to the functional unit if nothing changed beyond already implemented or decided policies”. This involves the definition of a baseline that evolves over time (e.g., as a result of changes in framework conditions from the implementation of existing policies) in the absence of new initiatives (EC, 2015b): e.g., involving gradual changes in collection and recycling efficiency, technology development, and energy supply. A single baseline scenario may be appropriate in the case of relatively short timeframes, but multiple baseline scenarios are advised to encompass distinct future developments of system (and framework) conditions as part of a sensitivity assessment, as in S¨oderman et al. (2016). i As also reflected in other frameworks (Ekvall, 2000; Schrijvers et al., 2020), an increase in recycled content affects the demand for secondary material that can either increase recycling of the waste providing the secondary material and/or shift the use of secondary materials from other products. Similarly, an increase in the collection of material for recycling affects the supply of secondary material. Depending on market dynamics, this might increase the recycled content in some products, and/or reduce the supply of secondary materials from other sources. Decisions on recycling made in one product life cycle affect wider markets and the circularity of other products. To account for these ef fects, we need to evaluate the interactions between several different markets. Alternative scenarios (Fig. 1, d) represent specific alternatives implementating the policy target in question, and here reflect how the full system reacts to an external “impulse” such as increasing or reducing 363 Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. 2.5. System boundaries Waste Management 135 (2021 Consumption Waste - - + - - + + + - Waste providing the secondary material for the recycled content Products absorbing the secondary material from the recycling - - + - - + + + - + + + + - Consumption Waste - - + - - + + + - Waste providing the secondary material for the recycled content Products absorbing the secondary material from the recycling - - + - - + + + - + + + + - a) b) System boundaries (red dotted lines) of an LCA assessing a change in the recycled content (ΔRIN∕= 0) and in the recycling rate (ΔROUT∕= 0) displ recycling processes (green boxes), primary material production (yellow boxes), energy recovery and disposal activities (grey boxes), and do s (orange boxes). a) without allocation between life cycles; b) with allocation between life cycles. The processes are explained in Sections 2.6.1 to 2 erms are described in Table 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thi si Bassi et al. Consumption Waste - - + - - + + + - Waste providing the secondary material for the recycled content Products absorbing the secondary material from the recycling - - + - - + + + - + + + + - a) a) Products absorbing the secondary material from the recycling Consumption Waste - - + - - + + + - Waste providing the secondary material for the recycled content Products absorbing the secondary material from the recycling - - + - - + + + - + + + + - b) - Waste providing the secondary material for the recycled content Products absorbing the secondary material from the recycling Consumption Waste Fig. 3. System boundaries (red dotted lines) of an LCA assessing a change in the recycled content (ΔRIN∕= 0) and in the recycling rate (ΔROUT∕= 0) displaying the affected recycling processes (green boxes), primary material production (yellow boxes), energy recovery and disposal activities (grey boxes), and downstream processes (orange boxes). a) without allocation between life cycles; b) with allocation between life cycles. The processes are explained in Sections 2.6.1 to 2.6.7, and all the terms are described in Table 1. literature because of the non-linearity of this framework. literature because of the non-linearity of this framework. The red lines in Fig. 2 show the system boundaries without allocation between different life cycles (recommended when the full impacts of a policy target are assessed), while the gray lines show the system boundaries in the case of allocation. By subtracting the alternative scenarios from the baseline, it is possible to identify those processes that are small- or large-scale compared to the market of interest. Fig. 3 shows the system bound aries and the affected process of an LCA assessing a change in the recycled content (ΔRIN∕= 0) and the recycling rate (ΔROUT∕= 0) without (Fig. 3,a) and with (Fig. 3, b) allocation. 2.6. Modeling and calculating the environmental impacts The equations in Sections 2.6.1 to 2.6.9 can be used for both small- and large-scale changes in the foreground system, but the input data can vary according to the magnitude of the investigated change. As described in Section 2.4, we are interested in studying the dif ference between the baseline system and alternative scenarios, and including only the affected processes. The environmental impact of the difference between the baseline and alternative scenarios ΔEtot can be summarized with the following equation: ΔEtot = ΔEIN + ΔEOUT + ΔED, where the impacts due to a change in the recycled content (ΔEIN) are summed to the impacts related to a change in the recycling rate (ΔEOUT) and to a change in the demand (ΔED). All the terms presented in the equations in Sections 2.6.1 to 2.6.9 are explained in Table 1. To note that the terms describing the life cycle impact assessment are not specific (i.e., given per kg) as in other 2.6.1. Environmental impacts due to a change in recycled content (ΔEIN) i 2.5. System boundaries (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) literature because of the non-linearity of this framework. 2.6.1. Environmental impacts due to a change in recycled content (ΔEIN) i Recycled content (RIN) is defined here as the ratio between the input secondary material and the total input material required to make a product. The impact of a ΔRIN ∕= 0, if the goal avoids allocation, is described in Eq. (1) (for a unit of analysis), which shows the two possible chain ef fects illustrated by the two lines in the equation. All the terms of Eq. (1) 364 Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. Table 1 Description of the terms used in Sections 2.6.1 to 2.6.9. Term Description Unit ΔEtot Difference between the environmental impacts of the alternative scenarios and in the baseline scenario. impact FU ΔEIN Difference between the environmental impacts of the alternative scenarios and in the baseline scenario due to a change of the recycled content (RIN). impact FU ΔEOUT Difference between the environmental impacts of the alternative scenarios and in the baseline scenario due to a change of the recycling rate (ROUT). impact FU ΔED Difference between the environmental impacts of the alternative scenarios and in the baseline scenario due to a change of the demand to fulfill the service of the functional unit. impact FU Alternative scenarios with different recycled content. ΔRIN Difference of the recycled content in the alternative scenarios and in the baseline scenario. The recycled content is defined as the ratio between the input secondary material and the total input material. kg kg or% XIN, XOUT Effectiveness factors. Number between 0 and 1. No- dimension Erec,IN Total environmental impacts of recycling the waste that provides the additional input secondary material. impact FU Ed,IN Total environmental impacts of the avoided energy recovery and disposal* of the waste providing the additional input secondary material. impact FU Ev,IN Total environmental impacts of the avoided production of primary materials in case the increased recycled content causes a reduction of primary material in the market. impact FU QIN Ratio between the quality (quantified with technical characteristics or market values) of the secondary material produced to increase the recycled content and of the primary material used in the baseline scenario. * ΔEα,IN Difference between the downstream environmental impacts of the use of the input secondary material instead of the input primary material. impact FU Ec v,IN Total environmental impacts of the induced production of primary materials in case the increased recycled content causes a reduction of recycling in a competing market. 2.6.1. Environmental impacts due to a change in recycled content (ΔEIN) i impact FU Ec rec,IN Total environmental impacts of the avoided recycling of the competing waste that was recycled in secondary material in the baseline scenario in case the increased recycled content causes a reduction of recycling in a competing market. impact FU Ec d,IN Total environmental impacts of the induced energy recovery and disposal* of the competing waste that was recycled in secondary material in the baseline scenario in case the increased recycled content causes a reduction of recycling in a competing market. impact FU ΔEc α,IN Difference between the downstream impacts of using the competing secondary material instead of the primary material in case the increased recycled content causes a reduction of recycling in a competing market (e.g., use, transport) impact FU Table 1 (continued) Term Description Unit Difference between the downstream environmental impacts of the production of secondary material in the alternative scenarios and in the baseline scenario. Ec v,OUT Total environmental impacts of the induced production of primary materials in case the increased recycling rate causes a reduction of recycling in a competing market. impact FU Ec rec,OUT Total environmental impacts of the avoided recycling of the competing waste that was recycled in secondary material in the baseline scenario in case the increased recycling rate causes a reduction of recycling in a competing market. impact FU Ec d,OUT Total environmental impacts of the induced energy recovery and disposal* of the competing waste that was recycled in secondary material in the baseline scenario in case the increased recycling rate causes a reduction of recycling in a competing market. impact FU ΔEc α,OUT Difference between the downstream impacts of using the competing secondary material instead of the primary material in case the increased recycled content causes a reduction of recycling in a competing market (e.g., use, transport). impact FU Table 1 (continued) Table 1 Description of the terms used in Sections 2.6.1 to 2.6.9. Table 1 * The units of QIN and QOUT depend on how these factors are calculated. Recycling here includes collection, sorting, transportation, recycling pro cesses, and the fate of residues from such processes. i *** Disposal here includes incineration without energy recovery, landfilling, and environmental dispersion. are explained in Table 1. The effectiveness factor XIN is a number between 0 and 1 and splits the mass between the two chain effects, depending on the forecasted market reaction. are explained in Table 1. The effectiveness factor XIN is a number between 0 and 1 and splits the mass between the two chain effects, depending on the forecasted market reaction. ΔEIN = ΔRINXIN ( Erec,IN −Ed,IN −Ev,INQIN + ΔEα,IN ) + ΔRIN(1 −XIN)* (( Erec,IN −Ec rec,IN ) + (−Ev,INQIN + Ec v,INQc IN)+(−Ed,IN + Ec d,IN) + (ΔEα,IN −ΔEc α,IN) ) (1) In the first case, the increased secondary material is coupled with an increase in recycling in the previous life cycle of the waste providing the secondary material, and it is quantified by summing the direct impacts of recycling (Erec,IN), the avoided energy recovery and disposal of waste that is now recycled ( −Ed,IN), and the avoided production of primary material ( −Ev,IN). Since the secondary material often has lower func tionality than the avoided primary material, Ev,IN is multiplied by a factor QIN, representing the ratio between the quality of the secondary material and of the primary material. Quality can be based either on technical characteristics (Rigamonti et al., 2020; Zink et al., 2016) or market value (Allacker et al., 2014; Schrijvers et al., 2016). Further more, differences in the downstream impacts between the use of sec ondary versus primary material are considered (ΔEα,IN) and described in Section 2.6.7. Alternative scenarios with different recycling rates. ΔROUT Difference of the recycling rate in the alternative scenarios and in the baseline scenario. The recycling rate is defined as the ratio between the output secondary material (after collection, sorting, and recycling losses) and the total generated waste. kg kgor % Erec,OUT Total environmental impacts of the additional waste recycling. impact FU Ed,OUT Total environmental impacts of the avoided energy recovery and disposal* of the waste being recycled in the alternative scenarios. impact FU Ev,OUT Total environmental impacts of the avoided production of primary materials in case the increased recycling rate causes a reduction of primary material in the market. 2.6.2. Environmental impacts due to a change in the recycling rate (ΔEOUT) 2.6.2. Environmental impacts due to a change in the recycling rate (ΔEOUT) The recycling rate (ROUT), also called the end-of-life (EoL) recycling rate (Graedel et al., 2011), is defined as the ratio between secondary material after collection, sorting, and recycling losses and the waste generated. i The choice of XIN can be supported by quantifying the mass available for additional recycling, e.g., how much waste is it possible to collect from the residual waste, how easily accessible is such a mass, what are the political and economic side policies that could be implemented to support such a collection (e.g., extended producer responsibility, deposit systems). Second, competing markets supplying the secondary material The impact of a ΔROUT ∕= 0, if the goal avoids allocation, is described in Eq. (2) (for a unit of analysis), which shows the two possible chain effects illustrated by the two lines in the equation. All the terms of Eq. (2) are explained in Table 1. The effectiveness factor XOUT is a number between 0 and 1 and splits the mass between the two chain effects, depending on the forecasted market reaction. ΔEOUT = ΔROUT* ( (1 −XOUT)* ( Erec,OUT −Ed,OUT −Ev,OUTQOUT + ΔEα,OUT ) ) + ΔROUTXOUT* (( Erec,OUT −Ec rec,OUT ) + (−Ev,OUTQOUT + Ec v,INQc OUT)+(−Ed,OUT + Ec d,OUT) + (ΔEα,OUT −ΔEc α,OUT) ) (2) to fulfill the functional unit should be identified (e.g., composting could be the competing market of suppling organic fertilizers instead of the digestate). Third, the own-price elasticity of supply for the recyclable material could be used to indicate the direction of the market. Similarly to the recycled content (Section 2.6.1), in the first case, the produced secondary material avoids some primary material that would have been produced without recycling ( Ev,OUTQOUT ) , and the impacts of recycling ( Erec,OUT ) are added to the avoided energy recovery and disposal ( −Ed,OUT ) and to the downstream impacts (ΔEα,OUT). In the second case, additional recycling simply replaces a competitive material that is now not recycled but sent to energy recovery and disposal. Also in this case, if the processes related to the functional unit are the same as the ones in the competitive market ( Erec,OUT = Ec rec,OUT; Ed,OUT = Ec d,OUT; The choice of XOUT should be made after collecting data on end- market (markets absorbing the secondary materials) types, size, and trends. 2.6.4. The effectiveness factors XIN and XOUT As highlighted in Eqs. (1) and (2), the impacts can be quite different, 2.6.3. Demand (ΔED) The expected future demand of the functional unit can be affected by different strategies, as in the case of waste prevention measures (e.g., a ban on plastic bags) or due to a change in consumer behavior. The ΔED (see Eq. (3)) can be calculated as a sum of avoided material production, including both primary and secondary material, as described in Section 2.6.1, and of the avoided waste management stage, including both the direct burdens and savings that would have happened in the case of recycling (Section 2.6.2) in the baseline scenario. ΔED = ΔD(EIN + EOUT + Eα) (3) ΔED = ΔD(EIN + EOUT + Eα) (3) 2.6.2. Environmental impacts due to a change in the recycling rate (ΔEOUT) A first estimate could be done by looking for products already advertised in the market. End-markets are important because they highlight not only which markets absorb the secondary material, but also why and under what conditions (e.g., the color and/or purity of a material). Another relevant characteristic of end-markets is the current recycled content and saturation level. The potential volume of the market can be calculated by multiplying the size of the end-market by the maximum (current or potential) recycled content. Unsaturated vol ume is obtained by subtracting the current use of secondary material from this potential volume. Maximum recycled content depends on several factors, such as technical limitations, quality of the secondary material, color, etc. the ones in the competitive market ( Erec,OUT = Ec rec,OUT; Ed,OUT = Ec d,OUT; Ev,OUT = Ec v,OUT; QOUT = Qc OUT; ΔEα,OUT = ΔEc α,OUT ) , increased recycling does not have any net environmental burden/ saving. If allocation is needed, XOUT becomes an allocation factor (see Section 1.2, Supplementary Material). More information on XOUT, Erec,OUT, Ed,OUT, QOUT, ΔEα,OUT can be found in the Sections 2.6.4 to 2.6.7. Another indication of short-term possible market saturation can be forecasted by looking at the response of the market, in particular to crisis conditions. Information on imported/exported waste can provide several indications on market health, the geographical location where potential environmental burdens or savings will take place, and the presence of environmental risks (e.g., environmental dispersion). For example, the recent Chinese ban on low-quality plastic and paper, and the following bans set in place by several other Asian countries, pin pointed the materials for which European recycling industry capacity was saturated or non-existent (i.e., plastic and low-quality paper). Finally, known market bottle-necks or specific weaknesses in the value chains of interest could be relevant. Table 1 impact FU QOUT Ratio between the quality (quantified with technical characteristics or market values) of the additional secondary material and of the primary material used in the baseline scenario. ΔE O impact In the second case, the increased secondary material simply reduces the recycled content in other markets, e.g., increasing the use of recycled nutrients from food waste could simply reallocate these nutrients, rather than sourcing additional food waste from the mixed municipal waste sent to energy recovery and disposal. In this case, we are certainly avoiding the use of primary material to fulfill our functional unit (−Ev,IN), but we are also inducing the production of a competitive pri mary material market from which we are diverting the secondary ma terial (Ec v,IN). Note that the competitive market can be composed of different products/markets. Furthermore, we should consider the dif ference between the collecting/sorting/recycling processes needed for our functional unit (Erec,IN) compared to the collecting/sorting/recycling processes required to fulfill competitive demand ( −Ec rec,IN), as well as the difference between avoided energy recovery and disposal ( −Ed,INP) and induced energy recovery and disposal (Ec d,IN). If the processes avoided 365 S. Andreasi Bassi et al. Waste Management 135 (2021) 360–371 dependent on the effectiveness factors XIN and XOUT that describe to what extent the additional recycled content and recycling rate avoid primary material production, or to what extent they simply compete with other secondary materials in the market. The difference between these effectiveness factors and the more common allocation factors is discussed in Section 4.1. and induced are the same (Erec,INP = Ec rec,IN; Ev,IN = Ec v,IN; QP = Qc IN, ΔEα,IN = ΔEc α,IN), increased recycling does not bring any net environ mental burden/saving. In case allocation is needed, XIN becomes an allocation factor that can be based on the PEF (EC, 2013; Zampori and Pant, 2019) or other market-based methods (see Section 1.2, Supplementary Material). The effectiveness factors quantify the full reaction of the market and forecast the consequences of changes therein. This is a complex and uncertain task, especially in the case of large-scale changes. Due to the uncertainty surrounding these factors, several scenarios should be modeled to evaluate the sensitivity of these assumptions. We do not provide a mathematical equation on how to calculate the effectiveness factors, but we do emphasize the critical data that should be collected for their quantification. Table 1 More information on XIN, Erec,IN, Ed,IN, QIN, ΔEα,INcan be found in the Sections 2.6.4 to 2.6.7. 2.6.9. The dimension of time As mentioned earlier, disposal is defined in this paper as including incineration without energy recovery, landfilling, and environmental dispersion. Since assessing large-scale changes in material circularity is often used to support policy targets, which are defined for a precise future year, we incentivize LCA practitioners to model a dynamic (i.e., time- dependent) LCA instead of a static LCA, where possible. This will allow for comparing the results of different ways of transitioning to reach such policy targets. This aspect is often under-evaluated in other methods for modeling waste management and recycling. i The impacts of energy recovery and disposal (Ez d,y, being y either IN or OUT and z the market objective of the study or the competitive market c) can be calculated as in Eq. (4): Ey d,x = Rinc* ( Ez coll,y + Ez inc,y −Ez energyinc,y ) + Rland* ( Ez coll,y + Ez land,y −Ez energyland,y ) + (1 −ROUT −Rinc −Rland)Ez other,y (4) There is no clear definition in the literature of what constitutes a dynamic LCA, and there is no consensus on how to deal with the issues of different time horizons and discounting (Lueddeckens et al., 2020), even though there is a growing interest in the topic (Sohn et al., 2020). In general, dynamic LCAs can include the dynamic modeling of goal and scope, inventory analysis, dynamic impact assessment (i.e., using time- dependent characterization factors), and interpretation with time- dependent weighted factors (Sohn et al., 2020). (4) Eq. (4) shows that the total environmental impacts of the collection Ez coll,y[impact FU ] (e.g., the collection of mixed residual waste) is added to the total direct environmental impacts of incineration (Ez inc,y[impact FU ]) and land filling (Ez land,y[impact FU ]), and subtracted from the avoided impact of the energy (electricity and heat) that would have been produced without energy re covery and disposal technologies (Ez energyinc,y[impact FU ]; Ez energyland,y[impact FU ]). Note that energy recovery also occurs when landfilling, for example, organic waste. The factors ROUT (recycling rate), Rinc (% of waste sent to inciner ation), and Rland (% of waste sent to landfill) are mass factors. We also added the impacts of waste that is neither recycled, incinerated, nor landfilled (Ez other,y[impact FU ]), as highlighted for environmental dispersion/lit tering in (Andreasi Bassi et al., 2020). 3. Application on PET tray circularity The application of the framework is illustrated for the hypothetical evaluation of an EU-wide policy target on PET trays. PET trays are defined as all the thermoforms packaging made of PET (Petcore Europe, 2016) and represent one of the plastic circular economy bottlenecks because they currently absorb a high quantity of secondary PET while having a very low source-separation and recycling rate (Plastics Re cyclers Europe, 2020). The framework is illustrated by showing the goal and scope, the life cycle inventory, and the mass balance. No life cycle impact assessment results are provided because it is beyond the scope of this paper. 2.6.7. Downstream impacts Downstream impacts (ΔEα,IN, ΔEc α,IN, ΔEα,OUT, ΔEc α,OUT) can happen every time a primary material is avoided or induced (Ev,IN, Ec v,IN, Ev,OUT, Ec v,OUT). They include all of the direct and indirect impacts that are caused by using secondary material instead of primary material (Schrijvers et al., 2020), such as different manufacturing processes (e.g., the need to use more additives), different impacts in the use phase (e.g., emissions from the use on land of organic fertilizers versus mineral fertilizers, or the combustion of natural gas versus biogas), and differ ences in waste management (e.g., incineration or landfilling of fossil plastic versus biobased plastic, or where increased use of secondary material lowers the recyclability of products). Such a dynamic goal and scope and system inventory could then be followed by the dynamic accounting of emissions, in order, for example, to capture the time-related effects associated with biogenic carbon flows (Brand˜ao et al., 2013; Cherubini et al., 2011; Faraca et al., 2019; Tonini et al., 2021). Among the downstream indirect impacts, one could also include (market-mediated) rebound effects since the reduced demand for pri mary material might lead to increased consumption of other goods using that material. Macro-economic models (e.g., partial or general equilib rium models) can be used to forecast such responses of the economy (Almeida et al., 2020). 2.6.9. The dimension of time Even if we do not aim at covering all of the challenges of time ho rizons and dynamic modeling, we suggest having a dynamic goal and scope that leads to a dynamic system inventory. In relation to policies involving circularity, the choice of a dynamic goal and scope helps investigate time-related consumption trends, policy implementation pathways (for example, constant improvements in time or fast invest ment in the last year), side policies and their time frames, monitoring indicators, technological improvements, and system developments. The results could also point out the years where an unwanted market response is more likely to happen. For example, since many databases provide yearly datasets, all of the processes included in Eqs. (1) to (4) should be assigned the year they are most likely to happen, and each year could have a different life cycle inventory, for example, a different energy mix composition, technological efficiencies (e.g., sorting and recycling efficiencies), consumption rates, bio-based content, etc. An example of a dynamic life cycle inventory can be found in Andreasi Bassi et al. (2020). 2.6.6. Energy recovery and disposal (material exiting the loop) 2.6.6. Energy recovery and disposal (material exiting the loop) i 2.6.6. Energy recovery and disposal (material exiting the loop) 2.6.8. Background processes LCA modeling involves several other multifunctional processes running in the background that needs to be solved with system expan sion (e.g., energy generation from incineration plants, ancillary material consumption). Changes in background systems, such as electricity pro duction, material production, etc., are often marginal, even when the foreground change is significant. To model marginal changes in back ground systems, we recommend using long-term marginal processes, although acknowledging that the uncertainty in actual marginal impacts is significant (Eriksson et al., 2007). Several systematic approaches exist for identifying marginal processes (Mathiesen et al., 2009; Mattsson et al., 2003; Palazzo et al., 2020; Weidema et al., 1999). 3.2. Functional unit 2.6.5. Recycling Note that environmentally important downstream impacts (Eα) should be considered if present (Section 2.6.7). The impacts of recycling (Erec,P; Ec rec,P; Erec,WM; Ec rec,WM) include collection, sorting, transportation, recycling processes, and the fate of residues from such processes (e.g., sorting rejects). The information gathered in Section 2.6.4 helps identify these activities. 2.6.4. The effectiveness factors XIN and XOUT As highlighted in Eqs. (1) and (2), the impacts can be quite different, 366 S. Andreasi Bassi et al. S. Andreasi Bassi et al. Waste Management 135 (2021) 360–371 Waste Management 135 (2021) 360–371 3.1. Goal The goal is “To quantify the environmental impact of implementing a new policy target increasing the recycled content of the PET trays consumed in the EU27+1 to 70% in 2025 (from the current 40%), combined with different changes in the waste management system to reach such recycled content”. The split of impacts between subsequent life cycles is not the focus of the policy-maker, as the interest is placed in a system-level assessment and in highlighting the hotspots and the risks that would prevent environmental improvements from occurring. The impacts of large-scale changes in the background system are more difficult to pinpoint. System dynamics models, technology choice models, or agent-based models - in principle - can be used for this pur pose (Palazzo et al., 2020). A mix of expertise on systems, technology development forecasting, market forecasting, technology cost modeling, and macro-economic models can also provide a basis for estimating large-scale impacts on background processes. 3.2. Functional unit The functional unit is the “consumption of PET trays in the EU27+1 367 Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. trays and source the remaining secondary material from PET trays. in 2025”. The total PET tray demand in 2018 was 0.9Mt (Plastics Re cyclers Europe, 2020), and the yearly growth rate is assumed to be 5.4% (Deloitte Sustainability, 2017; Plastics Recyclers Europe, 2020; Wood Mackenzie, 2017), meaning that, in 2025, the EU27+1 demand is ex pected be 1.3Mt PET trays. In total, 14% of the trays are assumed to be black (Eriksen and Astrup, 2019), meaning that only 14% of them can be manufactured from secondary flakes from colored PET bottles, while the rest can be made from clear PET bottles or PET trays. in 2025”. The total PET tray demand in 2018 was 0.9Mt (Plastics Re cyclers Europe, 2020), and the yearly growth rate is assumed to be 5.4% (Deloitte Sustainability, 2017; Plastics Recyclers Europe, 2020; Wood Mackenzie, 2017), meaning that, in 2025, the EU27+1 demand is ex pected be 1.3Mt PET trays. In total, 14% of the trays are assumed to be black (Eriksen and Astrup, 2019), meaning that only 14% of them can be manufactured from secondary flakes from colored PET bottles, while the rest can be made from clear PET bottles or PET trays. 3.5. System boundaries 3.5. System boundaries 3.5. System boundaries Fig. 3.3. Baseline scenario In the alternative scenario I, the additional 0.39 Mt of secondary granules are simply shifted from the manufacturing of the competitive market to trays. Due to the high pressure on PET bottle producers who have to increase their recycled content after the EU Directive 2019/804 (EC, 2019), it is more likely that the competitive market would be polyester used in textile. If polyester is the competitive market, the recycled content of European polyester becomes almost non-existent because the new policy target would divert the secondary material from polyester producers to PET trays producers. The only processes that count in the environmental assessment would be the difference between the primary materials avoided Ec v,IN −Ed,IN (i.e., is the primary PET used in the trays the same as in polyester?) and the recycling processes Ec rec,IN −Erec,IN (i.e., is the recycling of PET in secondary material for PET trays the same as recycling PET in secondary material for polyester?). However, the alternative scenario I could be tested to quantify the error of identifying the competing market in bottles instead of polyester. In this case, the recycled content of PET bottles would decrease from 11% in the baseline (EPBP, 2021) to 1% in the alternative scenario. Note that the different recyclability of bottles, trays, and polyester would not affect the results, since these products would be produced and handled as waste, with or without the use of a secondary material instead of a primary material. The secondary material used in PET trays is assumed to come from a combination of three markets (56% from clear bottles, 31% from mixed colored bottles, and 13% from trays (Andreasi Bassi et al., 2020)). The recycling of PET trays back into PET trays is a very niche market, due to the very low demand, low capacity of dedicated recycling facilities, and a high percentage of multi-polymeric trays. 3.1. Goal 4 shows the system boundaries of the alternative scenarios I (a) and II (b). 3.4. Alternative scenarios To reach the desired target (i.e, 70% recycled content in 2025), more than 0.46 Mt of food-grade secondary PET granules need to be produced. Two alternative scenarios were modeled based on two potential imple mentation scenarios for PET packaging. The alternative scenario I assumes no change in the PET waste management, meaning that the additional secondary granules are sim ply shifted from other markets that previously absorbed these (i.e., bottles and polyester). The alternative scenario II models a consorted legislative effort to increase the overall PET recycling rate by increasing the source- separation of bottles and trays and incentivizing sorting and recycling facilities in Europe. In this case, the additional secondary material for PET trays is derived from the increased source-separation of PET bottles and PET trays. Due to the high demand for clear secondary PET (as previously mentioned), it is more likely that the PET trays would absorb secondary PET from colored bottles to fulfill the demand for black PET The alternative scenario II assumes that the additional 0.39 Mt of secondary granules required to increase the recycled content of trays come from increased source-separation and recycling of PET bottles. However, only 14% of this 0.39 Mt can originate from colored bottles, - : Avoided production of primary PET trays1 - : Avoided energy recovery and disposal of PET bottles + : Collection, sorting and recycling of PET bottles in PET trays Waste providing the secondary material for the recycled content a) - : Avoided production of primary PET trays1 - : Avoided energy recovery and disposal of PET bottles/trays + : Collection, sorting and recycling of PET bottles/trays in PET trays + : Induced production of primary PET bottles1 + : Induced energy recovery and disposal of PET bottles - : avoided collection, sorting and recycling of PET bottles in PET bottles Waste providing the secondary material for the recycled content b) PET trays consumption in 2025 in EU PET trays consumption in 2025 in EU Fig. 4. System boundaries (dotted red lines) for the alternative scenarios I (a) and II (b), assuming all quality factors equal to 1 and no downstream impacts. EU indicates EU27+1. All the terms are described in Table 1. 3.4. Alternative scenarios (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) - : Avoided production of primary PET trays1 - : Avoided energy recovery and disposal of PET bottles/trays + : Collection, sorting and recycling of PET bottles/trays in PET trays + : Collection, sorting and recycling of PET bottles/trays in PET trays - : Avoided production of primary PET trays1 Fig. 4. System boundaries (dotted red lines) for the alternative scenarios I (a) and II (b), assuming all quality factors equal to 1 and no downstream impacts. EU indicates EU27+1. All the terms are described in Table 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 4. System boundaries (dotted red lines) for the alternative scenarios I (a) and II (b), assuming all quality factors equal to 1 and no downstream impacts. EU indicates EU27+1. All the terms are described in Table 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 368 S. Andreasi Bassi et al. Waste Management 135 (2021) 360–371 because only 14% of the trays are assumed to be black and can absorb this material (Eriksen and Astrup, 2019). In this alternative, the source- separation rate of bottles (excluding impurities) would have to increase from 57% in the baseline (ICIS, 2018; Plastics Recyclers Europe, 2020; Wood Mackenzie, 2017) to around 75% (depending on the type of source-separation and on the level of impurities). The increase in bottle source-separation also leads to an additional source-separation of 0.22 Mt colored bottles, 0.13 Mt of which cannot be absorbed by the tray manufacturing process, due to the constraint on the 14% maximum black trays. Colored bottles are usually used for strapping manufacturing and for black polyester; however, these are already saturated markets in Europe. Since in the baseline 25% of PET bales made from colored bottles are already exported outside EU27+1, it is unlikely that a new market able to absorb this quantity will be created in EU27+1 in the short time horizon considered herein (i.e, 2020–2025). 4.2. Differences with other frameworks While Eqs. (1) and (2) may appear similar to the circular footprint formula proposed by the PEF method and reported in the SM (EC, 2013; Zampori and Pant, 2019), and to the consequential LCA applied to marginal changes (Ekvall, 2000; Schrijvers et al., 2020), all of these methods require allocation between life cycles and rely on factors “A” indicating whether a secondary material market is constrained (i.e., saturated, characterized by a high offer and a low demand) or uncon strained (i.e., not saturated, characterized by a low offer and a high demand). Third, an assessment of products or company-level impacts should follow different approaches as allocation between individual life cycles becomes essential. Relevant methods for such product-oriented LCAs already exist in the literature (Allacker et al., 2014; Ekvall, 2000; Schrijvers et al., 2016; Zampori and Pant, 2019). As described previously, we do not recommend allocation when assessing large-scale changes in material circularity (notably, waste policy targets); instead, we suggest the use of XIN and XOUT as parameters that expand the system to jointly assess the actions of recycling material from one life cycle and using recycled material in the next life cycle. We support the idea that, in the case of large-scale studies, modeling recy cled content or waste management activities separated from each other is as analyzing the effect on applauses from “clapping with one hand” (Ekvall et al., 2021b, 2021a). However, Eqs. (1) and (2) can be trans formed to include allocation, if needed. Comparing Fig. 3a with Fig. 3b, it appears clear that the LCA avoiding allocation would not give the same results as summing the different life cycles in the case of allocation. Unlike others, our framework does not change in the case of a closed- 4.1. Applicability of the framework Three main limitations are found to be associated with the frame work: i) the intrinsic uncertainty of consequential LCA, ii) the chal lenging use of macro-economic models for definition of market responses, and iii) the incompatibility of the framework for product LCAs. The application of the framework to an EU-policy target on PET trays showed that collection rates, recyclability, and market absorption are equally important to consider, as well as how side policies (i.e., Euro pean targets for the source separation collection rate) could be employed to avoid unintended system responses that would reduce or cancel off the environmental savings. First, while a common critique of consequential LCAs is uncertainty in market responses (Plevin et al., 2014; Zamagni et al., 2012), which certainly increases when addressing future consequences, we believe that such uncertainty can be captured with an appropriate set of baseline scenarios encompassing potential developments in framework condi tions and selection of alternative scenarios reflecting potential effects of the investigated policies. Defining a set of relevant “what-if” or “likely” scenarios can inform decision-makers about the consequences from specific actions and initiatives, while systematic analysis of un certainties through parameter uncertainty propagation, data quality evaluation (e.g., pedigree matrix), and sensitivity analyses on frame work conditions support more robust results and interpretations thereof. The framework provided additional information on the potential responses of the market. Further analyses could focus on analyzing the impacts of reducing the dependency on fossil PET through, for example, alternative bio-based feedstock. Several other alternative scenarios could be tested (e.g., changing only the collection system, increasing the recyclability of products, removing colored bottles from the market, etc.). While it was not focus of the current study, we recommend that uncertainty analysis should also be performed, to increase the robust ness of results and interpretation. Second, the suggested use of macro-economic models (Sections 2.6.7 and 2.6.8) is often challenging due to their “coarse” disaggregation of economic sectors (Mattila, 2017). Yet, advances have been made to adapt some models, such as GTAP, to better support individual product types such as biofuels (Dandres et al., 2011; Igos et al., 2015), indicating that similar advancements are possible for other sectors such as waste management. To improve relevance for LCA, further flexibility in disaggregation within these models is required. 3.4. Alternative scenarios This means that either corrective policies will be put in place to increase the share of clear material, or colored bottles will likely continue to be exported off EU27+1 or sent to energy recovery and disposal in the sorting step. definition (Geyer et al., 2016) and in itself does not provide any addi tional information on the environmental effects connected to market demand, to the risk of market saturation, and to which materials are avoided (Andreasi Bassi et al., 2020; Geyer et al., 2016; Lonca et al., 2020). Moreover, as described in Section 2.5, it is highly unlikely that the different markets to be analyzed will coincide. Furthermore, we consider our framework to be more complete than the others, as it allows LCA practitioners to include all the overall interactions between several different markets. This is demonstrated by the higher number of pro cesses included (e.g., downstream impacts, maintained mass balance in recycling). To our knowledge, this is the only proposed framework for LCAs that can be applied to any size of change in material circularity, like other frameworks that focus only on marginal changes. We recommend applying the developed framework to large-scale case studies where decision-makers have an influence on the investigated policy targets and side-policies. Finally, the framework allows dynamic modeling, in particular relevant with respect to time-dependent life cycle inventories and the life cycle impact assessment stage. 5. Conclusions This paper describes a methodological framework for the definition and modeling of large-scale consequential life cycle assessments aiming at quantifying potential environmental impacts of policy targets focusing on increasing material circularity. Two types of scenarios are proposed, (multiple) baseline scenarios and alternative scenarios representing potential effects of an imple mented policy target. The difference in environmental performance between these two scenarios represents the consequential impacts associated with the implementation of the policy. Detailed recommen dations for the goal and scope phase of LCAs are provided, combined with mathematical formulations of how to calculate the environmental Unlike others, our framework does not change in the case of a closed- loop. Even if the term closed-loop recycling has been utilized in several papers (e.g. Marie and Quiasrawi, 2012), it does not have a clear 369 Waste Management 135 (2021) 360–371 S. Andreasi Bassi et al. consequences, and are supplemented with an illustrative example of applying the framework for EU target setting of recycled content in PET trays. Compared with previous frameworks in literature, allocation of impacts between individual life cycles is not recommended to support a system-level focus reflecting the legislative scope of the policies in question. The framework can accommodate changes and systems of various sizes within the suggested calculation approach, and also allows for non-linear market responses and dynamic modeling (i.e., time- dependent) when relevant. Applying the framework on an illustrative example of increasing the recycled content of PET trays consumed in the EU27+1 from 40% to 70% in 2025 (combined with required changes in the waste management system to reach this level of recycled content), demonstrated that such a policy target has to be supported by side policies (i.e., parallel initiatives) to also increase recycling of PET trays themselves. If not, secondary material already recycled in polyester textiles and bottles are likely to be diverted to trays. The likely conse quence of this is a considerable drop in recycled content of polyester to negligible levels, or for PET bottles a decrease from 11% to 1%, thereby not leading to the desired environmental impacts at system level. This illustrates that singular recycling targets may not be sufficient and that consequences throughout the full system are essential when assessing impacts from policy targets. Bao, Z., Lu, W., Chi, B., Yuan, H., Hao, J., 2019. Procurement innovation for a circular economy of construction and demolition waste : Lessons learnt from Suzhou. China. Declaration of Competing Interest EC, 2018a. PEFCR Guidance document, - Guidance for the development of Product Environmental Footprint Category Rules (PEFCRs) - version 6.3. European Commission, Bru. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EC, 2018b. 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https://openalex.org/W2395764130	https://biblio.ugent.be/publication/7225718/file/7225733.pdf	English	null	A Discussion on the Interpretation of the Darcy Equation in Case of Open-Cell Metal Foam Based on Numerical Simulations	Materials	2,016	cc-by	10,637	materials materials materials Article Sven De Schampheleire 1,, Kathleen De Kerpel 1, Bernd Ameel 1, Peter De Jaeger 2, Ozer Bag and Michel De Paepe 1 1 Department of Flow, Heat and Combustion Mechanics, Ghent University, Sint-Pietersnieuwstraat 41, Ghent 9000, Belgium; Kathleen.DeKerpel@ugent.be (K.D.K.); Bernd.Ameel@ugent.be (B.A.); Ozer.Bagci@UGent.be (O.B.); michel.depaepe@ugent.be (M.D.P.) 2 NV Bekaert SA Bekaertstraat 1 Zwevegem 8500 Belgium; Peter DeJaeger@bekaert com 1 Department of Flow, Heat and Combustion Mechanics, Ghent University, Sint-Pietersnieuwstraat 41, Ghent 9000, Belgium; Kathleen.DeKerpel@ugent.be (K.D.K.); Bernd.Ameel@ugent.be (B.A.); Ozer.Bagci@UGent.be (O.B.); michel.depaepe@ugent.be (M.D.P.) 2 NV Bekaert SA, Bekaertstraat 1, Zwevegem 8500, Belgium; Peter.DeJaeger@bekaert.com Correspondence: Sven.DeSchampheleire@ugent.be; Tel.: +32-9-2643289 1 Department of Flow, Heat and Combustion Mechanics, Ghent University, Sint-Pietersnieuwstraat 4 Ghent 9000, Belgium; Kathleen.DeKerpel@ugent.be (K.D.K.); Bernd.Ameel@ugent.be (B.A.); Ozer.Bagci@UGent.be (O.B.); michel.depaepe@ugent.be (M.D.P.) 2 NV Bekaert SA, Bekaertstraat 1, Zwevegem 8500, Belgium; Peter.DeJaeger@bekaert.com * Correspondence: Sven.DeSchampheleire@ugent.be; Tel.: +32-9-2643289 * Correspondence: Sven.DeSchampheleire@ugent.be; Tel.: +32-9-2643289 Academic Editor: Thomas Fiedler Academic Editor: Thomas Fiedler Received: 8 April 2016; Accepted: 20 May 2016; Published: 25 May 2016 Received: 8 April 2016; Accepted: 20 May 2016; Published: 25 May 2016 Abstract: It is long known that for high-velocity ﬂuid ﬂow in porous media, the relation between the pressure drop and the superﬁcial velocity is not linear. Indeed, the classical Darcy law for shear stress dominated ﬂow needs to be extended with a quadratic term, resulting in the empirical Darcy–Forchheimer model. Another approach is to simulate the foam numerically through the volume averaging technique. This leads to a natural separation of the total drag force into the contribution of the shear forces and the contribution of the pressure forces. Both representations of the total drag lead to the same result. The physical correspondence between both approaches is investigated in this work. The contribution of the viscous and pressure forces on the total drag is investigated using direct numerical simulations. Special attention is paid to the dependency on the velocity of these forces. The separation of the drag into its constituent terms on experimental grounds and for the volume average approach is uniﬁed. It is shown that the common approach to identify the linear term with the viscous forces and the quadratic term with the pressure forces is not correct. Keywords: volume averaging theory; permeability; inertial coefﬁcient; Darcy; Forchheimer; metal foam; pressure drop Materials 2016, 9, 409; doi:10.3390/ma9060409 1.1. Open-Cell Metal Foam There is a continuous search for new ﬁn designs and materials. One of these designs is open cell-metal foam [1]. Despite the quite labor-intensive process of making open-cell metal foam, it has found its way to an increasing number of applications [2,3]. There are numerous types of open-cell metal foams, two of which are especially common. The ﬁrst type is made through an investment casting process, usually based on a polyurethane preform. The second common type is based on a similar preform, but is made through an electrophoretic deposition process. To compare those two types of foam, not only the materials out of which they are made, but also the inner volumes of the strut are different. As a result of the casting process, the strut is solid, while the strut is hollow after the electrophoretic process. This has an impact on the thermal conductivity of the resulting foam material. Furthermore, in case of the casted manufacturing procedure, the polyurethane can ﬁrst be waxed in order to increase the strut’s equivalent diameter [4]. This results in slightly different foam topologies compared to those of commercial foam samples [5]. www.mdpi.com/journal/materials Materials 2016, 9, 409; doi:10.3390/ma9060409 www.mdpi.com/journal/materials 2 of 15 Materials 2016, 9, 409 The nomenclature of open-cell metal foam is illustrated in Figure 1. Struts interconnect the nodes and form both cells and pores. The shape of the struts themselves depends on the porosity. The thickness varies along the axial direction as illustrated in Figure 1. If the replication process is based on a polyurethane preform, then the cells are elongated in one direction (d2 in Figure 1) due to the effect of gravity in manufacturing the preform [6]. Designing new applications with foam requires a profound knowledge of both the heat transfer coefﬁcient and the ﬂow resistance [7,8]. In this paper, only the ﬂow resistance will be discussed. The nomenclature of open-cell metal foam is illustrated in Figure 1. Struts interconnect the nodes and form both cells and pores. The shape of the struts themselves depends on the porosity. The thickness varies along the axial direction as illustrated in Figure 1. If the replication process is based on a polyurethane preform, then the cells are elongated in one direction (݀ଶ in Figure 1) due to the effect of gravity in manufacturing the preform [6]. 1.2. Determination of Flow Resistance in Open Literature Th i fl th h di i d 1.2. Determination of Flow Resistance in Open Literature The creeping flow through porous media is described by the Darcy equation, which relates pressure drop to velocity (Equation (1)). In Equation (1), ߢ is the permeability of the porous medium. After the transitional regime, it is experimentally shown that the pressure drop becomes quadratic with velocity (Equation (2)). The equation to capture this behavior is called the Darcy–Forchheimer equation (or Hazen–Dupuit–Darcy). In this equation, ߚ is known as the inertial coefficient. Both permeability and inertial coefficient are originally seen as material properties [9]. They are exclusively related to the structure of the porous medium. ߤ The creeping ﬂow through porous media is described by the Darcy equation, which relates pressure drop to velocity (Equation (1)). In Equation (1), κ is the permeability of the porous medium. After the transitional regime, it is experimentally shown that the pressure drop becomes quadratic with velocity (Equation (2)). The equation to capture this behavior is called the Darcy–Forchheimer equation (or Hazen–Dupuit–Darcy). In this equation, β is known as the inertial coefﬁcient. Both permeability and inertial coefﬁcient are originally seen as material properties [9]. They are exclusively related to the structure of the porous medium. ∇ܲ= ߤ ߢܸ (1) ∇P “ µ κ V (1) ∇ܲ= ߤ ߢܸ (1) ∇ܲ= ߤ ߢܸ+ ߩߚܸ² (2) ∇P “ µ κ V (1) ∇P “ µ κ V ` ρβV2 (2) 1) (1) ∇ܲ= ߤ ߢܸ+ ߩߚܸ² (2) ∇P “ µ κ V ` ρβV2 (2) 2) (2) Dukhan et al. [10] mentioned that the Darcy equation accounts for the viscous drag while the Forchheimer term (ߩߚܸ²) corresponds to the form drag. This is also linked with the generally known interpretation of the Reynolds number. The Reynolds number represents the ratio of the momentum flux to the viscous stress. For many (but not all) applications, this can also be interpreted as the ratio between the inertial forces and the viscous forces. For low Reynolds number, the viscous forces are dominant and for high Reynolds numbers the inertial forces will become dominant. It is expected that when viscous stress dominates, that the relation between pressure drop and velocity is linear. H f hi h fl l i i h li d d f ll h D i D Dukhan et al. [10] mentioned that the Darcy equation accounts for the viscous drag while the Forchheimer term (ρβV2) corresponds to the form drag. 1.1. Open-Cell Metal Foam Designing new applications with foam requires a profound knowledge of both the heat transfer coefficient and the flow resistance [7,8]. In this paper, only the flow resistance will be discussed. Figure 1. The nomenclature of an open-cell metal foam and its strut thickness variation. Figure 1. The nomenclature of an open-cell metal foam and its strut thickness variation. Figure 1. The nomenclature of an open-cell metal foam and its strut thickness variation. Figure 1. The nomenclature of an open-cell metal foam and its strut thickness variation. 1.2. Determination of Flow Resistance in Open Literature Th i fl th h di i d 1.2. Determination of Flow Resistance in Open Literature This is also linked with the generally known interpretation of the Reynolds number. The Reynolds number represents the ratio of the momentum ﬂux to the viscous stress. For many (but not all) applications, this can also be interpreted as the ratio between the inertial forces and the viscous forces. For low Reynolds number, the viscous forces are dominant and for high Reynolds numbers the inertial forces will become dominant. It is expected that when viscous stress dominates, that the relation between pressure drop and velocity is linear. 3 of 15 Materials 2016, 9, 409 However, for higher ﬂow velocities, the linear pressure drop does not follow the Darcy equation. Du Plessis and Woudberg [11] provided an expression for the critical Reynolds number for departure from the Darcy regime, which only depends on the porosity of the porous medium (cd in Equation (3) is 1.9). Rec “ 50.8 φ p1 ´ φq 1 3 cd ” 1 ´ p1 ´ φq 1 3 ı (3) (3) In contrast, authors like Zeng and Grigg [12] illustrated that rather a Forchheimer number (Equation (4)) should be used to deﬁne the departure from Darcy regime. This number is deﬁned as the ratio of the pressure gradient to the viscous resistance. As the Forchheimer number is related to the non-Darcy effect, a non-Darcy effect of 10% gives a Forchheimer number of 0.11. This is called the critical Forchheimer number. β V Fo “ κβρV µ (4) Fo “ κβρV µ (4) In open literature, there is a large discrepancy in results for permeability and inertial factor. Antohe et al. [3] reported differences when using different working ﬂuids. This clearly indicates that the permeability and loss factor do not just depend on the structure of the porous medium, but on the ﬂuid as well. Bonnet et al. [13] has shown that for a given pore size, the pressure drop coefﬁcients are dispersed over at least one order of magnitude for high ﬂow rates (Redp ą 1000), and over more than two orders of magnitude for low ﬂow rates (1 ă Redp ă 10). These large differences can be attributed to: 1. The pore diameter dp being insufﬁcient to properly describe the foam structure. 1. The pore diameter dp being insufﬁcient to properly describe the foam structure. 2. The process of determining the permeability and inertial coefﬁcient. As discussed in De Schampheleire et al. 2. Experimental Approach 2. Experimental Approach The characteristic length for the Reynolds number is the average strut diameter: ݀௦= 4(1 −߶)/ߪ଴ which can be interpreted as the diameter of a cylinder with a length equal to the total strut length and a volume equal to the solid phase volume [20]. The relative uncertainty is calculated through the error propagation rules as described in the textbook from Taylor [21]. It varies between 4.5% and 10%. Figure 2. Contribution of the Darcy and Forchheimer term to the pressure gradient for a foam with following dimensions: ݀ଵ: 4.22 ݉݉, ݀ଶ: 6.23 ݉݉ and ܣ଴: 0.0988 ݉݉². Figure 2. Contribution of the Darcy and Forchheimer term to the pressure gradient for a foam with following dimensions: d1 : 4.22 mm, d2 : 6.23 mm and A0 : 0.0988 mm2. Figure 2. Contribution of the Darcy and Forchheimer term to the pressure gradient for a foam with following dimensions: ݀ଵ: 4.22 ݉݉, ݀ଶ: 6.23 ݉݉ and ܣ଴: 0.0988 ݉݉². Figure 2. Contribution of the Darcy and Forchheimer term to the pressure gradient for a foam with following dimensions: d1 : 4.22 mm, d2 : 6.23 mm and A0 : 0.0988 mm2. In the region of the low Reynolds numbers, corresponding to creeping flow, the Darcy term in Equation (2) is dominant and shows a nearly linear relation with the Reynolds number. The Forchheimer term becomes significant (more than 3% of the total pressure gradient) when the transition to the steady laminar regime occurs [22]. It indicates that inertial forces start to become significant. The share of the Forchheimer term increases significantly, until it contributes approximately 63% of the total pressure gradient [23]. The corresponding Reynolds number indicates the onset for the formation of regular vortex shedding from the struts, i.e., transition to the unsteady laminar regime. The onset of transitional flow regime is clearly observed when the Forchheimer term contribution is approximately 83% [24]. For the transition to the turbulent flow regime, however, a study of Seguin et al. [24] states that the inception takes place when the Forchheimer contribution reaches 91% of the total pressure gradient. Th D t t f th i d hil th F hh i t d t th In the region of the low Reynolds numbers, corresponding to creeping ﬂow, the Darcy term in Equation (2) is dominant and shows a nearly linear relation with the Reynolds number. 2. Experimental Approach 2. Experimental Approach A ﬁrst approach to investigating the effect of velocity on the permeability and the inertial factor is by performing experiments. Foam with a porosity of 93.2% (d1 : 4.22 mm, d2 : 6.23 mm and A0 : 0.0988 mm2) with a thickness of 40 mm is placed in a wind tunnel with a cross dimensional test section of 256 mm on 447 mm. The construction of the wind tunnel is explained in De Schampheleire et al. [19]. Pressure drop data for a velocity range between 0 and 26 m/s are gathered and ﬁtted to a second order polynomial to determine κ and β. For this speciﬁc case these values were, respectively, 5.77 ˆ 10´6 m2 and 118.59 1 m. With these values the Darcy term ( µ κ V) and Forchheimer term (ρβV2) are determined and compared to the global pressure drop in Figure 2. The characteristic length for the Reynolds number is the average strut diameter: ds “ 4 p1 ´ φq {σ0 which can be interpreted as the diameter of a cylinder with a length equal to the total strut length and a volume equal to the solid phase volume [20]. The relative uncertainty is calculated through the error propagation rules as described in the textbook from Taylor [21]. It varies between 4.5% and 10%. A first approach to investigating the effect of velocity on the permeability and the inertial factor is by performing experiments. Foam with a porosity of 93.2% ( ݀ଵ: 4.22 ݉݉, ݀ଶ: 6.23 ݉݉ and ܣ଴: 0.0988 ݉݉²) with a thickness of 40 mm is placed in a wind tunnel with a cross dimensional test section of 256 mm on 447 mm. The construction of the wind tunnel is explained in De Schampheleire et al. [19]. Pressure drop data for a velocity range between 0 and 26 m/s are gathered and fitted to a second order polynomial to determine ߢ and ߚ. For this specific case these values were, respectively, 5.77 × 10ି଺ ݉² and 118.59 ଵ ௠. With these values the Darcy term ( ఓ ఑ܸ) and Forchheimer term (ߩߚܸ²) are determined and compared to the global pressure drop in Figure 2. 1.2. Determination of Flow Resistance in Open Literature Th i fl th h di i d 1.2. Determination of Flow Resistance in Open Literature Materials 2016, 9, 409 Materials 2016, 9, 409 4 of 15 4 of 14 1.2. Determination of Flow Resistance in Open Literature Th i fl th h di i d 1.2. Determination of Flow Resistance in Open Literature [14], most manufacturers characterize their metal foam samples by reporting both the number of pores per (linear) inch (PPI) and the volumetric porosity φ. The porosity can be measured easily, and the same is true for the PPI value. However, because the cells are elongated in an anisotropic manner, there are at least two PPI values, depending on the direction of elongation. Furthermore, the ﬂow behavior will also depend on the thickness of the struts. The effort to represent these three degrees of freedom with a single degree of freedom results in the large dispersion observed in the literature. When the foam is characterized with two cell diameters (d1 and d2 as illustrated in Figure 1) and the area in the middle of the strut (A0), De Jaeger et al. [15] showed that the order of magnitude of dispersion on the experimental data becomes smaller than one. Besides, manufacturers do not give any information on the microscopic nature of the foam itself. As illustrated in Figure 1, the thickness and the shape of the strut vary over its length. This distribution depends on the applied waxing process. Even the description of the foam with three degrees of freedom is therefore still not complete. Since the shape and the thickness distribution of the struts inﬂuence the pressure drop, this results in a residual dispersion of the results. Most of the time, κ and β are determined experimentally. The pressure drop over the metal foam is determined and the quadratic ﬁt is compared with Equation (2) to determine the permeability and inertial coefﬁcient. A lot of issues arise from this method. First of all, the foam has to be so large that there are no wall effects [16] nor entrance/exit effects [17]. Furthermore, there are also large differences observed in κ and β depending on over which velocity range the quadratic ﬁt is made. Innocentinni et al. [18] and Dukhan et al. [10] studied this and observed differences up to 75% in the value for permeability. Based on these results, Dukhan et al. [10] suggested the use of two permeability values depending on the ﬂow regime: one for the Darcy regime and one for the Forchheimer regime. In this work, the velocity dependence of the permeability (and inertial factor) will be investigated for a broad velocity range, covering both the Darcy regime and the Forchheimer regime. 2. Experimental Approach 2. Experimental Approach The Forchheimer term becomes signiﬁcant (more than 3% of the total pressure gradient) when the transition to the steady laminar regime occurs [22]. It indicates that inertial forces start to become signiﬁcant. The share of the Forchheimer term increases signiﬁcantly, until it contributes approximately 63% of the total pressure gradient [23]. The corresponding Reynolds number indicates the onset for the formation of regular vortex shedding from the struts, i.e., transition to the unsteady laminar regime. The onset of transitional ﬂow regime is clearly observed when the Forchheimer term contribution is approximately 83% [24]. For the transition to the turbulent ﬂow regime, however, a study of Seguin et al. [24] states that the inception takes place when the Forchheimer contribution reaches 91% of the total pressure gradient. The Darcy term accounts for the viscous drag, while the Forchheimer term corresponds to the form drag [10]. However, using numerical simulations, it is also possible to directly evaluate the viscous drag and the form drag separately and support this statement. The Darcy term accounts for the viscous drag, while the Forchheimer term corresponds to the form drag [10]. However, using numerical simulations, it is also possible to directly evaluate the viscous drag and the form drag separately and support this statement. 5 of 15 Materials 2016, 9, 409 3.1. Equations and Calculation Method Metal foam based on a polyurethane preform, for example, is hierarchically structured as shown by Lakes [25]. This means that the complete structure of the foam can be represented by its representative elementary volume (REV) and there exists a clear-cut length scale separation between the microscopically- and macroscopically-scaled physics [25]. The continuum and momentum equations can be averaged over that representative elementary volume, based on the volume averaging process as presented by Whitaker [26]. The REV consists of two phases, which Whitaker indicates by σ (solid) and ζ (ﬂuid). An arbitrary quantity ψ of the ζ phase is indicated by ψζ. The intrinsic average of ψζ, evaluated at a location Ñx c, is then given by xψζy ´Ñx c ¯ “ ş Vζ m ´Ñx c ´ Ñr ¯ ψζ ´Ñr ¯ dV. Here, m ´Ñx c ´ Ñr ¯ is a ﬁlter function, which is normalized on the REV volume Vm and which is zero outside of the REV. This volume consists of both the σ and the ζ phases. Using Gray’s decomposition [27], the arbitrary quantity can be written as a function of the porosity φ, the intrinsic average and the microscopic scale deviation from the average: ψζ “ φxψζy ` rψζ. Combining the volume averaged equations with the decomposition results in equations for the spatial deviation terms, given by Equations (5) and (6). The constraints for these equations are given in Whitaker [28]. ∇¨ r Ñv “ 0 (5) (5) ρ Ñv ¨ ∇r Ñv “ ´∇rP ` µ∇2 r Ñv ´ 1 φ ş A f s mp Ñ xc ´ Ñr q rP ´Ñr ¯ Ñn f sdA ` 1 φ ş A f s mp Ñ xc ´ Ñr qµ∇r Ñv ´Ñr ¯ ¨ Ñn f sdA (6) (6) These deviation terms must also exhibit periodic behavior, which is mathematically expressed by Equations (7) and (8). The vector Ñ l n expresses the translation of a REV over its dimensions in three directions. The spatial deviation terms in Equations (5) and (6) are mapped by Whitaker [28,29] on the intrinsically averaged velocity according to Equations (9) and (10). Equations (7) and (8) are based on Gray’s decomposition. 3.1. Equations and Calculation Method Here a different approach is followed, where the ߢ̿∗ ିଵterm is the viscous part φx Ñv y i ¨ “κ ´1 ˚ “ ´ ż A f s mp Ñ xc ´ Ñr qµ∇r Ñv ´Ñr ¯ ¨ Ñn f sdA (14) φx Ñv y i ¨ “ β˚ “ 1 ρ\|φx Ñv y i\| ż A f s mp Ñ xc ´ Ñr q rP ´Ñr ¯ Ñn f sdA (15) part due to pressure forces on the interface between the two phases. It is also terms need to be tensors, as the pressure drop can be expected to depend on ow. This is due to the internal morphology of the metal foam, which is not n in Figure 1. viscous and pressure forces needs to be done over a REV. The derivation of lained in the work of De Jaeger et al [15] As explained earlier it is possible to (14) s also nd on φx Ñv y i ¨ “ β˚ “ 1 ρ\|φx Ñv y i\| ż A f s mp Ñ xc ´ Ñr q rP ´Ñr ¯ Ñn f sdA (15) p gy , n in Figure 1. viscous and pressure forces needs to be done over a REV. The derivation of lained in the work of De Jaeger et al [15] As explained earlier it is possible to (15) on of Note that there is only one equation, which is used to determine two closure functions. Assuming that the real pressure drag behavior is in fact given by a second order polynomial, in the Darcy–Forchheimer equation (Equation (2)), the κ´1 term is the ﬁrst-order part and the β term is the second-order part. Here a different approach is followed, where the “κ ´1 ˚ term is the viscous part and the “ β˚ term is the part due to pressure forces on the interface between the two phases. It is also apparent that the two terms need to be tensors, as the pressure drop can be expected to depend on the direction of the ﬂow. This is due to the internal morphology of the metal foam, which is not isotropic, as can be seen in Figure 1. the used volume is explained in the work of De Jaeger et al. [15]. As explained earlier, it is possible to derive a representative volume for the foam based on three parameters: ݀ଵ, ݀ଶ and ܣ଴. 3.1. Equations and Calculation Method ˆ ˙ y p rP ˆ Ñr ` Ñ l n ˙ “ rP ´Ñr ¯ (7) rv ˆ Ñr ` Ñ l n ˙ “ rv ´Ñr ¯ (8) r Ñv “ “ M¨ x Ñv y (9) rP “ µ Ñ m¨ x Ñv y (10) rP ˆ Ñr ` Ñ l n ˙ “ rP ´Ñr ¯ (7) rv ˆ Ñr ` Ñ l n ˙ “ rv ´Ñr ¯ (8) r Ñv “ “ M¨ x Ñv y (9) rP “ µ Ñ m¨ x Ñv y (10) (7) (8) (9) (10) (9) (9) (10) (10) Substituting these mapping functions in the closure problem and recognizing that x Ñv y is quasi-constant in a REV, the closure term problem can be re-written such that it requires solving for the two mapping functions “ M and Ñ m. An additional decomposition was done by Whitaker [28,29], which provides a theoretical framework for the derivation of the earlier-mentioned Darcy–Forchheimer equation. The ﬁrst mapping function is fully related to the viscous drag, while the second mapping is related to the form drag effect. Therefore, the mapping functions need to be split and blended into new mapping functions in order to mimic the phenomenologically deﬁned permeability and inertial loss factor [28,29]. The objective is to rewrite the average pressure gradient as a function of the average velocity. Inspired by the Darcy–Forchheimer relation (Equation (2)), a vectorial relation between the gradient of the average pressure and a second order polynomial function of the velocity can be imposed. A subscript asterisk is used to clearly distinguish the permeabilities from the Darcy–Forchheimer relation and the closure terms that are derived now, which will be called the direct formulation (Equation (11)). Materials 2016, 9, 409 Equation (13) [28] 6 of 15 The link between the Darcian and superﬁcial average velocity and the interfacial velocity is given by Equation (12). 1 −∇〈ܲ〉௜= −න݉(ݔ௖−ݎ)ߤ∇ݒ(ݎ) ∙݊௙௦݀ܣ ஺೑ೞ + ߶න݉(ݔ௖−ݎ)ܲ(ݎ) ஺೑ೞ ݊௙௦݀ܣ (13) A term-to-term comparison between Equation (11) and Equation (13) allows deriving the direct The link between the Darcian and superﬁcial average velocity and the interfacial velocity is given by Equation (12). 3.1. Equations and Calculation Method −∇〈ܲ〉= −න݉(ݔ௖−ݎ)ߤ∇ݒ(ݎ) ∙݊௙௦݀ܣ ஺೑ೞ + ߶න݉(ݔ௖−ݎ)ܲ(ݎ) ஺೑ೞ ݊௙௦݀ܣ (13) A term to term comparison between Equation (11) and Equation (13) allows deriving the direct ´ ∇xPyi “ µ “κ ´1 ˚ ¨ x Ñv y s ` ρ “ β˚¨ ˇˇˇx Ñv y sˇˇˇ x Ñv y s (11) rison between Equation (11) and Equation (13) allows deriving the direct ility and inertial coefficient [22]: (11) direct x Ñv y s “ φx Ñv y i (12) = −න݉(ݔ௖ ሬሬሬԦ −ݎԦ)ߤ∇ݒԦ෨(ݎԦ) ∙݊ሬԦ௙௦݀ܣ ஺ (14) (12) (14) The momentum balance for the deviations can be rearranged into a similar form, resulting in Equation (13) [28]: ஺೑ೞ ߶〈Ԧ〉௜ ߚ̿ 1 න (ሬሬሬԦ Ԧ) ෨(Ԧ) ሬԦ ݀ The momentum balance for the deviations can be rearranged into a similar form, resulting in Equation (13) [28]: ஺೑ೞ ߶〈Ԧ〉௜ ߚ̿ 1 න (ሬሬሬԦ Ԧ)ܲ෨(Ԧ) ሬԦ ݀ܣ (15) ´ ∇xPyi “ ´ ż A f s mp Ñ xc ´ Ñr qµ∇r Ñv ´Ñr ¯ ¨ Ñn f sdA ` 1 φ ż A f s mp Ñ xc ´ Ñr q rP ´Ñr ¯ Ñn f sdA (13) ߶〈ݒ〉 ߚ∗ ߩ\|߶〈ݒԦ〉௜\| න݉(ݔ௖ ݎ)ܲ(ݎ) ஺೑ೞ ݊௙௦݀ܣ (15) te that there is only one equation, which is used to determine two closure functions. h h l d b h i i i f i b d d l i l i h (13) A term-to-term comparison between Equation (11) and Equation (13) allows deriving the direct formulation of the permeability and inertial coefﬁcient [22]: Assuming that the real pressure drag behavior is in fact given by a second order polynomial, in the Darcy–Forchheimer equation (Equation (2)), the ߢିଵ term is the first-order part and the ߚ term is the second-order part. Here a different approach is followed, where the ߢ̿∗ ିଵterm is the viscous part A term-to-term comparison between Equation (11) and Equation (13) allows deriving the direct formulation of the permeability and inertial coefﬁcient [22]: Assuming that the real pressure drag behavior is in fact given by a second order polynomial, in the Darcy–Forchheimer equation (Equation (2)), the ߢିଵ term is the first-order part and the ߚ term is the second-order part. 3.1. Equations and Calculation Method The calculation method discussed previously will be applied for the foam mentioned in Section 2. The REV for this volume is determined by the model of De Jaeger et al. [15] and is shown in Figure 3a. In the PhD of De Jaeger [22] it is also shown that a quarter of this REV in the direction of the cell gives approximately the same results (within 12.5%) for pressure and viscous forces as the full REV, if the Reynolds number based on the specific cell diameter is lower than 200. This quarter REV is also called a periodic unit cell (PUC) and is shown in Figure 3b, where the cell is slightly modified by using small spheres in order to increase the quality of the computational grid. Figure 3. Illustration of the REV (a) and PUC (b) of the foam with dimensions: ݀ଵ: 4.22 ݉݉, ݀ଶ: 6.23 ݉݉ and ܣ଴: 0.0988 ݉݉². Figure 3. Illustration of the REV (a) and PUC (b) of the foam with dimensions: d1 : 4.22 mm, d2 : 6.23 mm and A0 : 0.0988 mm2. Figure 3. Illustration of the REV (a) and PUC (b) of the foam with dimensions: ݀ଵ: 4.22 ݉݉, ݀ଶ: 6.23 ݉݉and ܣ଴: 0.0988 ݉݉² Figure 3. Illustration of the REV (a) and PUC (b) of the foam with dimensions: d1 : 4.22 mm, d2 : 6.23 mm and A0 : 0.0988 mm2. Calculation of the viscous and pressure forces needs to be done over a REV. The derivation of the used volume is explained in the work of De Jaeger et al. [15]. As explained earlier, it is possible to derive a representative volume for the foam based on three parameters: d1, d2 and A0. The calculation 7 of 15 Materials 2016, 9, 409 method discussed previously will be applied for the foam mentioned in Section 2. The REV for this volume is determined by the model of De Jaeger et al. [15] and is shown in Figure 3a. In the PhD of De Jaeger [22] it is also shown that a quarter of this REV in the direction of the cell gives approximately the same results (within 12.5%) for pressure and viscous forces as the full REV, if the Reynolds number based on the speciﬁc cell diameter is lower than 200. 3.2. Calculation of Closure Terms The microscopic quantities that are monitored are the viscous and form drag force densities and the intrinsically averaged velocity components. The former are denoted by Ñ f v and Ñ f p, respectively, and computed in the CFD software as: Ñ f v “ ´ 1 Vm ż A f s µ∇r Ñv ´Ñr ¯ ¨ Ñn f sdA (16) Ñ f p “ 1 Vm ż A f s rP ´Ñr ¯ Ñn f sdA (17) (16) Ñ f p “ 1 Vm ż A f s rP ´Ñr ¯ Ñn f sdA (17) (17) This corresponds to setting the ﬁlter mp Ñ xc ´ Ñr q in Equations (6) and (13) equal to the reciprocal of the volume Vm (covering both solid and ﬂuid phase) when inside of the REV and equal to zero when outside of the REV. This corresponds to a block ﬁlter, but other ﬁlters could be chosen as well. The corresponding direct formulations of the permeability and inertial loss factor can be calculated through: φx Ñv y ¨ “κ ´1 ˚ “ Ñ f v (18) Ñ “ 1 Ñ φx Ñv y ¨ “κ ´1 ˚ “ Ñ f v (18) φx Ñv y ¨ “ β˚ “ 1 ρ ˇˇˇφx Ñv y ˇˇˇ Ñ f p (19) (18) φx Ñv y ¨ “ β˚ “ 1 ρ ˇˇˇφx Ñv y ˇˇˇ Ñ f p (19) (19) Both tensors are symmetrical and the non-diagonal components are zero [20,28,30]. For orthotropic media, Scheidegger [30] analyzed experimental data and revealed the symmetric behavior. This was later proven by Whitaker [28]. Thus, when permeability is determined along the principal directions, only the three diagonal components need to be determined. For the inertial loss factor though, symmetry is not guaranteed. Magnico [20], however, investigated this factor for shear-deformed open-cell nickel foams and found that the eigenvectors were nearly orthogonal and they followed the shear angles. It led to the conclusion that the inertial loss factor of foams could practically be considered symmetrical. Furthermore, the permeability and inertial coefﬁcient along the z-direction is the same as the one along the x-direction (κ˚,xx “ κ˚,zz and β˚,xx “ β˚,zz with x, y and z as indicated in Figure 3b). There are therefore just two unknown components in each tensor, which can be obtained imposing ﬂow in two different directions. 3.1. Equations and Calculation Method This quarter REV is also called a periodic unit cell (PUC) and is shown in Figure 3b, where the cell is slightly modiﬁed by using small spheres in order to increase the quality of the computational grid. q y p g Closer examination of the PUC reveals that a sub-volume bears additional periodicity. The green faces show geometrical periodicity, when a translation is performed in both the z and y direction. The red faces are also geometrically periodic in z direction, when a secondary translation in negative y direction is imposed. The same holds for the x direction. In the y direction, no secondary translation is needed to impose periodicity. Depending on the ﬂow direction that is studied (see latter), different periodic boundary conditions (different ∆p) were applied. 3.2. Calculation of Closure Terms Pressure gradients of different magnitudes are imposed, once in the x direction and once in the y direction. The calculations are done using a commercial CFD software package. The convective terms are discretized using a second-order upwind scheme and a coupled pressure-velocity scheme is used. No turbulence model is used. All residuals have to be lower than 10´6 before accepting the solution. Materials 2016, 9, 409 Coarse M 8 of 15 ࢍ࢘࢏ࢊ Shear stress calculation in laminar ﬂow ( Ñ f v) requires a sufﬁciently ﬁne mesh at the boundary layer to accurately resolve the gradient. In order to be certain that changing the size of the cells of ߢ∗,௫௫ 1.584 × 10 ݉ 1.610 × 10 ݉ 1.632 × 10 ݉ 3% 7.2% ߢ∗,௬௬ 6.318 × 10ି଻ ݉² 6.527 × 10ି଻݉² 6.700 × 10ି଻݉² 6% 15.4% ߚ∗௫௫ 72 36 1 72 66 1 73 74 1 1 9% 8 7% Shear stress calculation in laminar ﬂow ( Ñ f v) requires a sufﬁciently ﬁne mesh at the boundary layer to accurately resolve the gradient. In order to be certain that changing the size of the cells of “ ∗,௫௫ 1.584 × 10 ݉ 1.610 × 10 ݉ 1.632 × 10 ݉ 3% 7.2% ߢ∗,௬௬ 6.318 × 10ି଻ ݉² 6.527 × 10ି଻݉² 6.700 × 10ି଻݉² 6% 15.4% ߚ∗௫௫ 72.36 1 72.66 1 73.74 1 1.9% 8.7% Shear stress calculation in laminar ﬂow ( f v) requires a sufﬁciently ﬁne mesh at the boundary layer to accurately resolve the gradient. In order to be certain that changing the size of the cells of the computational grid does not inﬂuence the results for “κ˚ and “ β˚, a grid discretization study is performed. In this work the Roache’s grid convergence index (GCI) is used to estimate of the grid discretization error [31,32]. Three different grid sizes are tested each with a 10% reﬁnement of all cells in each direction. For the ﬁnest mesh, the ﬁrst boundary layer cell was 4 µm thick. The growth ratio was taken as 1.1 and a maximum size cell of 60 µm is imposed. For the PUC reported in Figure 3b this leads to a computational domain of 10.5 million cells. In Table 1 the GCI for the ﬁnest grid is reported. Even for this ﬁne grid, the relative uncertainty on κ˚,yy is quite high (15.4%). 3.2. Calculation of Closure Terms However, the uncertainties are acceptable in comparison to the experimental results, where uncertainties over one order of magnitude are reported (see Bonnet et al. [13]). ߢ∗,௬௬ 6.318 × 10ି଻ ݉² 6.527 × 10ି଻݉² 6.700 × 10ି଻݉² 6% 15.4% ߚ∗,௫௫ 72.36 1 ݉ 72.66 1 ݉ 73.74 1 ݉ 1.9% 8.7% ߚ∗,௬௬ 142.75 1 ݉ 143.70 1 ݉ 144.54 1 ݉ 1.2% 3.44% 3.3. Results in Laminar Regime The results for the different pressure gradients that were simulated are reported in Table 2 and Figures 4 and 5. The focus of this paper is low velocities: only steady calculations were performed. The permeability in both directions remains constant for ܴ݁ௗೞ< 0.25, see Figure 4 and Table 2. The flow regime here is creeping flow. For higher Reynolds numbers, the viscous force (shear stress) will start to increase and by observing the trend it can be stated that the viscous force increases Table 1. Determination of grid discretization error for a pressure gradient of 100 Pa over the PUC. Coarse Mesh (Start Size: 5 µm) Finer Mesh (Start Size: 4.5 µm) Finest Mesh (Start Size: 4 µm) ∆coarse´finest GCIfinest grid κ˚,xx 1.584 ˆ 10´6 m2 1.610 ˆ 10´6 m2 1.632 ˆ 10´6 m2 3% 7.2% κ˚,yy 6.318 ˆ 10´7 m2 6.527 ˆ 10´7 m2 6.700 ˆ 10´7 m2 6% 15.4% β˚,xx 72.36 1 m 72.66 1 m 73.74 1 m 1.9% 8.7% β˚,yy 142.75 1 m 143.70 1 m 144.54 1 m 1.2% 3.44% 3 3 R lt i L i R i slightly more than linearly with the Darcian velocity. According to Equation (18), this results in decrease of the permeability. The inertial coefficient in the direct formulation is defined as the ratio of the (volume averaged pressure force to the kinetic energy of the fluid (see Equation (15)). Upstream of a strut, there is stagnation zone where kinetic energy descends to zero, which results in a region with high pressure Downstream, a distinction has to be made between flow regimes with or without recirculatio regions in the wakes behind struts. In case of no recirculation (ܴ݁ௗೞ< 10, see Figure 5), the inertia loss factor decreases with increasing Reynolds number. This means that the pressure force increase at a rate lower than the average kinetic energy in the flow domain. For higher Reynolds numbers, Table 1. Determination of grid discretization error for a pressure gradient of 100 Pa over the PUC. 3.2. Calculation of Closure Terms tly more than linearly with the Darcian velocity. According to Equation (18), this results ease of the permeability. 3.3. Results in Laminar Regime at a rate lower than the averag is expected that the increment o 3.3. Results in Laminar Regime at a rate lower than the averag is expected that the increment This means that the pressure force increases at a rate lower than the average kinetic energy in the ﬂow domain. For higher Reynolds numbers, it is expected that the increment of pressure force will be balanced by the increase of velocity. This will be again characterized by a nearly constant inertial coefﬁcient. This can be again observed in Table 2 for the Reynolds numbers in laminar regime. Materials 2016, 9, 409 9 of 14 Figure 5. The inertial coefficient in the x and y direction (ߚ∗,௫௫ and ߚ∗,௬௬) determined through numerical calculations plotted against the Reynolds number. Figure 5. The inertial coefﬁcient in the x and y direction (β˚,xx and β˚,yy) determined through numerical calculations plotted against the Reynolds number. Figure 5. The inertial coefficient in the x and y direction (ߚ∗,௫௫ and ߚ∗,௬௬) determined through numerical calculations plotted against the Reynolds number. Figure 5. The inertial coefﬁcient in the x and y direction (β˚,xx and β˚,yy) determined through numerical calculations plotted against the Reynolds number. Table 2. Results for the permeability and inertial coefficient based on the numerical calculation method. ࡾࢋࢊ࢙,࢞ ࡾࢋࢊ࢙,࢟ ࣄ∗,࢞࢞ (m²) ࣄ∗,࢟࢟(m²) ࢼ∗,࢞࢞ (1/m) ࢼ∗,࢟࢟ (1/m) 0.02244 0.02982 1.682 × 10−6 8.600 × 10−7 28,744 28,062 0.04581 0.05889 1.682 × 10−6 8.600 × 10−7 14,374 14,037 0.08834 0.1178 1.682 × 10−6 8.600 × 10−7 7191.5 7029.9 0.2225 0.2977 1.681 × 10−6 8.580 × 10−7 2888.6 2843 0.4450 0.5857 1.680 × 10−6 8.510 × 10−7 1464.6 1472.7 2.0384 2.4474 1.664 × 10−6 7.820 × 10−7 360.79 444.38 3.7659 4.2338 1.663 × 10−6 7.380 × 10−7 218.26 311.63 5.3462 5.7618 1.654 × 10−6 7.130 × 10−7 165.99 260.38 6.8218 7.1327 1.649 × 10−6 6.950 × 10−7 138.24 231.67 8.2059 8.3926 1.645 × 10−6 6.810 × 10−7 120.93 212.72 11.9227 11.7460 1.639 × 10−6 6.530 × 10−7 94.22 180.00 14.0919 13.7320 1.637 × 10−6 6.400 × 10−7 85.39 167.31 22.5039 21.8332 1.632 × 10−6 5.960 × 10−7 69.85 138.94 33.5792 33.0721 1.620 × 10−6 5.430 × 10−7 65.65 126.75 Table 2. Results for the permeability and inertial coefﬁcient based on the numerical calculation method. 3.3. Results in Laminar Regime at a rate lower than the averag is expected that the increment The results for the different pressure gradients that were simulated are reported in Table 2 and Figures 4 and 5. The focus of this paper is low velocities: only steady calculations were performed. is expected that the increment of pressure force will be balanced by the increase of velocity. This will be again characterized by a nearly constant inertial coefficient. This can be again observed in Table 2 for the Reynolds numbers in laminar regime. The results for the different pressure gradients that were simulated are reported in Table 2 and Figures 4 and 5. The focus of this paper is low velocities: only steady calculations were performed. p p y y be again characterized by a nearly constant inertial coefficient. This can be again observed in Table 2 for the Reynolds numbers in laminar regime. Figure 4. The permeability in the x and y direction (ߢ∗,௫௫ and ߢ∗,௬௬) determined through numerical calculations plotted against the Reynolds number. Figure 4. The permeability in the x and y direction (κ˚,xx and κ˚,yy) determined through numerical calculations plotted against the Reynolds number. Figure 4. The permeability in the x and y direction (ߢ∗,௫௫ and ߢ∗,௬௬) determined through numerical calculations plotted against the Reynolds number. Figure 4. The permeability in the x and y direction (κ˚,xx and κ˚,yy) determined through numerical calculations plotted against the Reynolds number. The permeability in both directions remains constant for Reds ă 0.25, see Figure 4 and Table 2. The ﬂow regime here is creeping ﬂow. For higher Reynolds numbers, the viscous force (shear stress) will start to increase and by observing the trend, it can be stated that the viscous force increases slightly Materials 2016, 9, 409 9 of 15 more than linearly with the Darcian velocity. According to Equation (18), this results in a decrease of the permeability. The inertial coefﬁcient in the direct formulation is deﬁned as the ratio of the (volume averaged) pressure force to the kinetic energy of the ﬂuid (see Equation (15)). Upstream of a strut, there is a stagnation zone where kinetic energy descends to zero, which results in a region with high pressure. Downstream, a distinction has to be made between ﬂow regimes with or without recirculation regions in the wakes behind struts. In case of no recirculation (Reds ă 10, see Figure 5), the inertial loss factor decreases with increasing Reynolds number. 3.3. Results in Laminar Regime at a rate lower than the averag is expected that the increment Reds, x Reds, y κ˚,xx (m2) κ˚,yy (m2) β˚,xx (1/m) β˚,yy (1/m) 0.02244 0.02982 1.682 ˆ 10´6 8.600 ˆ 10´7 28,744 28,062 0.04581 0.05889 1.682 ˆ 10´6 8.600 ˆ 10´7 14,374 14,037 0.08834 0.1178 1.682 ˆ 10´6 8.600 ˆ 10´7 7191.5 7029.9 0.2225 0.2977 1.681 ˆ 10´6 8.580 ˆ 10´7 2888.6 2843 0.4450 0.5857 1.680 ˆ 10´6 8.510 ˆ 10´7 1464.6 1472.7 2.0384 2.4474 1.664 ˆ 10´6 7.820 ˆ 10´7 360.79 444.38 3.7659 4.2338 1.663 ˆ 10´6 7.380 ˆ 10´7 218.26 311.63 5.3462 5.7618 1.654 ˆ 10´6 7.130 ˆ 10´7 165.99 260.38 6.8218 7.1327 1.649 ˆ 10´6 6.950 ˆ 10´7 138.24 231.67 8.2059 8.3926 1.645 ˆ 10´6 6.810 ˆ 10´7 120.93 212.72 11.9227 11.7460 1.639 ˆ 10´6 6.530 ˆ 10´7 94.22 180.00 14.0919 13.7320 1.637 ˆ 10´6 6.400 ˆ 10´7 85.39 167.31 22.5039 21.8332 1.632 ˆ 10´6 5.960 ˆ 10´7 69.85 138.94 33.5792 33.0721 1.620 ˆ 10´6 5.430 ˆ 10´7 65.65 126.75 Table 2. Results for the permeability and inertial coefficient based on the numerical calculation method. Table 2. Results for the permeability and inertial coefﬁcient based on the numerical calculation method. Materials 2016, 9, 409 10 of 15 However, recalling the discussion in Section 2, a well-known interpretation of the Reynolds number is that for low velocities (Reds ă 1), one should expect a primarily viscous ﬂow. However, looking to the inﬂuence of the pressure force compared to the viscous force, the pressure inﬂuence cannot be neglected. To provide more detail, Table 3 reports the viscous and pressure forces over the simulated range of Reynolds numbers in the x-direction. Similar results hold for the y-direction. Next to both the viscous and pressure forces, the inﬂuence of the viscous force to the total force ( fv,x fv,x`fp,x ) is also reported in Table 3. The maximum inﬂuence of the viscous forces is only 32%. Although both forces increase with increasing Reynolds numbers, the relative inﬂuence of the viscous forces rapidly decreases. As expected, for high Reynolds numbers the inertial contribution to the drag becomes constant. Table 3. Results for the pressure and viscous forces acting on the PUC for different Reynolds numbers. Table 3. Results for the pressure and viscous forces acting on the PUC for different Reynolds numbers. 3.3. Results in Laminar Regime at a rate lower than the averag is expected that the increment Reds, x fp,x (N) fv,x (N) fv,x fv,x`fp,x 0.02243 1.77 ˆ 10´9 8.14 ˆ 10´10 0.315 0.0458 3.54 ˆ 10´9 1.63 ˆ 10´9 0.315 0.0883 7.08 ˆ 10´9 3.26 ˆ 10´9 0.315 0.2225 1.77 ˆ 10´8 8.13 ˆ 10´9 0.315 0.4450 3.55 ˆ 10´8 1.62 ˆ 10´8 0.313 2.0384 1.84 ˆ 10´7 7.48 ˆ 10´8 0.290 3.7659 3.79 ˆ 10´7 1.38 ˆ 10´7 0.267 5.3462 5.8 ˆ 10´7 1.95 ˆ 10´7 0.251 6.8219 7.87 ˆ 10´7 2.47 ˆ 10´7 0.239 8.2059 9.96 ˆ 10´7 2.96 ˆ 10´7 0.229 11.9227 1.64 ˆ 10´6 4.29 ˆ 10´7 0.208 14.0920 2.07 ˆ 10´6 5.1 ˆ 10´7 0.197 22.5040 4.33 ˆ 10´6 8.42 ˆ 10´7 0.163 33.5792 9.05 ˆ 10´6 1.28 ˆ 10´6 0.124 Furthermore, from Figure 6 and Table 3 it is also clear that the pressure force varies linearly with velocity for small Reynolds numbers (Reds ă 2). For this velocity range, β˚,xx and β˚,yy can be written as a constant value divided by the velocity (see Equation (15) with Ñ fp „ Ñv q. In the case of the studied foam, β˚,xx “ 19.76{vx for Reds ă 2 and β˚,yy “ 25.68{vy for Reds ă 2. Materials 2016, 9, 409 10 of 14 Figure 6. The pressure force in the x-direction is plotted against the Reynolds number. Figure 6. The pressure force in the x-direction is plotted against the Reynolds number. Figure 6. The pressure force in the x-direction is plotted against the Reynolds number. Figure 6. The pressure force in the x-direction is plotted against the Reynolds number. Table 3. Results for the pressure and 3.4. Discussion on the Darcy Equation ࡾࢋࢊ࢙,࢞ ࢌ࢖,࢞ (N) ࢌ࢜,࢞ (N) ࢌ࢜,࢞ ࢌ࢜,࢞+ ࢌ࢖,࢞ 0.02243 1.77 × 10−9 8.14 × 10−10 0.315 The large inﬂuence of pressure forces at low velocities can be explained by looking to the theory of Stokes ﬂow. Rewriting the momentum equations in dimensionless form, it can be shown that for Materials 2016, 9, 409 11 of 15 very low Reynolds numbers, the material derivative of the velocity can be neglected. Equivalently, this means that inertial effects are neglected. However, it is important to note that the pressure gradient can still be signiﬁcant in comparison to the viscous term. Only neglecting the inertial term but keeping the pressure term results in the so-called Stokes equation [33] (Equation (20)). In this equation, the pressure is made dimensionless with respect to µU{L, where U is the free stream velocity and L is a characteristic length scale. ∇p ´ µ ∇2Ñv “ 0 (20) (20) For a 3D sphere, there exists an exact analytical solution for the drag. With a, the radius of the sphere and V, the unidirectional incoming velocity, it is given by Equation (17) [33]: D “ 3πµaV ż π 0 cos2θsinθ dθ loooooooooooooomoooooooooooooon pressure ` 3πµaV ż π 0 sin3θdθ loooooooooomoooooooooon viscous “ 2πµaV ` 4πµaV “ 6πµaV (21) (21) In the case of a 3D sphere, one-third of the drag is due to pressure forces and two-thirds is due to viscous forces. This is also veriﬁed with the CFD software used in this work. However, from Table 3, even higher inﬂuences of the pressure forces are observed in the case of ﬂow around metal foam. This is because the struts themselves do not have the shape of a sphere. They can however be approximated by ﬂow around a cylinder or a triangular prism. Furthermore, the ﬂow is not around a single strut, but around a staggered array of struts, which has different ﬂow characteristics. Materials 2016, 9, 409 11 of 14 approximated by flow around a cylinder or a triangular prism. Furthermore, the flow is not around a single strut, but around a staggered array of struts, which has different flow characteristics. To illustrate the inﬂuence of the pressure forces, some additional calculations are performed on the following geometries: (1) a standalone circle; and (2) three circles in staggered layout (see Figure 7). Table 3. Results for the pressure and 3.4. Discussion on the Darcy Equation For these simulations, the solution techniques and discretization of the geometry is done exactly the same as in case of the ﬁnest mesh discussed above. Of course, instead of using the volume-averaged equations, the classical Navier–Stokes equations are used here. If the strut diameter is D, the surroundings are 10D (see Figure 7). The inﬂuence of the viscous force on the total force is reported in Figure 8 for a single circle and three circles placed in a staggered conﬁguration. As can be seen, the staggered layout of the circles results in a lower relative inﬂuence of the viscous forces. Furthermore, the middle circle of the staggered layout experiences an even lower inﬂuence of viscous forces, namely only 37%. These observations are consistent with the observations from Table 3: the contributions of the viscous forces are very low in a real foam. g gg y To illustrate the influence of the pressure forces, some additional calculations are performed on the following geometries: (1) a standalone circle; and (2) three circles in staggered layout (see Figure 7). For these simulations, the solution techniques and discretization of the geometry is done exactly the same as in case of the finest mesh discussed above. Of course, instead of using the volume-averaged equations, the classical Navier–Stokes equations are used here. If the strut diameter is D, the surroundings are 10D (see Figure 7). The influence of the viscous force on the total force is reported in Figure 8 for a single circle and three circles placed in a staggered configuration. As can be seen, the staggered layout of the circles results in a lower relative influence of the viscous forces. Furthermore, the middle circle of the staggered layout experiences an even lower influence of viscous forces, namely only 37%. These observations are consistent with the observations from Table 3: the contributions of the viscous forces are very low in a real foam. Figure 7. Illustration of the boundary conditions for the staggered case with circles. Figure 7. Illustration of the boundary conditions for the staggered case with circles. Figure 7. Illustration of the boundary conditions for the staggered case with circles. Figure 7. Illustration of the boundary conditions for the staggered case with circles. 12 of 15 Materials 2016, 9, 409 Figur Figure 8. Table 3. Results for the pressure and 3.4. Discussion on the Darcy Equation Illustration of the influence against the velocity of the viscous forces to the total forces acting on the surface of the foam. Figure 8. Illustration of the inﬂuence against the velocity of the viscous forces to the total forces acting on the surface of the foam. Figure 8. Illustration of the influence against the velocity of the viscous forces to the total forces on the surface of the foam. Figure 8. Illustration of the inﬂuence against the velocity of the viscous forces to the total forces a on the surface of the foam. Figure 8. Illustration of the influence against the velocity of the viscous forces to the total forces acting on the surface of the foam. Figure 8. Illustration of the inﬂuence against the velocity of the viscous forces to the total forces acting on the surface of the foam. As illustrated in Figure 8, the pressure influences are not negligible at low velocities. Furthermore, from Figure 6 it is clear that the pressure drop for ܴ݁ௗೞ< 2 varies linearly with the As illustrated in Figure 8, the pressure inﬂuences are not negligible at low velocities. Furthermore, from Figure 6 it is clear that the pressure drop for Reds ă 2 varies linearly with the velocity. Thus, the pressure drop over the foam can be written as a combination of viscous and pressure forces (over a microscopic element): dp dx “ µ κvis v loomoon viscous ` C v loomoon pressure (22) (22) Note again that this equation is different compared to the classical Darcy equation where the permeability is a combination of viscous and inertial inﬂuences. C in Equation (22) is a constant parameter representing the inﬂuence of the pressure drag on the pressure drop. In order to rewrite Equation (22) to the generally know Darcy equation, C should vary linearly with the molecular viscosity, such that it can be written as C “ µ κinertial . e a To investigate this, the simulations of the staggered layout are repeated but with an increase of the ﬂuid viscosity with a factor of 2. Increasing the viscosity will also increase the viscous forces with the same factor, since there is a linear relation between both. From the dimensionless relation for the pressure in the Stokes ﬂow, it is expected that the pressure gradient will also get scaled linearly with the viscosity. The results are depicted in Figure 9. Table 3. Results for the pressure and 3.4. Discussion on the Darcy Equation It is conﬁrmed that in creeping ﬂow where inertial effects are negligible and Stokes ﬂow is valid, for Reds ă 2, the pressure drag indeed varies linearly with the viscosity. This means that the Darcy law is still valid, see Equation (23), with κclassical “ κ as reported in open literature. However, one needs to be careful with the interpretation of the Darcy equation. In Dukhan et al. [10], it was mentioned that the Darcy equation was representing the viscous drag, but the permeability as reported in the Darcy equation is really a combination of a viscous and pressure drag component. dp dx “ µ κvis v ` µ κpressure v “ µ κclassical v (23) (23) The direct formulation of the permeability and the inertial coefﬁcient shows velocity-dependent behavior, which is due to two reasons. Firstly, the linear term of the pressure drop is not purely due to the shear stress, since the pressure drag also exhibits linear behavior for low Reynolds numbers. Similarly, it is not really correct to lump the pressure drag into the inertial term for these low velocities, as it really has a linear behavior and not a quadratic behavior. Secondly, the real pressure drag versus velocity behavior is not exactly given by a second order polynomial, which 13 of 15 ese low pressure Materials 2016, 9, 409 Similarly, it is no velocities as it rea results in velocity-dependent values for the permeability and the inertial coefﬁcient even in the phenomenological approach of the Darcy law. g y y g y p y velocity-dependent values for the permeability and the inertial coefficient even in the phenomenological approach of the Darcy law. Figure 9. Illustration of the influence against the velocity of the viscous forces to the total forces acting on the surface of the foam for the staggered circle layout and two different viscosities. Figure 9. Illustration of the inﬂuence against the velocity of the viscous forces to the total forces acting on the surface of the foam for the staggered circle layout and two different viscosities. Figure 9. Illustration of the influence against the velocity of the viscous forces to the total forces acting on the surface of the foam for the staggered circle layout and two different viscosities. Figure 9. Table 3. Results for the pressure and 3.4. Discussion on the Darcy Equation Illustration of the inﬂuence against the velocity of the viscous forces to the total forces acting on the surface of the foam for the staggered circle layout and two different viscosities. 4. Conclusions This work has pointed out that there is another way to calculate permeability and inertial coefﬁcient. Based on a numerical approach, both closure terms are calculated depending on, respectively, the viscous and pressure forces acting on a representative elementary volume of the studied open-cell foam. It was shown that in the creeping ﬂow regime, the linear term in the Darcy law was due to both pressure forces and viscous forces. 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https://openalex.org/W4287799446	https://zenodo.org/record/5141642/files/study-of-birth-complications-in-diabetic-mothers%20%282%29.pdf	English	null	Study of Birth Complications in Diabetic Mothers	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	12,460	Abstract Corresponding author: Hafiz Hafeez Ullah, Doctor of Pharmacy the Indus Hospital Lahore, Kahna Nau, Pakistan, Tel: +923364794595; E-mail: hafizhafeez54@gmail.com The current study was being performed to evaluate the birth complications in diabetic mothers; including both maternal and fetal complications; miscarriages. The nature of study was observational cross-sectional study. The study was being taken place at different hospitals, clinical settings, and maternity homes of Lahore during September 2016 - November 2016. The demographic data, family history, socio-economic history, indications, examination findings, results, lab findings etc. were recorded. Total 200 pregnant diabetic patients were evaluated for this study. The age limit for this study was 18-40 years. The patients were being analyzed for their FBS/ BSR or HbA1c findings and the type of diabetes was being record- ed. Out of 200 patients, 81% had GDM while the remaining patients were being presented with pregestational diabetes (type I 5%, type II 14% patients). Most of the GDM cases were being diagnosed during 5th to 8th week of pregnancy. Out of 200 pregnancies, 20.5% (41) of these patients had normal pregnancies, and had no major fetal complications except uncontrolled sugar level in mothers. Remain- ing 79.5% (159) pregnancies/deliveries were associated with some major complications including respiratory distress, macrosomia, hy- poglycemic babies, CVS malformations and still births/miscarriages. The ratio of normal vaginal delivery to CS was found out to be 29% to & 76%. The major indications for these CS deliveries were placen- tal abruption (19.74%), dystocia (14.47%), uterine rupture (13.16%), breech position (6.58%), fetal distress (46.05%) and to some extent previous CS. The miscarriages were being associated with hyper- tension (41.5%), polyhydramnios (22%), Hughes syndrome (12.2%), and uncontrolled sugar level (24.3%). In our study population TT immunization status was good i.e. 76%. Diabetes is still a major problem of birth complications and miscarriages. Public awareness program is required to educate the people about reproductive health and to motivate them to undergo BSR/FBS during pregnancy prior to 24thgestational weeks to diagnose for GDM. Received Date: March 14, 2020 Accepted Date: April 24, 2020 Published Date: May 01, 2020 Published Date: May 01, 2020 Citation: Ullah HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Com- plications in Diabetic Mothers, Dietary Supplements and their Associated Risks. J Diab Meta Syndro 3: 008. Copyright: © 2020 Ullah HH, et al. Nomenclature and classification of diabetes The first widely accepted classification of diabetes mellitus was published by WHO in 1980 and, in modified form, in 1985. The 1980 Expert Committee proposed two major classes of diabetes mellitus and named them, IDDM or Type 1, and NIDDM or Type 2. In the 1985 Study Group Report the terms Type 1 and Type 2 were omit- ted, but the classes IDDM and NIDDM were retained, and a class of Malnutrition–Related Diabetes Mellitus (MRDM) was introduced. In both the 1980 and 1985 reports other classes of diabetes included Other Types and Impaired Glucose Tolerance (IGT) as well as GDM. These were reflected in the subsequent International Nomenclature of Diseases (IND) in 1991, and the tenth revision of the International Classification of Diseases (ICD–10) in 1992. The 1985 classification was widely accepted and is used internationally [2]. Keywords: Complications; Diabetes; Foetus; GDM; Miscarriage; Neonate; Pregnancy The nomenclature of human DM has been revised, and this clas- sification has been accepted throughout the medical world and litera- ture. The major categories of diabetes are [3]. Definition • Insulin-dependent DM, type I or IDDM • Noninsulin-dependent DM, type II or NIDDM • Secondary DM or type S • Impaired glucose tolerance, IGT • Gestational diabetes or GDM • Previous abnormality of glucose tolerance PrevAGT • Insulin-dependent DM, type I or IDDM • Noninsulin-dependent DM, type II or NIDDM • Secondary DM or type S • Impaired glucose tolerance, IGT • Gestational diabetes or GDM • Previous abnormality of glucose tolerance PrevAGT • Insulin-dependent DM, type I or IDDM “Diabetes Mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period”. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. the context of coexisting insulin resistance. The effect of insufficient insulin plays a primary role in the metabolic derangements linked to diabetes; hyperglycemia, in turn, plays an important role in dis- ease-related complications [1]. Study of Birth Complications in Diabetic Mothers Hafiz Hafeez Ullah1, Hafiz Faheem Ullah Khan2, Hafiz Ghulam Murtaza Saleem3 and Wali Ullah4 1Doctor of Pharmacy the Indus Hospital Lahore, Kahna Nau, Pakistan 2Department of Biochemistry the University of Lahore, Pakistan 3Department of Molecular Biology & Genetics the University of Lahore, Pakistan 4Department of physiology, Pakistan Red Crescent Medical and Dental College Lahore, Pakistan Overview • Previous abnormality of glucose tolerance PrevAGT DM is a chronic disorder characterized by hyperglycemia and the late development of vascular and neuropathic complications. Regard- less of its cause, the disease is associated with a common hormonal defect namely insulin deficiency that may be absolute or relative in Ullah HH, et al., J Diab Meta Syndro 2020, 3: 008 Journal of Diabetes & Metabolic Syndrome Research Article Henry Publishing Groups © Ullah HH, et al., 2020 Type-1 Diabetes The major genetic susceptibility to IDDM is determined by genes in the HLA chromosomal region. An increased relative risk for de- veloping the disease is observed in subjects who are HLA A1, A2, B8, B18, B15, B40, CW3, Bfs, DW3, DW4, DRW3, and DRW4 positive. There is an additive relative risk in subjects who possess two “high risk” HLA B alleles which has an important influence on the preva- lence of the disease in sibships and possibly on the concordance rate in diabetic identical twins [6]. Table 1.2: Secondary Diabetes and its Classification. Major Class Cause Examples Sec- ondary Diabetes Genetic defects of β-cell function Maturity-onset diabetes of the young, types 1 to 9; point mutations in mitochondrial DNA Genetic defects in insulin action Type A insulin resistance, leprechaunism, Rab- son-Mendenhall syndrome, lipoatrophic diabe- tes Disease of the exocrine pancreas Pancreatitis, trauma, pancreatectomy, neoplasia, cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy Endocrinopathies Acromegaly, Cushing’s syndrome, hyperthyroid- ism, pheochromocytoma, glucagonoma, soma- tostatinoma, aldosteronoma Drug or chemical induced Diabetes Vacor, pentamidine, nicotinic acid, glucocorti- coids, thyroid hormone, diazoxide, β-adrenergic agonists, thiazides, phenytoin, interferon-α Infections Congenital rubella, cytomegalovirus Uncommon forms of im- mune-mediated diabetes Stiff man syndrome, anti-insulin receptor anti- bodies Other genetic syndromes Down syndrome, Klinefelter’s syndrome, Turn- er’s syndrome, Wolfram’s syndrome, Friedreich’s ataxia, Huntington’s disease, Laurence-Moon- Biedl syndrome, myotonic dystrophy, porphyria, Prader-Willi syndrome GDM Etiology The vast majority of human diabetics show no clear etiological analogy. But certain rare cases are clearly due to primary pancreatic disease which destroys insulin production, and to other endocrine factors which induce a diabetic state. These include: Embryonic stem (ES) cells display the ability to differentiate in vi- tro into a variety of cell lineages. Using a cell-trapping system, we have obtained an insulin-secreting cell clone from undifferentiated ES cells. Clusters obtained from this clone were implanted (1x10(6) cells) in the spleen of streptozotocin-induced diabetic animals. Transplanted animals correct hyperglycemia within 1 week and restore body weight in 4 weeks. Whereas an intraperitoneal glucose tolerance test showed a slower recovery in transplanted versus control mice, blood glucose normalization was same after a challenge meal. This approach opens new possibilities for tissue transplantation in the treatment of type-1 and type-2 diabetes and offers an alternative to gene therapy [8]. Risk Factors Table 1.1: Primary Diabetes and its Classification. Major Class Sub-Class Cause Primary Diabetes Type 1 diabetes (IDDM or juvenile-onset diabetes) β-cell destruction, usually leading to absolute insulin deficiency Immune mediated Idiopath- ic Type 2 diabetes (NIDDM or adult-onset diabetes) Range from predominant insulin resistance with relative insulin deficiency to predomi- nant secretory defect with insulin resistance Types Major Class Sub-Class Cause Primary Diabetes Type 1 diabetes (IDDM or juvenile-onset diabetes) β-cell destruction, usually leading to absolute insulin deficiency Immune mediated Idiopath- ic Type 2 diabetes (NIDDM or adult-onset diabetes) Range from predominant insulin resistance with relative insulin deficiency to predomi- nant secretory defect with insulin resistance Major Class Cause Examples Sec- ondary Diabetes Genetic defects of β-cell function Maturity-onset diabetes of the young, types 1 to 9; point mutations in mitochondrial DNA Genetic defects in insulin action Type A insulin resistance, leprechaunism, Rab- son-Mendenhall syndrome, lipoatrophic diabe- tes Disease of the exocrine pancreas Pancreatitis, trauma, pancreatectomy, neoplasia, cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy Endocrinopathies Acromegaly, Cushing’s syndrome, hyperthyroid- ism, pheochromocytoma, glucagonoma, soma- tostatinoma, aldosteronoma Drug or chemical induced Diabetes Vacor, pentamidine, nicotinic acid, glucocorti- coids, thyroid hormone, diazoxide, β-adrenergic agonists, thiazides, phenytoin, interferon-α Infections Congenital rubella, cytomegalovirus Uncommon forms of im- mune-mediated diabetes Stiff man syndrome, anti-insulin receptor anti- bodies Other genetic syndromes Down syndrome, Klinefelter’s syndrome, Turn- er’s syndrome, Wolfram’s syndrome, Friedreich’s ataxia, Huntington’s disease, Laurence-Moon- Biedl syndrome, myotonic dystrophy, porphyria, Prader-Willi syndrome GDM Eti l In a recent study the basic classification of diabetes was described as follows (Tables 1.1 & 1.2): [1]. Type- 2 diabetes Type II diabetes is a common disorder whose prevalence is in- creasing in the United States and throughout the world. Type II di- abetes is also associated with several other metabolic abnormalities such as central obesity, hypertension, and dyslipidemia, which con- tributes to the very high rate of cardiovascular morbidity and mor- tality. The main pathologic defects in diabetes consist of excessive he- patic glucose production, peripheral insulin resistance, and defective beta-cell secretory function. Treatment of type-1 and type-2 DM Initiation of non-pharmacologic therapy should be started as soon as the diagnosis is made. Pharmacologic agents should be initiated if the glycemic goals are not met with a 3-month trial of diet and exer- cise. The cornerstone of therapy consists of a regular exercise routine along with a diet consisting of 40% to 50% complex carbohydrates, 10% to 20% protein, and monounsaturated fats such as canola oil and olive oil. If non-pharmacologic therapy does not achieve adequate glycemic control, initiation of an oral anti-diabetic agent is warranted. In addi- tion to the sulfonylureas, which work by stimulating insulin secretion, we now have metformin, which inhibits excessive hepatic glucose pro- duction; acarbose, which delays the absorption of carbohydrates in the gut; and troglitazone, which reduces insulin, resistance primarily in skeletal muscle [7]. Henry Publishing Groups © Ullah HH, et al., 2020 Types The vast majority of cases of diabetes fall into two broad etiopatho- genetic categories. In one category, type-1 diabetes, the cause is an Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 1 of 13 1 of 13 Citation: Ullah HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. J Diab Meta Syndro 3: 008. FK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. Primary disturbance of other endocrines (Table 2): Table 2: Endocrinal disturbances during Diabetes [5]. Endocrinal Disturbance Disease Pituitary Disease Acromegaly; severe diabetes. Cushing’s basophilism; milder. Adrenal disease Pheochromocytoma; variable severity. Basophilism; variable severity. Thyrogenic diabetes With Disturbances of Fat Storage absolute deficiency of insulin secretion. Individuals at increased risk of developing this type of diabetes can often be identified by sero- logical evidence of an autoimmune pathologic process occurring in the pancreatic islets and by genetic markers. In the other, much more prevalent category, type-2 diabetes, the cause is a combination of re- sistance to insulin action and an inadequate compensatory insulin secretory response [4]. absolute deficiency of insulin secretion. Individuals at increased risk of developing this type of diabetes can often be identified by sero- logical evidence of an autoimmune pathologic process occurring in the pancreatic islets and by genetic markers. In the other, much more prevalent category, type-2 diabetes, the cause is a combination of re- sistance to insulin action and an inadequate compensatory insulin secretory response [4]. sistance to insulin action and an inadequate compensatory insulin secretory response [4]. In a recent study the basic classification of diabetes was described as follows (Tables 1.1 & 1.2): [1]. Table 1.1: Primary Diabetes and its Classification. Table 1.2: Secondary Diabetes and its Classification. Primary pancreatic destruction • Total surgical pancreatectomy for widespread carcinoma or hyper- insulinism • calculus disease and chronic relapsing diabetes • haemochromatosis (these three forms are severely insulin-defi- cient) Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 2 of 13 Signs and symptoms GDM represents a heterogeneous group of metabolic disorders, which result in varying degrees of maternal hyperglycemia and preg- nancy-associated risk. The frequency of GDM is rising globally and may also increase further as less-stringent criteria for diagnosis are adopted [9]. GDM typically does not cause any noticeable signs or symp- toms. This is why screening tests are so important. Rarely, an in- creased thirst or increased urinary frequency may be noticed. GDM has serious, long-term consequences for both baby and mother, in- cluding a predisposition to obesity, metabolic syndrome and diabetes later in life. Early detection and intervention can greatly improve out- comes for women with this condition and their babies. Unfortunate- ly, screening and diagnostic tests are not uniform worldwide, which could lead not only to under diagnosis but also under management of the illness [13] (Table 3) (Figure 3). Etiology During pregnancy, the placenta makes hormones that can lead to a buildup of sugar in blood. Usually, pancreas can make enough in- sulin to handle that. If not, blood sugar levels will rise and can cause GDM. A transient physiologic insulin resistance and hyperinsulin- emia are characteristic of normal pregnancy. In women with β-cells that are not capable of maintaining the high insulin production, GDM develops. A restriction of high-insulinogenic carbohydrates may help to prevent the development of GDM [11]. In pregnancy, several phys- iologic changes take place, the sum of which tends to reset the glucose homeostasis in the direction of diabetes. About 1-2% of all pregnant women develop an abnormal glucose tolerance in pregnancy, but most often glucose tolerance returns to normal postpartum. This condition is called GDM [12] ) (Figure 2.1). Some of the signs and symptoms commonly experienced include (Figure 1) • Frequent urination • Excessive thirst • Increased hunger • Weight loss, Tiredness • Lack of interest and concentration • A tingling sensation or numbness in the hands or feet • Blurred vision Figure 1: Main Symptoms of Diabetes. Gestational Diabetes Mellitus (GDM) • Frequent urination • Excessive thirst • Increased hunger • Weight loss, Tiredness • Lack of interest and concentration • A tingling sensation or numbness in the hands or feet • Blurred vision • Frequent urination Figure 1: Main Symptoms of Diabetes. Figure 2.1: Etiology of GDM. Figure 1: Main Symptoms of Diabetes. Figure 1: Main Symptoms of Diabetes. Gestational Diabetes Mellitus (GDM) Literature Review The current study suggested that although the excess risk for birth defects among children of mothers with diabetes mellitus is well doc- umented, there are few data concerning the risk for specific malforma- tions. In the Atlanta Birth Defects Case-Control Study, those risks for malformations were evaluated. The population-based study included 4929 live and stillborn babies with major malformations ascertained by the Metropolitan Atlanta Congenital Defects Program in the first year of life born to residents of Metropolitan Atlanta between 1968 and 1980. The study also included 3029 non-malformed live babies who were frequency-matched to case babies by race, period of birth, and hospital of birth. The relative risk for major malformations among infants of mothers with insulin-dependent diabetes mellitus (n=28) was 7. (5% Confidence Interval [CI] 1.9, 33.5) compared with infants of non-diabetic mothers. The relative risks for major central nervous system and cardiovascular system defects were 15.5 (95% CI=3.3, 73.8) and 18.0 (95% CI=3.9, 82.5), respectively. The absolute risks for major, central nervous system, and cardiovascular system malforma- tions among infants of diabetic mothers were 18.4, 5.3, and 8.5 per 100 live births, respectively. Infants of mothers with gestational diabetes mellitus who required insulin during the third trimester of pregnancy were 20.6 (95% CI=2.5, 168.5) times more likely to have major car- diovascular system defects than infants of non-diabetic mothers. The absolute risk for infants of this group of diabetic mothers was 9.7%. No statistically significant differences were found among infants of mothers with gestational diabetes mellitus who did not require insulin during pregnancy. These results suggested a stronger association than previously reported between maternal diabetes mellitus and specific categories of major malformations and implicate gestational diabetes mellitus as a risk factor for major cardiovascular system defects [14]. This study proposed that intrauterine exposure to diabetes is asso- ciated with an excess of diabetes and obesity in the offspring, but the effects of intrauterine exposure are confounded by genetic factors. To determine the role of the intrauterine diabetic environment per se, the prevalence of diabetes and the mean BMI were compared in siblings born before and after their mother was recognized as having diabetes. Nuclear families in which at least one sibling was born before and one after the mother was diagnosed with type 2 diabetes were selected. Consequently, the siblings born before and after differed in their ex- posure to diabetes in utero. Objectives Objectives • To determine birth complications in diabetic mothers • To determine the prevalence of GDM during pregnancy • To determine the ratio of CS and risk factors leading to CS • To determine reproductive health of females in our community Definition GDM is defined as “Glucose intolerance that is first detected during pregnancy”. This simple definition belies the complexity of a condition that spans a spectrum of glycaemia, pathophysiology, and clinical effects and for which there is a wide diversity of opinion re- garding detection and clinical management [10] (Figure 2). Table 3: Major complications associated with GDM. Major complications Description Hypoglycemic Babies Babies of GDM positive mothers develop hypoglycemia shortly after birth because their own insulin production is high. It may provoke seizures in the baby. Macrosomia High glucose from mother’s blood crosses the placenta, which trig- gers foetal pancreas to make extra insulin. This can cause the baby to grow too large (macrosomia). Very large babies (weigh 9 pounds or more) may become wedged in the birth canal, sustain birth injuries or require a C-section birth. High blood pressure and Pre-Eclampsia GDM raises risk of high blood pressure, as well as, preeclampsia. Respiratory distress It is a syndrome in premature infants caused by developmental in- sufficiency of pulmonary surfactant production and structural im- maturity in the lungs. Operative delivery due to macrosomia also increases the risk for transient tachypnea of the newborn, whereas polycythemia predisposes the infant to persistent pulmonary hyper- tension of the newborn. Cardiovascular anomalies Cardiomyopathy with ventricular hypertrophy and outflow tract obstruction may occur in as many as 30% of neonates of diabetic mothers. Figure 2: Main Symptoms of Diabetes. Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 3 of 13 Although our GDM patients were stringently managed with diet or diet plus insulin, as indicated and maintained almost euglycemic val- ues, these neonatal complications could not be eliminated. Our data may be consistent with observations published during the last decade that even subtle degrees of maternal hyperglycemia can have a det- rimental effect on perinatal outcome. Most neonatal complications readily respond to therapy if diagnosed and treated early and prompt- ly. Macrosomia can have a detrimental effect on delivery (trauma) and later long-term implications during childhood. Tight metabolic control with diet and, when indicated, insulin treatment may be ad- vantageous in reducing fetal birth weight. Criteria of how tight the metabolic control should be remain to be accurately defined [15]. Henry Publishing Groups © Ullah HH, et al., 2020 Definition Although our GDM patients were stringently managed with diet or diet plus insulin, as indicated and maintained almost euglycemic val- ues, these neonatal complications could not be eliminated. Our data may be consistent with observations published during the last decade that even subtle degrees of maternal hyperglycemia can have a det- rimental effect on perinatal outcome. Most neonatal complications readily respond to therapy if diagnosed and treated early and prompt- ly. Macrosomia can have a detrimental effect on delivery (trauma) and later long-term implications during childhood. Tight metabolic control with diet and, when indicated, insulin treatment may be ad- vantageous in reducing fetal birth weight. Criteria of how tight the metabolic control should be remain to be accurately defined [15]. Figure 3: Cause of Macrosomia. This study proposed that the perinatal mortality rate of Infants of Diabetic Mothers (IDMs) has declined dramatically from 250 per 1000 live births in the 1960s to a near-normal 20 per 1000 live births in the 1980s. Five to 8% of all IDMs suffer from major congenital mal- formations, and it is the latter that are responsible for 50% of these perinatal deaths. It has been shown that tight glycemic control prior to conception and during pregnancy can prevent an excess rate of con- genital malformations, fetal macrosomia, birth trauma, and neona- tal respiratory distress syndrome. We briefly reviewed the short- and long-range complications that occur in Offspring of Diabetic Mothers (ODMs) from gestation through young adulthood. Short-term neo- natal complications, such as hypoglycemia, hypocalcemia, hypomag- nesemia, hyperbilirubinemia, and polycythemia, are related mainly to fetal hyperinsulinemia, hypoxemia, and prematurity. They are readily controllable within the setup of modern neonatal intensive care units. Long-range complications include an increased rate of childhood and adolescent obesity, impaired glucose tolerance or diabetes mellitus, and subtle neuropsychological dysfunctions. These may be related to the severity of the maternal hyperglycemia during pregnancy, the consequent fetal hyperinsulinemia, and third trimester maternal lipid metabolism disturbances. Today we have at hand the knowledge and tools to properly treat both pregestational and gestational diabetes. Increased education of the general practitioner and the target popu- lation regarding early referral of pregestational diabetic mothers and the implementation of screening programs for gestational diabetes will further reduce diabetic pregnancy-related morbidity [16]. Figure 3: Cause of Macrosom Literature Review The aim of this study was to investigate that a transient physiologic insulin resistance and hyperinsulinemia are characteristic of normal pregnancy. This insulin action has evolved during a period of human evolution that was characterized by a very low-carbohydrate nutri- tion. The development of gestational diabetes mellitus (GDM) is pro- posed to result from a collision of this evolutionary inheritance with our “modern” nutrition: The “Western” high-insulinogenic nutrition increases the postprandial demand for insulin significantly during the insulin resistant state of late pregnancy. In women with β-cells that are not capable of maintaining the high insulin production, GDM de- velops. A restriction of high-insulinogenic carbohydrates may help to prevent the development of GDM [11]. This study suggested that Dietary vitamin D supplementation is as- sociated with reduced risk of type 1 diabetes in animals. Our aim was to ascertain whether or not vitamin D supplementation or deficiency in infancy could affect development of type 1 diabetes. A birth-cohort study was done, in which all pregnant women (n=12 055) in Oulu and Lapland, northern Finland, who were due to give birth in 1966 were enrolled. Data was collected in the first year of life about frequency and dose of vitamin D supplementation and presence of suspected rickets. Our primary outcome measure was diagnosis of type 1 diabetes by the end of December, 1997. 12,058 out of 12,231 represented live births, and 10,821 (91% of those alive) children were followed-up at age 1 year. Of the 10,366 children included in analyses, 81 were diagnosed with diabetes during the study. Vitamin D supplementation was asso- ciated with a decreased frequency of type 1 diabetes when adjusted for neonatal, anthropometric, and social characteristics (Rate Ratio [RR] for regular VS no supplementation 0·12, 95% CI=0·03–0·51, and irreg- ular VS no supplementation 0·16, 0·04-0·74. Children who regularly took the recommended dose of vitamin D (2000 IU daily) had a RR of 0·22 (0·05-0·89) compared with those who regularly received less than the recommended amount. Children suspected of having rickets during the first year of life had a RR of 3·0 (1·0-9·0) compared with those without such a suspicion. Dietary vitamin D supplementation is associated with reduced risk of type 1 diabetes. Ensuring adequate vitamin D supplementation for infants could help to reverse the in- creasing trend in the incidence of type 1 diabetes [19]. Literature Review A total of 58 siblings from 19 families in which at least one sibling had diabetes were examined at similar ages (within 3 years). The risk of diabetes was significantly higher in sib- lings born after the mother developed diabetes than in those born be- fore the mother’s diagnosis of diabetes (odds ratio 3.7, P=0.02). In 52 families, among 183 siblings without diabetes, the mean BMI was 2.6 kg/m2 higher in offspring of diabetic than in offspring of non-diabetic pregnancies (P=0.003). In contrast, there were no significant differ- ences in risk of diabetes or BMI between offspring born before and after the father was diagnosed with diabetes. Intrauterine exposure to diabetes per se conveyed a high risk for the development of diabetes and obesity in offspring in excess of risk attributable to genetic factors alone [17]. In this study Neonatal morbidity was assessed in the offspring of 878 mothers with Gestational Diabetes Mellitus (GDM), 132 mothers with pre-GDM, and 380 control subjects. Compared with the con- trol group, the GDM group had a higher incidence of complications, including macrosomia, hypoglycemia, hyperbilirubinemia, hypo- calcemia, polycythemia, and major congenital anomalies (P<0.05). Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 4 of 13 Citation: Ullah HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. J Diab Meta Syndro 3: 008. who became pregnant between 1 April 1999 and 1 April 2000. Main outcome measures were Maternal, perinatal, and neonatal outcomes of pregnancy. Results showed that 84% (n=271) of the pregnancies were planned. Glycaemic control early in pregnancy was good in most women (HbA1c  7.0% in 75% (n=212) of the population), and folic acid supplementation was adequate in 70% (n=226). 314 pregnancies that went beyond 24 weeks’ gestation resulted in 324 infants. The rates of pre-eclampsia (40; 12.7%), preterm delivery (101; 32.2%), caesare- an section (139; 44.3%), maternal mortality (2; 0.6%), congenital mal- formations (29; 8.8%), perinatal mortality (9; 2.8%), and macrosomia (146; 45.1%) were considerably higher than in the general population. Neonatal morbidity (one or more complications) was extremely high (260; 80.2%). Literature Review The incidence of major congenital malformations was significantly lower in planned pregnancies than in unplanned preg- nancies (4.2% (n=11) v 12.2% (n=6); relative risk 0.34, 95% confi- dence interval 0.13 to 0.88). This study concluded that despite a high frequency of planned pregnancies, resulting in overall good glycae- mic control (early) in pregnancy and a high rate of adequate use of folic acid, maternal and perinatal complications were still increased in women with type 1 diabetes. Neonatal morbidity, especially hypo- glycaemia, was also extremely high. Near optimal maternal glycaemic control (HbA1c  7.0%) apparently is not good enough [20]. The aim of the study was to examine the outcome of the pregnan- cy and neonatal period in women with gestational diabetes mellitus and non-diabetic pregnant women, and in women with early and late diagnosis of gestational diabetes mellitus. It included 327 wom- en with gestational diabetes mellitus and 295 non-diabetic women, who were screened with a 75 g oral glucose tolerance test because of risk factors for gestational diabetes. Women with gestational dia- betes mellitus were treated with a low-caloric diet and insulin when appropriate, while women in the control group received routine an- tenatal care. Gestational age at delivery was significantly lower in the group with gestational diabetes mellitus, both when considering all deliveries (39.1 ± 1.7 weeks versus 39.8 ± 2.0 weeks, p<0.05) and only those with spontaneous onset of labor (38.8 ± 2.0 weeks versus 40.0 ± 1.6 weeks, p<0.05). The frequency of macrosomia was in- creased, although not statistically significant (8% vs. 2%, p=0.07), and the rate of admission to the neonatal ward was significantly increased (18% vs. 9%, p<0.05) in the group with gestational diabe- tes. Women with early diagnosis of gestational diabetes mellitus had a significantly increased need for insulin treatment during pregnancy (36% vs. 9% p<0.05) and a significantly higher occurrence of diabetes mellitus at follow-up from two months until three years postpartum. This study of women with gestational diabetes mellitus and non-dia- betic pregnant women showed that gestational diabetes mellitus was associated with a significantly lower gestational age at delivery and an increased rate of admission to the neonatal ward. Women diagnosed with GDM before 20 weeks of gestation had an increased need for in- sulin treatment during pregnancy and a high risk of subsequent overt DM, compared with women diagnosed with GDM later in pregnancy [18]. Henry Publishing Groups © Ullah HH, et al., 2020 Literature Review The observation and quanti- fication of maternal outcomes with gestational diabetes mellitus were necessary, so that proper measures could be taken to reduce complica- tions during delivery and the neonatal period and thereby, minimize particularly NICU admission rate [23]. The current study was conducted to determine the range of com- plications occurring in Infants of Diabetic Mothers (IDMs). An obser- vational cross-sectional study was performed in Federal Government Services Hospital, Islamabad and National Institute of Child Health, Karachi, from August 1999 to January 2000. All IDMs born during the study period were immediately admitted to the neonatal intensive care unit after delivery. Maternal history was obtained and a detailed phys- ical examination was performed to detect congenital abnormalities. Babies were screened for hypoglycemia, hypocalcaemia, hyperbiliru- binemia, birth asphyxia, Respiratory Distress Syndrome (RDS) and birth trauma. Outcome of IDMs and relative frequencies of various complications were evaluated. Results were analyzed using Statistical Package for Social Sciences (SPSS) version 11. A total number of 40 babies with IDM were included in the study. Out of diabetic mothers, only 19 (47.5%) were taking insulin albeit irregularly. No mother was taking oral hypoglycemic agents, 5 (12.5%) were following only dietary advice while 16 (40%) were not following any advice for control of di- abetes. Twenty-two (55%) mothers were delivered by C-section and 18 (45%) had vaginal delivery. Seven (17.5%) mothers experienced birth injuries all of them were delivered vaginally and majority of them were large babies. Fifteen percent IDMs suffered from birth asphyxia. Most (82.5%) were delivered vaginally. Congenital anomalies were found in 10 (25%) babies. Eighteen (45%) were macrosomic, 20 (50%) were Appropriate for Gestational Age (AGA) and 02 (5%) were Small for Gestational Age (SGA) or growth retarded. Hypoglycemia was noted in 35% and hypocalcaemia in 15%. Hyperbilirubinemia was observed in 12 (30%) newborns. Mortality was 7.5%. The results of this study showed high frequency complications in IDMs. The diabetic mothers should have regular antenatal follow-up and maintain good glycemic control throughout pregnancy. Cesarean sections may be allowed more liberally, especially with clinical evidence of macrosomic babies, to avoid birth injury and asphyxia. All deliveries of diabetic mother should be attended by a pediatrician to minimize complications [22]. The current study proposed that Metformin is a logical treatment for women with gestational diabetes mellitus, but randomized trials to assess the efficacy and safety of its use for this condition are lacking. Literature Review They were subdivided into four groups according to maternal glucose metabolism during pregnancy and genetic predisposition to type 2 di- abetes: 1) offspring of women with diet-treated GDM (O-GDM), 2) offspring of genetically predisposed women with a normal OGTT (O-No GDM), 3) offspring of women with type 1 diabetes (O-type 1), and 4) offspring of women from the background population (O- BP).The prevalence of type 2 diabetes and pre-diabetes (impaired glucose tolerance or impaired fasting glucose) in the four groups was 21, 12, 11, and 4%, respectively. In multiple logistic regression analy- sis, the adjusted Odds Ratios (ORs) for type 2 diabetes/pre-diabetes were 7.76 (95% CI=2.58-23.39) in O-GDM and 4.02 (1.31-12.33) in O-type 1 compared with O-BP. In O-type 1, the risk of type 2 dia- betes/pre-diabetes was significantly associated with elevated maternal blood glucose in late pregnancy: OR 1.41 (1.04-1.91) per mmol/l. A hyperglycemic intrauterine environment appears to be involved in the pathogenesis of type-2 diabetes/pre-diabetes in adult offspring of primarily Caucasian women with either diet-treated GDM or type-1 diabetes during pregnancy [25]. The current study proposed that Carbohydrate intolerance is the most common metabolic complication of pregnancy. Gestational Di- abetes Mellitus (GDM) poses numerous problems for both mother and fetus. The objectives of this study were to find out the incidence of gestational diabetes mellitus in pregnant women and their preg- nancy outcomes. It was also to discover the risk factors for the ad- mission of neonates to the Neonatal Intensive Care Unit (NICU). A hospital-based prospective study performed at King Khalid University Hospital (KKUH), where 685 pregnant women who were diagnosed with gestational diabetes mellitus, out of 8000 pregnant women regis- tered during January 2000 - December 2001, were followed and their outcomes studied. The incidence of gestational diabetes mellitus was found to be 8.6% (95% CI=8.1, 9.3). There were 511 (74.6%) spon- taneous vertex deliveries, and 148 (21.6%) were delivered by lower segment cesarean section. Maternal morbidity in these women was 1.2%. A total of 697 babies were delivered by these 685 women, out of whom 675 were singleton pregnancies, 9 sets of twins and one set of quadruplets. Six-hundred-eighty-seven babies were born alive, 7 ba- bies died in utero and 3 died in the neonatal period. The incidence of neonatal intensive care admission was 4.9%. The mean length of stay in the NICU was 16 days. Literature Review This study randomly assigned 751 women with gestational diabetes mellitus at 20 to 33 weeks of gestation to open treatment with met- formin (with supplemental insulin if required) or insulin. The prima- ry outcome was a composite of neonatal hypoglycemia, respiratory distress, need for phototherapy, birth trauma, 5-minute Apgar score less than 7, or prematurity. The trial was designed to rule out a 33% increase (from 30% to 40%) in this composite outcome in infants of women treated with metformin as compared with those treated with insulin. Secondary outcomes included neonatal anthropometric mea- surements, maternal glycemic control, maternal hypertensive compli- cations, postpartum glucose tolerance, and acceptability of treatment. Of the 363 women assigned to metformin, 92.6% continued to receive metformin until delivery and 46.3% received supplemental insulin. The rate of the primary composite outcome was 32.0% in the group assigned to metformin and 32.2% in the insulin group (relative risk, 1.00; 95% confidence interval, 0.90 to 1.10). More women in the met- formin group than in the insulin group stated that they would choose to receive their assigned treatment again (76.6% vs. 27.2%, P<0.001). The rates of other secondary outcomes did not differ significantly be- tween the groups. There were no serious adverse events associated with the use of metformin. In women with gestational diabetes melli- tus, metformin (alone or with supplemental insulin) is not associated with increased perinatal complications as compared with insulin. The women preferred metformin to insulin treatment [24]. The main objective of this study was to determine the role of in- trauterine hyperglycemia and future risk of type 2 diabetes in human offspring is debated. It studied glucose tolerance in adult offspring of women with either Gestational Diabetes Mellitus (GDM) or type-1 di- abetes, taking the impact of both intrauterine hyperglycemia and ge- netic predisposition to type-2 diabetes into account. The glucose toler- ance status following a 2-h 75-g Oral Glucose Tolerance Test (OGTT) was evaluated in 597 subjects, primarily Caucasians, aged 18-27 years. Literature Review This study proposed that Pregnancies of women with type 1 dia- betes mellitus are associated with maternal and perinatal complica- tions. These complication rates remain elevated despite achievement of the treatment goals described in the widely used guidelines of the American Diabetes Association (i.e. HbA1c level ≤ 7.0%). Against this background, we sought to answer two questions: (1) are HbA1c levels within 1% above normal appropriate in pregnant women with type 1 diabetes or should treatment be aimed at normal HbA1c levels; and (2) how many Self-Monitored Blood Glucose (SMBG) levels are needed per day to obtain an adequate image of glycaemic control in pregnant women with type 1 diabetes? We asked 43 pregnant women with type 1 diabetes to use the Continuous Glucose Monitoring System (CGMS) once in each trimester of pregnancy, while continuing their SMBG measurements. Glucose levels measured with the CGMS were com- pared between patients with HbA1c levels of 4.0-6.0%, 6.0-7.0% and >7.0%. Self-monitored glucose levels and those measured with CGMS were compared between patients with four or five, six to nine and ten or more SMBG determinations daily. In patients with HbA1c levels ≤6.0%, the glucose levels obtained by CGMS were significantly better than in patients with HbA1c levels>6.0%. In women with HbA1c lev- els 6.0-7.0% and>7.0%, these levels did not differ. The detection rate of hyper- and hypoglycemic episodes was significantly higher in patients with ten or more SMBG determinations daily than in patients with fewer than ten. Treatment of diabetes in pregnant women should be aimed at achieving HbA1c levels within the normal range, i.e. ≤ 6.0%. The objective of the current study was to investigate maternal, perinatal, and neonatal outcomes of pregnancies in women with type 1 diabetes in the Netherlands. The study design was nationwide prospective cohort study. It was performed in 118 hospitals in the Netherlands. Participants include 323 women with type 1 diabetes Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 5 of 13 A minimum of ten SMBG determinations daily is necessary to obtain adequate information of all daily glucose fluctuations [21]. A minimum of ten SMBG determinations daily is necessary to obtain adequate information of all daily glucose fluctuations [21]. tion of labor (RR: 2.5, 95% CI=1.4, 4.5). Literature Review After women with preexisting diabetes were excluded, GDM was identified in 15,121 (7.6%) of 199,298 screened pregnancies. The age- and race/ethnicity-adjusted GDM prevalence remained constant at 7.5 per 100 in 1999 to 7.4 per 100 in 2005 (Ptrend=0.07). Among all deliveries to women with either form of diabetes, 10% were due to preexisting diabetes in 1999, rising to 21% in 2005, with GDM accounting for the remainder. The stable prevalence of GDM and increase in the prevalence of preexisting diabetes were independent of changes in the age and race/ethnicity of the population. The increase in preexisting diabetes, particularly among younger women early in their reproductive years, is of concern [26]. The current study proposed that Gestational Diabetes Mellitus (GDM) represents a heterogeneous group of metabolic disorders, which result in varying degrees of maternal hyperglycemia and preg- nancy-associated risk. The frequency of GDM is rising globally and may also increase further as less-stringent criteria for the diagnosis are potentially adopted. The additional burden placed on the health care system by increasing cases of GDM requires consideration of di- agnostic approaches and currently used treatment strategies. Debate continues to surround both the diagnosis and treatment of GDM despite several recent large-scale studies addressing these controver- sial issues. As many now have come to reassess their approach to the management of GDM, we provided information in this review to help guide this process. The goal for each health care practitioner should continue to be to provide optimum care for women discovered to have carbohydrate intolerance during pregnancy [9]. This current study proposed that Gestational diabetes mellitus is a substantial and growing health concern in many parts of the world. Certain populations are especially vulnerable to developing this con- dition because of genetic, social, and environmental factors. Gesta- tional diabetes has serious, long-term consequences for both baby and mother, including a predisposition to obesity, metabolic syndrome, and diabetes later in life. Early detection and intervention can greatly improve outcomes for women with this condition and their babies. Unfortunately, screening and diagnostic tests are not uniform world- wide, which could lead not only to under diagnosis but also under management of the illness. Here this study reported the controversies surrounding the causes, screening, diagnosis, management, and pre- vention of gestational diabetes, and gave specific recommendations for research studies to address the major issues of this medical condi- tion [13]. Literature Review The commonest cause of neonatal NICU admission was hyperbilirubinemia (41.2%). The risk factors for NICU admission were delivery by non SVD procedure (RR: 4.6, 95% CI= 2.8, 7.7), preterm deliveries, (RR: 4.6, 95% CI=2.7, 7.7), and induc- Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 6 of 13 Citation: Ullah HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. J Diab Meta Syndro 3: 008. changes. Among the long lasting effects of these phenomena are a high rate of overweight and obesity at childhood and a high tendency to develop the “metabolic syndrome” characterized by hypertension, cardio-vascular complications and type-2 diabetes. Similarly, mater- nal overweight and obesity during pregnancy or excessive weight gain are also associated with increased obesity and complications in the offspring. Although there are different causes for fetal growth restric- tion (FGR) or for fetal excessive growth (Macrosomia), paradoxically both are associated with the “metabolic syndrome” and its long term consequences. The exact mechanism(s) underlying these long term effects on growth are not fully elucidated, but they involve insulin re- sistance, fetal hyperleptinemia, hypothalamic changes and most prob- ably epigenetic changes. Preventive measures to avoid the metabolic syndrome and its complications seem to be a tight dietary control and physical activity in the children born to obese or diabetic mothers or who had antenatal growth disturbances for other known or unknown reasons [27]. The purpose of this study was to assess changes in the prevalence of preexisting diabetes (diabetes antedating pregnancy) and gesta- tional diabetes mellitus (GDM) from 1999 through 2005. In this ret- rospective study of 175, 249 women aged 13-58 years with 209,287 singleton deliveries of ≥ 20 weeks’ gestation from 1999 through 2005 in all Kaiser Permanente hospitals in southern California, information from clinical databases and birth certificates was used to estimate the prevalence of preexisting diabetes and GDM. Preexisting diabetes was identified in 2,784 (1.3%) of all pregnancies, rising from an age- and race/ethnicity-adjusted prevalence of 0.81 per 100 in 1999 to 1.82 per 100 in 2005 (Ptrend=0.001). Significant increases were observed in all age-groups and all racial/ethnic groups. Literature Review The aim of current study was to investigate the Gestational diabe- tes mellitus. It is defined as diabetes diagnosed during pregnancy that is not clearly overt diabetes, and is becoming more common as the epidemic of obesity and type-2 diabetes continues. Newly proposed diagnostic criteria will, if adopted universally, further increase the prevalence of this condition. Much controversy surrounds the diag- nosis and management of gestational diabetes. This review provided information regarding various approaches to the diagnosis of gesta- tional diabetes and the recommendations of a number of professional organizations. The implications of gestational diabetes for both the mother and the offspring were described. Approaches to self-moni- toring of blood glucose concentrations and treatment with diet, oral medications, and insulin injections were covered. Management of glucose metabolism during labor and the postpartum period were discussed, and an approach to determining the timing of delivery and the mode of delivery was outlined. This review provided an overview of current controversies as well as current recommendations for ges- tational diabetes care [28]. This study suggested that Gestational diabetes mellitus is a sub- stantial and growing health concern in many parts of the world. Cer- tain populations are especially vulnerable to developing this condition because of genetic, social, and environmental factors. Gestational dia- betes has serious, long-term consequences for both baby and mother, including a predisposition to obesity, metabolic syndrome, and diabe- tes later in life. Early detection and intervention can greatly improve outcomes for women with this condition and their babies. Unfor- tunately, screening and diagnostic tests are not uniform worldwide, which could lead not only to under diagnosis but also under man- agement of the illness. Here, we report the controversies surround- ing the causes, screening, diagnosis, management, and prevention of gestational diabetes, and give specific recommendations for research studies to address the major issues of this medical condition [13]. The current study was done to investigate life transitions associat- ed with high levels of stress affecting health behaviors among people with Type 1 diabetes. It suggested that Transition to motherhood is a major transition with potential complications accelerated by preg- nancy with risks of adverse childbirth outcomes and added anxiety and worries about pregnancy outcomes. Further, preparing and go- ing through pregnancy requires vigilant attention to a diabetes man- agement regimen and detailed planning of everyday activities with added stress on women. Literature Review Psychological and social well-being during This study proposed that Pregestational (PGDM) and gestational (GDM) diabetes may be associated with a variety of fetal effects includ- ing increased rate of spontaneous abortions, intrauterine fetal death, congenital anomalies, neurodevelopmental problems and increased risk of perinatal complications. Additional problems of concern are fetal growth disturbances causing increased or decreased birth weight. Optimal control of maternal blood glucose is known to reduce these Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 7 of 13 hort study involving pregnant women attending prenatal clinics from urban, semi-urban and rural areas in the greater Chennai region in South India. Around 9850 pregnant women was screened for GDM. Socio-economic status, demographic data, obstetric history, delivery and birth outcomes, perinatal and postnatal complications, neonatal morbidity, maternal postpartum and offsprings follow-up data will be collected. Those diagnosed with GDM were initially advised routine care. Those unable to reach glycaemic control with diet alone were advised to take insulin. Postpartum screening for glucose abnormal- ities were performed at months 3 and 6 and then every year for 10 years. The offsprings followed up every year for anthropometric mea- surements and growth velocity, as well as, plasma glucose, insulin and lipid profile. In addition, qualitative research carried out to identify barriers and facilitators for early GDM screening, treatment compli- ance and postpartum follow-up and testing, as well as, for continued adherence to lifestyle modifications. The study demonstrated whether measures to improve diagnosis and care of GDM mothers followed by preventive postpartum care are possible in the routine care setting. It also mapped out the barriers and facilitators for such initiatives and provided new evidence on the determinants and risk factors for both GDM development and occurrence of adverse pregnancy outcomes and development of future diabetes and metabolic abnormalities in the GDM mother and her offspring [31]. and after pregnancy are integral for good pregnancy outcomes for both mother and baby. The aim of this study was to establish the face and content validity of two novel measures assessing the well-being of women with type 1 diabetes in their transition to motherhood, 1) during pregnancy and 2) during the postnatal period. Inclusion criteria The current study suggested that women with Gestational Di- abetes Mellitus (GDM) and their offsprings are at increased risk of future type 2 diabetes and metabolic abnormalities. Early diagnosis and proper management of GDM, as well as, postpartum follow-up and preventive care is expected to reduce this risk. However, no large scale prospective studies have been done particularly from the devel- oping world on this aspect. The objective of this study was to identify and follow a cohort of pregnant women with and without GDM and their offspring to identify determinants and risk factors for GDM, for various pregnancy outcomes, as well as, for the development of fu- ture diabetes and metabolic abnormalities. This was a prospective co- In this study, we included all those female patients who gave in- formed consent to be a part of this study, with positive diagnostic tests for diabetes like BSR or FBS or whose HbA1c value indicated hypergly- cemia during pregnancy in the time period of September 2016- No- vember 2016. In this study, those female patients were also added with the age limit between 18-40 years. Study design The current study suggested that Macrosomia is defined as birth- weight over 4,000 g irrespective of gestational age and affects 3-15% of all pregnancies. The present study aimed to determine the relation- ship between mother’s characteristics and macrosomic births and also compare macrosomic and normal newborns regarding the maternal and offspring complications of diabetes during pregnancy. In this case control study, among the 420 consecutive births occurring in public and private hospitals of Shiraz, Iran from October 2006 to March 2007, the data of 32 macrosomic and 128 normal newborns were an- alyzed using t-test and chi square in bivariate and logistic regression in multivariate model. The mean (SD) of neonate weight, height, and head size was 3323.4 (709), 48.95 (3.2), and 34.9 (1.8), respectively. Re- gression analysis showed that gestational diabetes (Odds Ratio (OR): 11.9, Confidence Interval CI=4.6-30.3), preeclampsia in the pregnan- cy period due to diabetes (OR: 3.81, CI=1.1-13.2), and macrosomic birth history (OR: 3.3, CI=1.04-10.4) were the main predictors of macrosomia. Moreover, macrosomia increased neonate hypoglycemia (OR: 4.7, CI=1.4-15.8) and section delivery (OR: 4.1, CI=1.27-13.1). Gestational diabetes, preeclampsia due to diabetes, and history of macrosomic birth were the main predictors of macrosomia. More- over, macrosomia increased some delivery complications for both mothers and newborns [30]. The design of the current study was observational cross-sectional study. A questionnaire with the variables related to birth complica- tions in diabetic mothers was designed with consensus of gynecol- ogists/patients for the study. The demographic data, family history, previous medical history, reproductive history as well as the Tetanus Toxoid Vaccination status and drug profile was included in the ques- tionnaire. This study was conducted in different Hospitals, Clinical settings, Maternity homes in Lahore, Punjab Pakistan. Data collection The questionnaire was designed to collect information regarding various parameters associated with birth complications due to diabe- tes, directly by patient counseling. Records of patients having IDDM, NIDDM or GDM were evaluated for fetal status. The pathology reports like HbA1C were reviewed to determine the glucose control during previous months. The type of diabetes in patients, presenting signs and symptoms, family history of diabetes, other related pathological conditions, diabetes associated risk factors in mother and child, birth complications, treatment strategies, risk of diabetes in newborn, were evaluated. Literature Review The approach to the development of the Pregnancy and Postnatal Well-being in T1DM Transition questionnaires was based on a four-stage pre-testing pro- cess; systematic overview of literature, items development, piloting testing of questionnaire and refinement of questionnaire. The ques- tionnaire was reviewed at every stage by expert clinicians, researchers and representatives from consumer groups. The cognitive debriefing approach confirmed relevance of issues and identified additional items. The literature review and interviews identified three main ar- eas impacting on the Women’s postnatal self-management; (1) psy- chological well-being; (2) social environment, (3) physical (maternal and fetal) well-being. The cognitive debriefing in pilot testing of the questionnaire identified that immediate postnatal period was difficult, particularly when the women were breastfeeding and felt depressed. The questionnaires fill an important gap by systematically assessing the psychosocial needs of women with type 1 diabetes during preg- nancy and in the immediate postnatal period. The questionnaires can be used in larger data collection to establish psychometric properties. The questionnaires potentially play a key role in prospective research to determine the self-management and psychological needs of wom- en with type 1 diabetes transitioning to motherhood and to evaluate health education interventions [29]. Henry Publishing Groups © Ullah HH, et al., 2020 Results During the above mentioned period of September 2016- Novem- ber 2016, a total of 200 patients were included in this study out of which 162 women were being identified to have GDM. Among the screened GDM positive women, the majority of the women were in 5TH TO 8TH gestational week (2nd month of pregnancy) i.e. 65 cases as shown in (Figure 4.1). Then 34 cases were within 9th to 12th week, 28 cases were in1st to 4th week and19 cases were in17th to 20thgestation- al week. The least number of cases i.e. only 15 were being diagnosed during 17th to 20th week (Table 4.1). Figure 4.1: The percentage of patients being diagnosed with GDM and the time of diagnosis. Figure 4.2: Comparison of gestational and pregestational (Type-I & Type-II) diabetes. Table 4.3: Major feotal complications in diabetic mothers. Major complications No. of patients % of patients Respiratory distress 36 23 Hypoglycemic babies 82 52 Macrosomia 25 16 Cardiovascular malformations 12 7 Stillbirths/ Miscarriages 4 2 Table 4.3: Major feotal complications in diabetic mothers. Major complications No. of patients % of patients Respiratory distress 36 23 Hypoglycemic babies 82 52 Macrosomia 25 16 Cardiovascular malformations 12 7 Stillbirths/ Miscarriages 4 2 Figure 4.1: The percentage of patients being diagnosed with GDM and the time of diagnosis. Figure 4.1: The percentage of patients being diagnosed with GDM and the time of diagnosis. Figure 4.3: Majorfoetal complications in diabetic mothers. Table 4.1: The percentage of patients being diagnosed with GDM and the time of di- agnosis. Gestational week No. of patients % of patients 1st-4th 28 17.4 5th-8th 65 40.4 9th-12th 34 21.1 13th-16th 19 11.8 17th-20th 16 9.87 In the current study we compared the prevalence of gestational and pregestational diabetes in pregnant women. Out of 200 patients, the majority of the cases were of GDM i.e. 162 (81%). The remaining cases (19%) were of pregestational diabetes i.e. type I or type II DM. 28 (5%) cases were being presented with Type I DM while 10 (14%) patients with Type II DM as shown in (Table 4.2) (Figure 4.2). Figure 4.3: Majorfoetal complications in diabetic mothers. Out of 200 patients 41 patients had miscarriage due to one of the following reasons. The major reason of miscarriage in diabetic moth- ers was found to be hypertension and out of 41 cases there were 17 (41.5%) cases of miscarriage due to hypertension. Exclusion criteria In this study those female patients were not included who refused to give informed consent to be a part of this study and those who left against medical advice after provisional diagnosis were made. In this Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 8 of 13 study, those female patients were also excluded with the age less than 16 years or more than 45 years. the most frequently occurring complication was hypoglycemic babies i.e.52%. Then at second number was respiratory distress 23%, at third number was macrosomia 16%, at fourth number was CVS malforma- tions 7% and the least prevailing complication was still birth/miscar- riage i.e. 2% as shown in (Table 4.3) (Figure 4.3). Data analysis Information was compiled on the pre-designed questionnaire. Data was analyzed on MS Excel, 2010. Figure 4.2: Comparison of gestational and pregestational (Type-I & Type-II) diabetes. Henry Publishing Groups © Ullah HH, et al., 2020 in (Table 4.6) (Figure 4.6). Our study population of 200 patients was also being analyzed to check their TT status. So 76% (153) of the pregnant ladies were found to be vaccinated for tetanus while 24% (47) of the pregnant women Figure 4.4: Reasons of miscarriages in diabetic mothers. Table 4.7: Tetanus toxoid vaccination status analysis. No. of patients % of patients Vaccinated 153 76 Not vaccinated 47 24 Figure 4.7: Tetanus toxoid vaccination status analysis. were not being vaccinated as shown in (Table 4.7) (Figure.4.7). Figure 4.7: Tetanus toxoid vaccination status analysis. Table 4.5: Normal delivery VS C-section delivery. Mode of delivery No. of patients % of patients Normal vaginal 29 28 Cesarean section 76 72 re not being vaccinated as shown in (Table 4.7) (Figure.4.7) Figure 4.5: Normal delivery VS C-section delivery. Results There were 10 (24.3%) cases of miscarriage due extreme sugar level, 9 (22%) cases of miscarriage due to polyhydramnios and 5 (12.2%) cases of miscar- riage due to Hughes syndrome as shown in (Table 4.4) (Figure 4.4). Table 4.2: Comparison of pre-gestational (type I, type II) and gestational diabetes. Type of diabetes No. of patients % of patients Type I 10 5 Type II 28 14 GDM 162 81 In our study population, the ratio of the normal and CS delivery was being determined. Out of 200 patients who were included in this study, 105 were being delivered during our study period.28% (29) cas- es were of normal delivery whereas 72% (76) cases were of C-section as shown in (Table 4.5) (Figure 4.5). In our study population 159 patients had foetal/neonatal compli- cations while the remaining pregnancies were normal and controlled. The major complications found were macrosomia, hyperglycemia, CVS malformations, respiratory distress and stillbirth. Out of these, Out of 105 patients who were being delivered during our study Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 9 of 13 Citation: Ullah HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. J Diab Meta Syndro 3: 008. FK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Assoc dro 3: 008. period, there were 76 cases of CS. The major reasons of CS found out were as shown in the following figure. The major reason of CS was fetal distress and we found 35 (46.05%) such cases. Then at second place was the placental abruption with 15 (14.47%) cases, third major reason was dystocia with 11 (19.74%) cases and the fourth major rea- son was uterine rupture with 10 (13.16%) cases. The least reason of CS was breech position and we found only 5 (6.58%) such cases as shown Figure 4.6: Reasons for C-section delivery. Table 4.4: Reasons of miscarriages in diabetic mothers. Reasons of miscarriage No. of patients % of patients Hypertension 17 41.50 Polyhydramnios 9 22.00 Hughes syndrome 5 12.20 Uncontrolled sugar level 10 24.30 in (Table 4.6) (Figure 4.6). Results in (Table 4.6) (Figure 4.6). Discussion Patients who cannot control their diabetes with diet and exercise only, require insulin, which leads to state of hyperinsulinemia in the foetus of such mothers. This and many oth- er reasons lead to the foetal complications. Postnatal hypoglycemia, respiratory distress, macrosomia, CVS malformations and stillbirths are major complications in the babies born to the diabetic mothers. The current study has shown that these complications are frequently occurring in the neonates and foetus of diabetic mothers. In recent years a study was performed to determine the range of complications occurring in infants of diabetic mothers. A total number of 40 ba- bies were included in this study.35% new borns presented respiratory distress and8% were having CVS malformations. Hypoglycemia was noted in 50% [22]. Another study was being performed to compare the prevalence at live birth and the spectrum of cardiovascular mal- formations in infants born to diabetic mothers with that in infants of non-diabetic mothers. This study concluded that maternal diabetes is associated with a fivefold increase in risk of cardiovascular malfor- mations. Transposition of the great arteries, truncus arteriosus, and tricuspid atresia are overrepresented to produce a substantial excess of these malformations [35]. All these previous studies strongly support our results. The rise in the rate of cesarean sections in diabetic mothers is due to widening of the relative indications, such as dystocia, previous cesarean, fetal distress and breech presentation. In a recent study in- crease in the cesarean rate of 6.2% points was partitioned according to five complications of delivery including previous cesarean delivery, breech presentation, dystocia, fetal distress, and all other complica- tions. Nearly half (48%) of the increase was associated with previous cesarean delivery, 29 per cent with dystocia, 16 per cent with fetal dis- tress, 5 per cent with breech presentation, and 2 per cent with all other complications [39]. In the current study we examined the recorded indications of the CS and most common of them were fetal distress, placental abruption, dystocia, uterine rupture and breech position re- spectively. Previous CS was also a reason of CS in index deliveries. Tetanus is an acute, often fatal, disease caused by an exotoxin produced by Clostridium tetani. It occurs in newborn infants born to mothers, who do not have sufficient circulating antibodies to pro- tect the infant passively, by transplacental transfer. Discussion Prevention may be possible by the vaccination of pregnant or non-pregnant women, or both, with tetanus toxoid, and the provision of clean delivery ser- vices. Tetanus toxoid consists of a formaldehyde-treated toxin which stimulates the production of antitoxin. Recently a study was conduct- ed to determine the TT vaccination status for pregnant women, and to examine the effects of various factors on TT vaccination coverage during pregnancy in reproductive-age women in Turkey. Four-hun- dred and ninety-three postpartum women who had live births at a hospital in Ankara were interviewed and information was collected on the mothers’ sociodemographic characteristics, TT vaccination history, and prenatal care during the pregnancy studied. The rates for no vaccination, one-dose vaccination, and two-dose vaccination were 53.3%, 18.9%, and 27.8%, respectively [40]. While in the current study, 76% of the screened women were being vaccinated with tetanus toxoid. This fact shows that maternal vaccination status is much better in Pakistan, but still more public awareness is required to boost up the rate of maternal vaccination in our country. Pregnancy affects both the maternal and fetal metabolism and even in nondiabetic women exerts a diabetogenic effect. Pregnancy loss is significantly higher among women with diabetes compared to the nondiabetic population. Neonatal mortality is also higher among infants of diabetic mothers in approximately 15-fold when compared to the general population [36]. Recently, a population-based cohort study conducted in the UK has shown that women with type 1 diabe- tes have a higher risk of late fetal loss, presenting a four- to five-fold increase in perinatal death, and a four- to six-fold in stillbirth [37]. The major reasons of miscarriages in diabetic women include hyper- tension, Hughes syndrome, polyhydramnios and uncontrolled sugar level; hypertension and pre-eclampsia being the root cause. Discussion This study was conducted to determine the occurrence of GDM and the birth complications in diabetic mothers. In the estimation of the prevalence of GDM during different gestational weeks, it was found out that although GDM manifests in all trimesters of pregnan- cy, but in majority of the cases GDM occurs in early gestational weeks. Screening for GDM is usually performed around 24-28 weeks of ges- tational age. In a recent study a total of 4151 consecutive pregnant women irrespective of gestational weeks attending antenatal health posts across Chennai city underwent a 75 g OGTT (oral glucose tol- erance test) recommended by WHO and diagnosed GDM if 2hr PG value ≥ 140 mg/dl. Observation in this study was that 38.7% women developed GDM even prior to 24th week of gestation [32]. Our results are very much similar to this study. So the OGTT must be performed in pregnant women prior to 24 weeks of gestation for better hypergly- cemic control and to reduce the risk of birth complications. Figure 4.5: Normal delivery VS C-section delivery. Table 4.6: Reasons for C-section delivery. Reasons of CS No. of patients % of patients Placental abruption 15 14.47 Dystocia 11 19.74 Uterine rupture 10 13.16 Breech position 5 6.58 Foetal distress 35 46.05 During pregnancy, out of all types of diabetes most prevailing type is GDM. In the current study, 162 (81% of the total cases) wom- Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 10 of 13 10 of 13 ulation [38]. These results strongly support our current study which showed that 72% of the total deliveries were through CS. en were presented with GDM, while with type I only 5% and with type II 14%. So the risk of GDM is more in Pakistan. A recent study analyzed 1,729,225 Canadian women and found that the prevalence of gestational and pregestational diabetes in 1996 was 2.7% and 0.4%, respectively [33]. In another study, rates of gestational diabetes (Class A1 combined with Class A2) and pregestational diabetes were 2.0% and 0.3%, respectively [34]. Our results were similar to these studies in a sense that the ratio of gestational diabetes is more as compared to pregestational diabetes among pregnant women in Pakistan. 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(2007) Gestational diabetes mellitus manifests in all trimesters of pregnan- cy. Diabetes Research and Clinical Practice 77: 482-484. 15. Hod M, Merlob P, Friedman S, Schoenfeld A, Ovadia J (1991) Ges- tational diabetes mellitus: A survey of perinatal complications in the 1980s. Diabetes 40: 74-78. 33. Wen SW, Liu S, Kramer MS, Joseph K, Levitt C, et al. (2000) Impact of prenatal glucose screening on the diagnosis of gestational diabe- tes and on pregnancy outcomes. American journal of epidemiology, 152: 1009-1014. 16. Weintrob N, Karp M, Hod M (1996) Short-and long-range compli- cations in offspring of diabetic mothers. Journal of Diabetes and its Complications 10: 294-301. 34. Sheffield JS, Butler-Koster EL, Casey BM, Mcintire DD, Leveno KJ (2002) Maternal diabetes mellitus and infant malformations. Obstet- rics & Gynecology 100: 925-930. 17. Dabelea D, Hanson RL, Lindsay RS, Pettitt DJ, Imperatore G, et al. (2000), Intrauterine exposure to diabetes conveys risks for type 2 diabetes and obesity: A study of discordant sibships. Diabetes 49: 2208-2211. 35. Wren C, Birrell G, Hawthorne G (2003) Cardiovascular malforma- tions in infants of diabetic mothers. Heart 89: 1217-1220. 18. Svare JA, Hansen BB, Mølsted-Pedersen L (2001) Perinatal com- plications in women with gestational diabetes mellitus. Acta obste- tricia et gynecologica Scandinavica 80: 899-904. 36. Hawthorne G, Robson S, Ryall E, Sen D, Roberts S, et al. (1997) Prospective population based survey of outcome of pregnancy in diabetic women: Results of the Northern Diabetic Pregnancy Audit. Bmj 315: 279-281. 19. Hyppönen E, Läärä E, Reunanen A, Järvelin MR, Virtanen SM (2001) Intake of vitamin D and risk of type 1 diabetes: A birth-cohort study. The Lancet 358: 1500-1503. 37. Casson I, Clarke C, Howard C, Mckendrick O, Pennycook S, et al. Conclusion World Health Organization 1-65. 23. Al-Hakeem MM (2006) Pregnancy outcome of gestational diabetic mothers: Experience in a tertiary center. Journal of Family & Com- munity Medicine 13: 55-59. 3. Stogdale L (1986) Definition of diabetes mellitus. The Cornell veter- inarian 76: 156-174. 24. Rowan JA, Hague WM, Gao W, Battin MR, Moore MP (2008) Met- formin versus insulin for the treatment of gestational diabetes. New England Journal of Medicine 358: 2003-2015. 4. Association AD (2010) Diagnosis and classification of diabetes mel- litus. Diabetes care 33: S62-S69. 25. Clausen TD, Mathiesen ER, Hansen T, Pedersen O, Jensen DM, et al. (2008) High prevalence of type 2 diabetes and pre-diabetes in adult offspring of women with gestational diabetes mellitus or type 1 diabetes the role of intrauterine hyperglycemia. Diabetes care 31: 340-346. 5. Lawrence R (1951) Types of human diabetes. British medical jour- nal 1: 373. 6. Cudworth A (1978) Type I diabetes mellitus. Diabetologia 14: 281- 291. 26. Lawrence JM, Contreras R, Chen W, Sacks DA (2008) Trends in the prevalence of preexisting diabetes and gestational diabetes mellitus among a racially/ethnically diverse population of pregnant women, 1999–2005. Diabetes carec31: 899-904. 7. Edelman SV (1997) Type II diabetes mellitus. Advances in internal medicine 43: 449-500. 8. Soria B, Roche E, Berna G, León-Quinto T, Reig JA, et al. (2000) Insulin-secreting cells derived from embryonic stem cells normal- ize glycemia in streptozotocin-induced diabetic mice. Diabetes 49: 157-162. 27. Ornoy A (2011) Prenatal origin of obesity and their complications: Gestational diabetes, maternal overweight and the paradoxical ef- fects of fetal growth restriction and macrosomia. Reproductive tox- icology 32: 205-212. 9. Landon MB, Gabbe SG (2011) Gestational diabetes mellitus. Ob- stetrics & Gynecology 118: 1379-1393. 28. Coustan DR (2013) Gestational Diabetes Mellitus. Clinical Chemis- try 59: 1310-1321. 10. Kjos SL, Buchanan TA (1999) Gestational diabetes mellitus. New England journal of medicine 341: 1749-1756. 29. Rasmussen B, Dunning T, Hendrieckx C, Botti M, Speight J (2013) Transition to motherhood in type 1 diabetes: Design of the preg- nancy and postnatal well-being in transition questionnaires. BMC Pregnancy and Childbirth 13: 54. 11. Kopp W (2005) Role of high-insulinogenic nutrition in the etiology of gestational diabetes mellitus. Medical hypotheses 64: 101-103. 30. Mohammadbeigi A, Farhadifar F, Soufi Zadeh N, Mohammadsalehi N, Rezaiee M, et al. (2013) Fetal Macrosomia: Risk Factors, Mater- nal, and Perinatal Outcome. Ann Med Health Sci Res 3: 546-50. 12. Conclusion Diabetes is still a major problem of birth complications and mis- carriages in Pakistan. In the current study we concluded that during pregnancy GDM is the most prevailing type of diabetes and it is asso- ciated with major birth complications including respiratory distress, hypoglycemic babies, macrosomia, CVS malformations and even mis- carriages. Most of our patients were being diagnosed with GDM be- fore 24th week of gestation. So a public awareness program is required to educate the people about reproductive health and to motivate them to undergo BSR/FBS during pregnancy prior to 24th gestational weeks to diagnose for GDM. This will lead to better glycemic control during pregnancy and decreased incidence of birth complications and mis- carriages as according to our current study the major reason of mis- carriages in diabetic mothers are hypertension and uncontrolled sugar level. From the current study we also concluded that the women being vaccinated with TT, are still at risk of GDM. The prevalence of births worldwide complicated by DM is increas- ing. In the UK, for example, <25% of diabetic women have a non-in- strumental vaginal delivery. Strikingly, more than half the CS in these patients was non-elective, but the reasons for this are not understood. This recent study tested the hypothesis that poor myometrial contrac- tility as a consequence of the disease contributes to this high CS rate. There was significantly decreased contraction amplitude and dura- tion in uteri from diabetic compared with control patients. There is poorer contractility even in the presence of oxytocin. The underlying mechanism is related to reduced Calcium channel expression and in- tracellular calcium signals and a decrease in muscle mass. This study concluded that these factors significantly contribute to the increased emergency CS rate in diabetic patients. It showed a high induction of labor rate (39%) and a high C-section rate (67%) in women with type I and type II DM as compared to 21% of the general maternal pop- References Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 Henry Publishing Groups © Ullah HH, et al., 2020 Henry Publishing Groups © Ullah HH, et al., 2020 11 of 13 Citation: Ullah HH, Ullah HFK, Saleem HGM, Ullah W (2020) Study of Birth Complications in Diabetic Mothers, Dietary Supplements and their Associated Risks. J Diab Meta Syndro 3: 008. 2. Assal J, Groop L (1999) Definition, diagnosis and classification of diabetes mellitus and its complications. Henry Publishing Groups, 41341 Red Birch Dr, Aldie ,VA ,20105, USA, Tel: +1 571-275-4480; E-mail: contact@henrypublishinggroups.org https://www.henrypublishinggroups.com/ Conclusion (1997) Outcomes of pregnancy in insulin dependent diabetic wom- en: Results of a five year population cohort study. Bmj 315: 275- 278. 20. Evers IM, De Valk HW, Visser GH (2004) Risk of complications of pregnancy in women with type 1 diabetes: nationwide prospective study in the Netherlands. Bmj 328: 915. 38. Al-Qahtani S, Heath A, Quenby S, Dawood F, Floyd R, et al. (2012) Diabetes is associated with impairment of uterine contractility and high Caesarean section rate. Diabetologia 55: 489-498. 21. Kerssen A, De Valk HW, Visser GHA (2006) Do HbA1c levels and the self-monitoring of blood glucose levels adequately reflect gly- caemic control during pregnancy in women with type 1 diabetes mellitus?. Diabetologia 49: 25-28. 39. Taffel SM, Placek PJ, Liss T (1987) Trends in the United States cesarean section rate and reasons for the 1980-85 rise. American journal of public health 77: 955-959. 22. Alam M, Raza SJ, Sherali A, Akhtar A (2006) Neonatal complica- tions in infants born to diabetic mothers. Journal of the College of Physicians and Surgeons--Pakistan 16: 212-215. Henry Publishing Groups © Ullah HH, et al., 2020 Volume: 3 \| Issue: 1 \| 100008 ISSN: 2565-5795 12 of 13 12 of 13 Henry Journal of Acupuncture & Traditional Medicine Henry Journal of Anesthesia & Perioperative Management Henry Journal of Aquaculture and Technical Development Henry Journal of Cardiology & Cardiovascular Medicine Henry Journal of Case Reports & Imaging Henry Journal of Cell & Molecular Biology Henry Journal of Tissue Biology & Cytology Henry Journal of Clinical, Experimental and Cosmetic Dermatology Henry Journal of Diabetes & Metabolic Syndrome Henry Journal of Emergency Medicine, Trauma & Surgical Care Henry Journal of Haematology & Hemotherapy Henry Journal of Immunology & Immunotherapy Henry Journal of Nanoscience, Nanomedicine & Nanobiology Henry Journal of Nutrition & Food Science Henry Journal of Obesity & Body Weight Henry Journal of Cellular & Molecular Oncology Henry Journal of Ophthalmology & Optometry Henry Journal of Perinatology & Pediatrics Submit Your Manuscript: https://www.henrypublishinggroups.com/submit-manuscript/
https://openalex.org/W2162568227	https://discovery.dundee.ac.uk/ws/files/8026506/1_s2.0_S0092867414000233_main.pdf	English	null	Norspermidine Is Not a Self-Produced Trigger for Biofilm Disassembly	Cell	2,014	cc-by	10,266	Citation for published version (APA): Hobley, L., Kim, S. H., Maezato, Y., Wyllie, S., Fairlamb, A. H., Stanley-Wall, N. R., & Michael, A. J. (2014). Norspermidine is not a self-produced trigger for biofilm disassembly. Cell, 156(4), 844-854. https://doi.org/10.1016/j.cell.2014.01.012 Norspermidine is not a self-produced trigger for biofilm disassembly Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. University of Dundee University of Dundee orspermidine is not a self-produced trigger for biofilm disassembly SUMMARY ecules in Gram-positive and Gram-negative bacterial species, biosynthesis is tightly regulated (Joo and Otto, 2012; Vlamakis et al., 2013). The Gram-positive bacterium Bacillus subtilis has emerged as a model organism for deciphering the molecular basis of bioﬁlm development (Vlamakis et al., 2013). Formation of the extracellular matrix of B. subtilis bioﬁlms is dependent on production of an exopolysaccharide synthesized by the prod- ucts of the epsA-O operon and an amyloid-like protein compo- nent that is generated by products of the tapA-sipW-tasA operon (Branda et al., 2006; Romero et al., 2010). Assembly of the matrix also requires production of the bacterial hydrophobin BslA that forms a hydrophobic coat over the bioﬁlm surface (Hobley et al., 2013; Kobayashi and Iwano, 2012; Ostrowski et al., 2011). In the laboratory, bioﬁlms formed by B. subtilis manifest as robust surface-associated colonies and ﬂoating pellicles that both display a complex rugose architecture (Branda et al., 2001; Vlamakis et al., 2013). Formation of Bacillus subtilis bioﬁlms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the poly- amine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 mM prevents B. subtilis bioﬁlm formation, (3) endogenous norspermidine is present in bioﬁlms at 50–80 mM, and (4) norspermidine prevents bioﬁlm formation by condensing bioﬁlm exopolysaccharide. In contrast, we ﬁnd that, at concentrations up to 200 mM, exogenous norspermidine promotes bioﬁlm formation. We ﬁnd that norspermidine is absent in wild-type B. subtilis bioﬁlms at all stages, and higher concentrations of exogenous norspermidine eventu- ally inhibit planktonic growth and bioﬁlm formation in an exopolysaccharide-independent manner. More- over, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, conﬁrming that norspermidine is not physiologically relevant to bioﬁlm function in this species. Formation of robust colony bioﬁlms and pellicles in B. subtilis is dependent on the presence of the polyamine spermidine (Burrell et al., 2010). Indeed, externally supplied spermidine can restore bioﬁlm formation to a B. subtilis spermidine auxo- troph. In B. subtilis, the triamine spermidine is formed from the diamine putrescine (Figure 1A) through transfer of an amino- propyl group to putrescine by spermidine synthase encoded by speE (Sekowska et al., 1998). General rights i h d 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. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Matters Arising SUMMARY The donor of the aminopropyl group, decarboxylated S-adenosylmethionine, is produced by the activity of S-adenosylmethionine decarboxylase, encoded by speD (Figure 1A) (Sekowska et al., 2000). Putrescine and, most likely, spermidine are required for bioﬁlm formation in Yersinia pestis, the causative agent of bubonic plague (Patel et al., 2006; Wortham et al., 2010). However, for the cholera agent Vibrio cholerae, bioﬁlm formation is dependent on biosyn- thesis of the unusual shorter structural analog of spermidine, norspermidine (Figure 1B), and spermidine cannot replace the function of norspermidine in bioﬁlm formation in this species (Lee et al., 2009). Furthermore, external norspermidine is sensed by a substrate-binding protein homolog of a polyamine transporter, which then stimulates bioﬁlm formation (Karatan et al., 2005; Karatan and Michael, 2013), whereas uptake of Laura Hobley,1 Sok Ho Kim,2 Yukari Maezato,2 Susan Wyllie,3 Alan H. Fairlamb,3 Nicola R. Stanley-Wall,1,* and Anthony J. Michael2,* Sok Ho Kim,2 Yukari Maezato,2 Susan Wyllie,3 Alan H. Fairlamb,3 Nicola R. Stanley-Wall,1,* Laura Hobley,1 Sok Ho Kim,2 Yukari Maezato,2 Susan Wyllie,3 Alan H. Fairlamb,3 Nicola R. Stanley-Wall,1,* and Anthony J Michael2 * 1Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD15EH, UK 2Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 3Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD15EH, UK Correspondence: n.r.stanleywall@dundee.ac.uk (N.R.S.-W.), anthony.michael@utsouthwestern.edu (A.J.M.) http://dx.doi.org/10.1016/j.cell.2014.01.012 p g j 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. O d CC BY li Norspermidine Is Not a Self-Produced Trigger for Bioﬁlm Disassembly Laura Hobley,1 Sok Ho Kim,2 Yukari Maezato,2 Susan Wyllie,3 Alan H. Fairlamb,3 Nicola R. Stanley-Wall,1, and Anthony J. Michael2,* INTRODUCTION The ability of microbial cells to colonize a habitat is frequently dependent on bioﬁlm formation, a process by which microbial cells form a sessile community encased in a self-produced extracellular matrix (Costerton et al., 1995; Davey and O’Toole, 2000; Flemming and Wingender, 2010). This extracellular matrix is typically composed of extracellular DNA, proteins, and exopo- lysaccharides that together function to adhere cells to a surface and provide protection from external physical and chemical stresses (Branda et al., 2005; Flemming and Wingender, 2010). To ensure correct deployment of these extracellular macromol- 844 Cell 156, 844–854, February 13, 2014 ª2014 The Authors Figure 1. Spermidine and Norspermidine Biosynthetic Pathways (A) Spermidine biosynthetic pathway of B. subtilis NCIB3610. Figure 1. Spermidine and Norspermidine Biosynthetic Pathways (A) Spermidine biosynthetic pathway of B. subtilis NCIB3610. A B A B B A (B) Norspermidine biosynthetic pathway of V. cholerae. decarboxylase encoded by speA or S- adenosylmethionine decarboxylase en- coded by speD (Figure 1A) prevents development of the highly wrinkled col- ony bioﬁlm morphology of the wild-type B. subtilis strain NCIB3610 grown on solid polyamine-free MSgg growth me- dium (Figure 2A). Exogenous provision of the shorter spermidine structural analog norspermidine (H2N(CH2)3NH(CH2)3NH2) (Figure 1B) in the growth medium is more effective than exogenous spermidine in restoring the complex colony bioﬁlm phenotype to the speA and speD spermi- dine-deﬁcient mutants, whereas the longer structural analog homospermidine (H2N(CH2)4NH(CH2)4NH2) is ineffective at the same concentrations (Figure 2A). Spermidine, norspermidine, and homospermidine do not restore a normal complex colony bioﬁlm morphology to the tasA and eps mutants, which lack the bioﬁlm amyloid protein and exopolysaccharide, respectively. Formation of robust pellicle bioﬁlms of B. subtilis NCIB3610 that develop at the liquid-air interface is also spermidine dependent (Burrell et al., 2010). The wrinkled pellicle morphology of the B. subtilis NCIB3610 wild-type strain is absent in the spermidine biosynthetic mutants speA and speD (Figure 2B) after 2-day incubation. Exogenous provision of just 5 mM spermidine or norspermidine to the liquid MSgg growth medium at the start of incubation restores the wrinkled pellicle morphology to the speA and speD mutants, whereas homospermidine is ineffective even at 100 mM (Figure 2B). Moreover, we noted that when exog- enous norspermidine is provided to the B. subtilis wild-type NCIB3610 strain at higher concentrations (25 or 100 mM) for longer times (5 or 7 days), the pellicles become more wrinkled (Figure 2C). INTRODUCTION Our two laboratories each independently assayed the effect of exogenously supplied norspermidine on pellicle formation. In contrast to the ﬁnding of Kolodkin-Gal et al. (2012) that B. subtilis NCIB3610 pellicle bioﬁlm formation is inhibited by INTRODUCTION This phenotype may not be as apparent if the wild-type pellicle is already highly wrinkled. Increased wrinkling occurred with a B. subtilis NCIB3610 isolate maintained by us and also with an NCIB3610 isolate obtained from the Losick laboratory (referred to here as NCIB3610-H) (Figure 2C). exogenous spermidine inhibits bioﬁlm formation (McGinnis et al., 2009). The complete pathway for norspermidine biosynthesis in V. cholerae (Figure 1B) has been established only recently by Lee et al. (2009) and is enzymatically distinct from the spermi- dine biosynthetic pathway found in B. subtilis. During planktonic growth, spermidine is the only detectable polyamine in B. subtilis (Burrell et al., 2010), whereas in V. cholerae, the only detectable triamine is norspermidine (Lee et al., 2009). Recently, a study reported that B. subtilis synthesizes norsper- midine in 8-day-old pellicle bioﬁlms using the V. cholerae-type biosynthetic pathway in order to disassemble the bioﬁlm (Kolod- kin-Gal et al., 2012). The authors reported that B. subtilis bioﬁlms contain 50–80 mM norspermidine and that just 25 mM exogenous norspermidine added to the growth medium prior to inocula- tion fully inhibits bioﬁlm formation without inhibiting planktonic growth. It was proposed that the B. subtilis genes gabT and yaaO encode the norspermidine biosynthetic enzymes L-2,4-dia- minobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT) andcarboxynorspermidinedecarboxylase(CANSDC)(Figure1B), respectively, and that mutation of either of those genes abolished norspermidine biosynthesis and prevented bioﬁlm disassembly. The authors also proposed that norspermidine inhibited bioﬁlm formation by binding to the exopolysaccharide. Due to the impor- tant implications of these ﬁndings for understanding bioﬁlm phys- iology and the biosynthesis and function of polyamines, our laboratories each independently reexamined the key ﬁndings of Kolodkin-Gal et al. (2012). In contrast, we ﬁnd that norspermidine is not synthesized by B. subtilis, is not naturally present in bioﬁlms formed by B. subtilis, and orthologs of the V. cholerae norspermi- dine biosynthetic genes are absent from the B. subtilis genome. Therefore, norspermidine is unlikely to have a native role in bioﬁlm physiology of this species, whereas the related polyamine sper- midine is essential for robust bioﬁlm formation. Given the essential role of spermidine in formation of B. subtilis NCIB3610 robust colony and pellicle bioﬁlms and the ability of norspermidine to efﬁciently substitute for spermidine in this func- tion, we were surprised by the ﬁnding of Kolodkin-Gal et al. (2012) that norspermidine at a concentration of only 25 mM prevents formation of B. subtilis NCIB3610 pellicle bioﬁlms. Norspermidine Replaces the Essential Role of Spermidine in B. subtilis Bioﬁlm Formation The polyamine spermidine (H2N(CH2)3NH(CH2)4NH2) (Figure 1A) is essential for robust bioﬁlm formation in B. subtilis (Burrell et al., 2010). Deletion of the spermidine biosynthetic enzymes arginine RESULTS Cells of our laboratory B. subtilis wild-type isolate (NCIB3610) and the wild-type isolate from the Losick laboratory (NCIB3610-H) were grown for either 5 or 7 days in MSgg liquid medium with 0, 25, or 100 mM norspermidine. 25 mM norspermidine, we found that bioﬁlm formation was not inhibited until a concentration of 250 mM norspermidine (Figure 3A). We were concerned that our laboratory isolate of B. subtilis NCIB3610 might differ in behavior toward exoge- nously provided norspermidine compared with the NCIB3610- H isolate used by Kolodkin-Gal et al. (2012). However, analysis demonstrated that the two isolates behaved very similarly, with pellicle formation being prevented by a concentration of exoge- nous norspermidine of between 250 and 300 mM (Figure 3A). ence of spermidine was not reported. To determine whether spermidine was also present in the pellicles, we employed high-performance liquid chromatography (HPLC) with pure chemical standards to both detect and distinguish spermidine and norspermidine. Two different labeling methods were used to detect the polyamines, by attaching chemical tags that would then render them visible by ﬂuorescence detection. The ﬁrst approach used a commercial amino acid detection system (AccQ-Fluor Reagent; Waters) that derivatizes polyamines with the ﬂuorescent AccQ-Fluor tag (Figure 4), whereas the second approach derivatized polyamines by dansylation (Figure 5). p m ( g ) It was noted by Kolodkin-Gal et al. (2012) that the wispy fragments of ﬂoating material present in an exopolysaccharide mutant were unaffected by addition of norspermidine at the same concentrations that inhibited pellicle formation of B. subtilis NCIB3610. They postulated that norspermidine in- hibited bioﬁlm formation by interfering with the exopolysacchar- ide component of the bioﬁlm matrix. In contrast, we found that at a concentration of 300 mM norspermidine, the wispy fragments of ﬂoating material in the exopolysaccharide mutant eps (A-O) strain no longer formed, and at 500 mM norspermidine, no resid- ual pellicle material could be detected with the B. subtilis NCIB3610 eps (A-O) mutant (Figure 3B). These results sug- gested to us that norspermidine might be inhibiting the growth of planktonic cells, which in turn would lead to the bioﬁlm defects observed. Therefore, the effect of increasing concentrations of norspermidine on planktonic growth of B. subtilis NCIB3610 (Fig- ures 3C and 3E) and the eps (A-O) exopolysaccharide mutant (Figures 3D and 3F) was examined. RESULTS Norspermidine Replaces the Essential Role of Spermidine in B. subtilis Bioﬁlm Formation The polyamine spermidine (H2N(CH2)3NH(CH2)4NH2) (Figure 1A) is essential for robust bioﬁlm formation in B. subtilis (Burrell et al., 2010). Deletion of the spermidine biosynthetic enzymes arginine The polyamine spermidine (H2N(CH2)3NH(CH2)4NH2) (Figure 1A) is essential for robust bioﬁlm formation in B. subtilis (Burrell et al., 2010). Deletion of the spermidine biosynthetic enzymes arginine Cell 156, 844–854, February 13, 2014 ª2014 The Authors 845 Figure 2. Norspermidine Replaces the Function of Spermidine in Bioﬁlm Formation (A) Complex colony bioﬁlms of B. subtilis strains were grown on solid MSgg medium with or without polyamines indicated. MSgg, polyamine- free chemicallydeﬁned growth medium; Spd, spermidine; Nspd, norspermidine; Hspd, homo- spermidine. Strains of B. subtilis included NCIB3610 (wild-type strain), tasA (mutant of the bioﬁlm amyloid protein [NRS2415]), eps(A-O) (mutant of the exopolysaccharide biosynthetic gene cluster [NRS2450]), speA (mutant of argi- nine decarboxylase [putrescine and spermidine auxotroph, NRS3089]), and speD (mutant of S-adenosylmethionine decarboxylase [spermi- dine auxotroph, NRS4005]). Strains were grown at 30C for 48 hr prior to imaging. Scale bars represent 2.5 mm. (B) Pellicle bioﬁlms of B. subtilis strains formed at the liquid-air interface on liquid growth medium after 2 days of incubation at 25C. Strains of B. subtilis included NCIB3610, eps(A-O), speA, and speD. (C) Norspermidine promotes B. subtilis wild-type pellicle bioﬁlm formation and rugosity. Cells of our laboratory B. subtilis wild-type isolate (NCIB3610) and the wild-type isolate from the Losick laboratory (NCIB3610-H) were grown for either 5 or 7 days in MSgg liquid medium with 0, 25, or 100 mM norspermidine. Figure 2. Norspermidine Replaces the Function of Spermidine in Bioﬁlm Formation (A) Complex colony bioﬁlms of B. subtilis strains were grown on solid MSgg medium with or without polyamines indicated. MSgg, polyamine- free chemicallydeﬁned growth medium; Spd, spermidine; Nspd, norspermidine; Hspd, homo- spermidine. Strains of B. subtilis included NCIB3610 (wild-type strain), tasA (mutant of the bioﬁlm amyloid protein [NRS2415]), eps(A-O) (mutant of the exopolysaccharide biosynthetic gene cluster [NRS2450]), speA (mutant of argi- nine decarboxylase [putrescine and spermidine auxotroph, NRS3089]), and speD (mutant of S-adenosylmethionine decarboxylase [spermi- dine auxotroph, NRS4005]). Strains were grown at 30C for 48 hr prior to imaging. Scale bars represent 2.5 mm. q q g medium after 2 days of incubation at 25C. Strains of B. subtilis included NCIB3610, eps(A-O), speA, and speD. (C) Norspermidine promotes B. subtilis wild-type pellicle bioﬁlm formation and rugosity. RESULTS A concentration of 300 mM norspermidine had a marked inhibitory effect on growth in both strains, and a concentration of 1 mM norspermidine essentially abolished growth of both strains (Figures 3C and 3D). There was little effect on planktonic growth in the presence of 1 mM spermidine (Figures 3E and 3F). Analysis of 3-day-old and 8-day-old B. subtilis NCIB3610 pel- licles grown in polyamine-free MSgg medium indicated the pres- ence of spermidine but the complete absence of norspermidine (red traces in Figures 4A and 4C). As expected, the spermidine peak was absent in residual pellicle samples derived from the spermidine biosynthetic mutants speA and speD (Figures 4D and 4F). The absence of norspermidine in 8-day-old pellicles was surprising given the ﬁnding of Kolodkin-Gal et al. (2012) that norspermidine was present at 50–80 mM. To exclude the possibility that our HPLC system was unable to detect a norsper- midine concentration of 50–80 mM, we determined the limit of detection of a pure norspermidine standard added to the B. subtilis NCIB3610 pellicle material before extraction with tri- chloroacetic acid. The limit of norspermidine detection from the pellicle material was found to be well below 0.5 mM (Fig- ure 4E). To conﬁrm that self-produced norspermidine could be detected by our analytical system, cells of Vibrio parahaemolyti- cus, which encodes a norspermidine biosynthetic pathway highly similar to that of V. cholerae, were analyzed and found to contain norspermidine (Figure 4B) and some spermidine. The spermidine was likely derived from the Luria Bertani (LB) growth medium used to grow the V. parahaemolyticus, whereas LB medium does not contain norspermidine (McGinnis et al., 2009). As an alternative ﬂuorescence-derivatization approach for labeling polyamines with a ﬂuorescent chemical tag, pellicle extracts were dansylated and then analyzed by HPLC. Analysis Figure 3. Higher Concentrations of Norspermidine Inhibit Growth and Pellicle Formation subtilis NCIB3610 gabT mutant with the native gabT gene under control of the of 1-, 3-, and 8-day-old pellicles indicated the presence of sper- midine increasing in concentration from day 1 to day 3 and then declining by day 8 (Figures 5A–5C). Norspermidine was not detected at any stage of pellicle development in the NCIB3610 strain, and spermidine was absent in the speA strain from the corresponding stages (Figures 5D–5F). Pure norspermidine and spermidine chemical standards were easily resolved by the solvent system (Figures 5G–5I). With the dansylation method, the limit of detection for norspermidine was below 0.3 mM. of 1-, 3-, and 8-day-old pellicles indicated the presence of sper- midine increasing in concentration from day 1 to day 3 and then declining by day 8 (Figures 5A–5C). Norspermidine was not detected at any stage of pellicle development in the NCIB3610 strain, and spermidine was absent in the speA strain from the corresponding stages (Figures 5D–5F). Pure norspermidine and spermidine chemical standards were easily resolved by the solvent system (Figures 5G–5I). With the dansylation method, the limit of detection for norspermidine was below 0.3 mM. Norspermidine Is Not Present in B. subtilis Pellicle The concentration of norspermidine in 8-day-old pellicles deter- mined by Kolodkin-Gal et al. (2012) was 50–80 mM, but the pres- 846 Cell 156, 844–854, February 13, 2014 ª2014 The Authors Figure 3. Higher Concentrations of Norspermidine Inhibit Growth and Pellicle Formation of 1-, 3-, and 8-day-old pellicles indicated the presence of sper- midine increasing in concentration from day 1 to day 3 and then declining by day 8 (Figures 5A–5C). Norspermidine was not detected at any stage of pellicle development in the NCIB3610 strain, and spermidine was absent in the speA strain from the corresponding stages (Figures 5D–5F). Pure norspermidine and spermidine chemical standards were easily resolved by Kolodkin-Gal et al. ( thesized in B. sub V. cholerae (Lee et orthologous gene e the gabT mutant str tion. This result is data demonstrated t Figure 3. Higher Concentrations of Norspermidine Inhibit Growth and Pellicle Formation (A) Effect of higher norspermidine concentrations on wild-type pellicle formation. Cells of our labo- ratory B. subtilis NCIB3610 isolate (3610) or an isolate from the Losick laboratory (3610-H) were grown for 2 days in increasing concentrations of norspermidine in liquid MSgg medium. (B) Norspermidine inhibits the formation of the vestigial pellicle fragments of an exopolysaccharide mutant. NCIB3610, wild-type B. subtilis; eps(A-O) (NRS2450), exopolysaccharide-deﬁcient mutant. (C) Norspermidine inhibits the growth of B. subtilis NCIB3610 planktonic cells. Growth of B. subtilis NCIB3610 planktonic cells grown in liquid MSgg at 37C with shaking at different concentrations of norspermidine is shown. (D) Norspermidine inhibits the growth of B. subtilis eps(A-O) exopolysaccharide-deﬁcient planktonic cells. Growth of B. subtilis eps(A-O) (NRS2450) planktonic cells grown in liquid MSgg at 37C with different concentrations of norspermidine is shown. (E) Norspermidine but not spermidine inhibits the growth of B. subtilis NCIB3610 planktonic cells. Growth of B. subtilis NCIB3610 planktonic cells grown in liquid MSgg at 37C with shaking and no polyamine, 1 mM norspermidine (repeated from C), or 1 mM spermidine is shown. (F) Norspermidine but not spermidine inhibits the growth of B. subtilis eps(A-O) exopolysaccharide- deﬁcient planktonic cells. Growth of B. subtilis eps(A-O) planktonic cells grown in liquid MSgg at 37C with shaking and no polyamine, 1 mM norspermidine (repeated from D), or 1 mM sper- midine is shown. V. cholerae norspermidine biosynthetic pathway is conversion of aspartate b-semialdehyde to 2,4-diaminobutyrate (Ikai and Yamamoto, 1997), catalyzed by the enzyme DABA AT (EC.2.6.1.76). Kolodkin-Gal et al. (2012) asserted that norspermidine is syn- thesized in B. subtilis via the same pathway employed in V. cholerae (Lee et al., 2009), and they selected gabT as the orthologous gene encoding DABA AT. They concluded that the gabT mutant strain was blocked in norspermidine produc- tion. This result is surprising because previously published data demonstrated that gabT encodes the enzyme 4-aminobu- tyrate:2-ketoglutarate aminotransferase (EC.2.6.1.19) that is involved in 4-aminobutyrate (GABA) catabolism (Dover and Halpern, 1974). Another study showed that in the B. subtilis strain SMY, mutation of the gabT gene prevented utilization of GABA as a sole nitrogen source (Belitsky and Sonenshein, 2002). Here, we constructed an in-frame mutant of gabT in the B. subtilis NCIB3610 strain (Figure 6A) and conﬁrmed a loss of growth on GABA as the sole nitrogen source (Fig- ure 6C). Complementation of the B. Cell 156, 844–854, February 13, 2014 ª2014 The Authors 847 B. subtilis Lacks a Norspermidine Biosynthetic Pathway Given our ﬁnding of a complete absence of detectable nor- spermidine in B. subtilis NCIB3610 pellicles of various ages, determined independently in each of our laboratories, and the obvious discrepancy with the ﬁndings of Kolodkin-Gal et al. (2012), we analyzed the genome of B. subtilis for the presence of norspermidine biosynthetic genes. The ﬁrst step in the Cell 156, 844–854, February 13, 2014 ª2014 The Authors 847 A B C D E F Figure 4. Norspermidine Is Not Present in B. subtilis NCIB3610 Pellicle Bioﬁlms B A B C D isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible pro- moter (Phy-spank) restored growth on GABA as the sole nitrogen source in the presence of IPTG (Figure 6D). Consistent with these ﬁndings, catabolism of GABA by GabT produces succi- nate semialdehyde, which is converted to succinate by succi- nate semialdehyde dehydrogenase (GabD). In B. subtilis, the gabT gene is found immediately upstream of gabD in a GABA catabolic operon (Figure 6A). Differences between the DABA AT reaction required for synthesis of 1,3-diaminopropane and norspermidine and the GabT reaction required for catabolism of 4-aminobutyric acid (GABA) are shown in Figure 6F. The key difference is that the product of GabT is succinate semialde- hyde, which is converted by GabD to succinate for entry into the tricarboxylic acid cycle, whereas the product of DABA AT is the amino acid L-2,4-diaminobutyrate, which is then committed to polyamine biosynthesis by the action of L-2,4-diaminobutyrate decarboxylase (DABA DC). B A D C D E F The B. subtilis NCIB3610 genome contains several genes en- coding proteins with some similarity to V. cholerae DABA AT, all of which belong to the aspartate aminotransferase family (Schneider et al., 2000). Most similar are argD encoding N-ace- tylornithine aminotransferase (34% aa identity), rocD encoding ornithine aminotransferase (30% aa identity), and as discussed above, gabT (30% aa identity). The Gram-positive B. subtilis GabT protein is more similar to GabT encoded by the Gram- negative Acinetobacter baumannii than to DABA AT from related Bacillus megaterium (Figure S1A available online). Furthermore, the B. subtilis, B. megaterium, and A. baumannii gabT open reading frame (ORF) is found clustered with a gabD ORF, whereas the DABA AT-encoding ORF from A. baumannii, B. megaterium, and V. cholerae is found adjacent or fused to a DABA DC-encoding ORF (Figure S1B). Thus, conﬁrmed enzy- matic activity (Belitsky and Sonenshein, 2002), sequence similar- ity, and operon context demonstrate that the B. B. subtilis Lacks a Norspermidine Biosynthetic Pathway subtilis GabT is involved in GABA catabolism and not biosynthesis of norspermi- dine. The next step in the V. cholerae norspermidine biosynthetic pathway is the conversion of L-2,4-diaminobutyrate produced by DABA AT to 1,3-diaminopropane, accomplished by DABA DC (EC.4.1.1.68). Sequence analysis indicates that there are no homologs of DABA DC in any sequenced B. subtilis genome except for a distant homolog (YP_005559741) encoded in B. subtilis subspecies Natto BEST195; however, this genome does not encode DABA AT. In conclusion, B. subtilis does not encode the biosynthetic pathway for the norspermidine precur- sor 1,3-diaminopropane. F E E F Figure 4. Norspermidine Is Not Present in B. subtilis NCIB3610 Pellicle Bioﬁlms HPLC analysis of polyamine content of pellicles. R, AccQ-Fluor reagent; IS, internal standard (1,7-diaminoheptane). Red trace indicates pellicle material, and black trace indicates pellicle material spiked with norspermidine standard before extraction with perchloric acid (except in E; see speciﬁc legend). The position of spermidine was conﬁrmed by spiking a separate pellicle sample with pure spermidine standard (data not shown). (A) Polyamines detected by HPLC analysis of wild-type B. subtilis NCIB3610 3- day-old pellicles grown in liquid MSgg medium. Representative analysis of three independent experiments is presented. (B) Endogenous norspermidine detected by HPLC analysis of V. parahaemolyticus RIMD 2210633. Cells of V. parahaemolyticus RIMD 2210633 were grown in MLB liquid growth medium. (C) Polyamines detected by HPLC analysis of wild-type B. subtilis NCIB3610 8- day-old pellicles grown in MSgg medium. Representative analysis of three independent experiments is presented. (C) Polyamines detected by HPLC analysis of wild-type B. subtilis NCIB3610 8- day-old pellicles grown in MSgg medium. Representative analysis of three independent experiments is presented. After conversion of L-2,4-diaminobutyrate to 1,3-diaminopro- pane, the next step in norspermidine production in V. cholerae is conversion of 1,3-diaminopropane to carboxynorspermidine by the enzyme carboxynorspermidine dehydrogenase (CANSDH) (Figure 1B). We ﬁnd that no homologous gene is present in any sequenced B. subtilis genome. Carboxynorspermidine is then converted to norspermidine by CANSDC. Kolodkin-Gal et al. (2012) selected the yaaO gene as the B. subtilis ortholog of CANSDC and found that a yaaO mutant strain produced a longer-lasting pellicle. However, our protein sequence com- parison analysis (Figure 7) indicates that YaaO exhibits negli- gible sequence similarity with the biochemically characterized CANSDC proteins from V. cholerae and Campylobacter jejuni. (D) Polyamines detected by HPLC analysis of B. subtilis speA (NRS3089) 8-day-old pellicles grown in MSgg medium. DISCUSSION Instead, YaaO is a homolog of the B. subtilis SpeA (YP_007533417) biosynthetic arginine decarboxylase (34.9% aa identity in 467 aa overlap) and the E. coli AdiA acid-inducible arginine decarboxylase protein (Burrell et al., 2010) and is there- fore from a different protein fold to CANSDC (Deng et al., 2010). In short, genes encoding CANSDH and CANSDC are absent from all B. subtilis genomes. An endogenous plasmid, pBS32 is present in B. subtilis NCIB3610 (Konkol et al., 2013), but DABA AT, DABA DC, CANSDH, and CANSDC are not encoded on this plasmid. Finally, we resequenced the genomes of the two isolates of B. subtilis used in our experiments (NCIB3610 and NCIB3610-H) and compared them with the reference genome of B. subtilis 168 (Srivatsan et al., 2008). There are 29 SNPs unique to the isolate of B. subtilis NCIB3610 from our lab- oratory and 28 SNPs unique to the B. subtilis NCIB3610-H isolate (Table S1). None of the SNPs in either NCIB3610 isolate affects any genes related to polyamine metabolism. Instead, YaaO is a homolog of the B. subtilis SpeA (YP_007533417) biosynthetic arginine decarboxylase (34.9% aa identity in 467 aa overlap) and the E. coli AdiA acid-inducible arginine decarboxylase protein (Burrell et al., 2010) and is there- fore from a different protein fold to CANSDC (Deng et al., 2010). In short, genes encoding CANSDH and CANSDC are absent from all B. subtilis genomes. An endogenous plasmid, pBS32 is present in B. subtilis NCIB3610 (Konkol et al., 2013), but DABA AT, DABA DC, CANSDH, and CANSDC are not encoded on this plasmid. Finally, we resequenced the genomes of the two isolates of B. subtilis used in our experiments (NCIB3610 and NCIB3610-H) and compared them with the reference genome of B. subtilis 168 (Srivatsan et al., 2008). There are 29 SNPs unique to the isolate of B. subtilis NCIB3610 from our lab- oratory and 28 SNPs unique to the B. subtilis NCIB3610-H isolate (Table S1). None of the SNPs in either NCIB3610 isolate affects any genes related to polyamine metabolism. The biosynthesis of norspermidine in V. cholerae is achieved by four different enzymatic steps that have been biochemically characterized and the corresponding V. cholerae genes identi- ﬁed (Ikai and Yamamoto, 1994, 1997; Lee et al., 2009; Nakao et al., 1991; Yamamoto et al., 1986). B. subtilis Lacks a Norspermidine Biosynthetic Pathway Representative analysis of three independent experiments is shown. (E) Limit of detection of norspermidine in 8-day-old pellicles of B. subtilis NCIB3610. Different concentrations of pure norspermidine standard were spiked into 8-day-old pellicles before extraction of polyamines from the pel- licles with trichloroacetic acid. The wild-type trace with no added norspermi- dine is colored black. (F) Polyamines detected by HPLC analysis of B. subtilis speD (NRS4005) 8-day-old pellicles grown in MSgg medium. Representative analysis of three independent experiments is presented. 848 Cell 156, 844–854, February 13, 2014 ª2014 The Authors Figure 5. Spermidine Is Present at All Stages of B. subtilis Pellicle Bioﬁlm Development Polyamines were detected by HPLC after derivatization with dansyl chloride. (A–C) Polyamine content in wild-type B. subtilis NCIB3610 pellicle bioﬁlms at days 1, 3, and 8 of development, respectively. (D–F) Polyamine content in pellicles of the B. subtilis speA polyamine auxotrophic mutant at days 1, 3, and 8 of development, respectively. (G–I) Pure spermidine, norspermidine, and combined spermidine and norspermidine chemical standards, respectively. Figure 5. Spermidine Is Present at All Stages of B. subtilis Pellicle Bioﬁlm Development Figure 5. Spermidine Is Present at All Stages of B. subtilis Pellicle Bioﬁlm Development Figure 5. Spermidine Is Present at All Stages of B. subtilis Pellicle Bioﬁlm Development Polyamines were detected by HPLC after derivatization with dansyl chloride. (A–C) Polyamine content in wild-type B. subtilis NCIB3610 pellicle bioﬁlms at days 1, 3, and 8 of development, respectively. (D–F) Polyamine content in pellicles of the B. subtilis speA polyamine auxotrophic mutant at days 1, 3, and 8 of development, respectively. (G–I) Pure spermidine, norspermidine, and combined spermidine and norspermidine chemical standards, respectively. Figure 5. Spermidine Is Present at All Stages of B. subtilis Pellicle Bioﬁlm Development Polyamines were detected by HPLC after derivatization with dansyl chloride. Polyamines were detected by HPLC after derivatization with dansyl chloride. (A–C) Polyamine content in wild-type B. subtilis NCIB3610 pellicle bioﬁlms at days 1, 3, and 8 of development, respectively. (D–F) Polyamine content in pellicles of the B. subtilis speA polyamine auxotrophic mutant at days 1, 3, and 8 of development (G–I) Pure spermidine, norspermidine, and combined spermidine and norspermidine chemical standards, respectively. DISCUSSION It is also unclear why spermidine was not detected because it is abundant in pellicles at all develop- mental stages and elutes from an LC column very close to norspermidine. erroneous. Not only does YaaO exhibit no homology to CANSDC (Figure 7), but B. subtilis gabT has been shown previously to be essential for GABA catabolism (Belitsky and Sonenshein, 2002). We cannot explain the discrepancies between our ﬁndings and those of Kolodkin-Gal et al. (2012), although we note that the mass spectrometric analysis of norspermidine presented by those authors was that of a pure norspermidine chemical standard from Sigma-Aldrich and not of a biological sample (Figure S1 of Kolodkin-Gal et al., 2012). Indeed, the only data presented that examined the presence of norspermidine within a biological sample are the liquid chromatography-mass spec- trometry (LC-MS) analysis of pellicle material presented in their Figure 1C, which presents only retention times and not mass analysis. Moreover, it should be noted that this ﬁgure does not include a control pellicle sample spiked with a pure norspermi- dine standard to indicate where the norspermidine elutes after extraction from the biological matrix. In the absence of any other supporting evidence, it is not clear why it was concluded by Kolodkin-Gal et al. (2012) that norspermidine was present in the pellicle material. It is also unclear why spermidine was not detected because it is abundant in pellicles at all develop- mental stages and elutes from an LC column very close to norspermidine. To ascertain the level of norspermidine in the B. subtilis 8-day- old pellicles, our two laboratories independently attempted to detect norspermidine at various stages of pellicle development. At physiological pH, the amine groups of polyamines such as norspermidine are fully protonated and bind strongly to polya- nionic molecules. To extract all noncovalently bound polyamines from pellicles, we used an aggressive extraction procedure using trichloroacetic acid (CCl3COOH). We note that Kolodkin-Gal et al. (2012) used PBS to extract polyamines, which may account for the lack of detection of spermidine. Nevertheless, we did not detect any norspermidine in 8-day-old pellicles or in pelli- cles at any stage, although our limit of detection was below 0.5 mM, i.e., less than 1% of the concentration reported by Kolodkin-Gal et al. (2012). In contrast, we found that spermi- dine was abundant at all stages of pellicle development but decreased with increasing age of the pellicle (Figures 4 and 5). shows B. subtilis gabT mutant expressing an IPTG-inducible wild-type gabT ORF from the heterologous amyE site on the chromosome (NRS4104). (C) The B. subtilis strains detailed above grown on MS medium containing 0.5% GABA as the sole nitrogen source and 0.5% glycerol as the carbon source. (D) Strains detailed above grown on MS medium containing 0.5% GABA as the sole nitrogen source, with induction of the complimenting gabT gene by 50 mM IPTG. (E) Strains detailed above grown on MS medium containing both 0.5% GABA and 0.5% glutamic acid as the nitrogen source. (F) The DABA AT and GabT reactions. DISCUSSION Based on our comparative genomic analyses described above, it is clear that there are no homologs of DABA DC, CANSDH, or CANSDC in B. subtilis NCIB3610. Distant homologs of DABA AT are present, but they have known functions and are not involved in norspermidine biosynthesis: argD encoding N-acetylornithine aminotransferase (Mountain et al., 1986), rocD encoding ornithine aminotrans- ferase (Gardan et al., 1995), and gabT encoding GABA amino- transferase (Belitsky and Sonenshein, 2002). Thus, the claim by Kolodkin-Gal et al. (2012) that the genes gabT and yaaO encode orthologs of V. cholerae DABA AT and CANSDC is Cell 156, 844–854, February 13, 2014 ª2014 The Authors 849 Figure 6. GabT Is Involved in GABA Catabolism and Not Biosyn- thesis of Norspermidine (A) Schematic of the B. subtilis GABA catabolic operon. The transcription factor-encoding gabR ORF is transcribed divergently from the gabT and gabD ORFs. Arrows and ﬁlled circles represent transcriptional promoters and terminators, respectively. (B) Schematic of the arrangement of B. subtilis strains grown on solid MSgg growth medium plates. Top-left view shows the wild-type B. subtilis NCIB3610, top-right view shows B. subtilis gabT mutant (NRS4007), and bottom view erroneous. Not only does YaaO exhibit no homology to CANSDC (Figure 7), but B. subtilis gabT has been shown previously to be essential for GABA catabolism (Belitsky and Sonenshein, 2002). We cannot explain the discrepancies between our ﬁndings and those of Kolodkin-Gal et al. (2012), although we note that the mass spectrometric analysis of norspermidine presented by those authors was that of a pure norspermidine chemical standard from Sigma-Aldrich and not of a biological sample (Figure S1 of Kolodkin-Gal et al., 2012). Indeed, the only data presented that examined the presence of norspermidine within a biological sample are the liquid chromatography-mass spec- trometry (LC-MS) analysis of pellicle material presented in their Figure 1C, which presents only retention times and not mass analysis. Moreover, it should be noted that this ﬁgure does not include a control pellicle sample spiked with a pure norspermi- dine standard to indicate where the norspermidine elutes after extraction from the biological matrix. In the absence of any other supporting evidence, it is not clear why it was concluded by Kolodkin-Gal et al. (2012) that norspermidine was present in the pellicle material. DISCUSSION Gaps in the sequence alignment are highlighted in gray, red highlights the identity within the CANSDC/CASDC family of proteins, blue highlights the identity in all four sequences, and black highlights the identity in three out of four sequences regardless of family. See also Figure S1. g g Alignment (CLUSTALW) is shown for the amino acid sequences of the C. jejuni CANSDC (GenBank WP_002855819 for Cjej CASDC), B. halodurans CANSDC (GenBank NP_244826) homolog (Bhal CASDC), V. cholerae CANSDC (GenBank NP_231262 for Vcho CANSDC), and the B. subtilis YaaO protein (GenBank NP_387908 for Bsub YaaO). Gaps in the sequence alignment are highlighted in gray, red highlights the identity within the CANSDC/CASDC family of proteins, blue highlights the identity in all four sequences, and black highlights the identity in three out of four sequences regardless of family. See also Figure S1. midine, a concentration 30-fold higher than their reported MIC for bioﬁlm development. Our results indicate that exogenous norspermidine does not act through the targeting of exopolysac- charide; instead, norspermidine inhibits growth per se, which is likely to be the predominant inhibitory factor preventing pellicle development. targeting exopolysaccharide. This idea was based on their observation that the residual pellicles of wild-type cells that developed in the presence of norspermidine resembled the wispy fragments of ﬂoating material seen for a mutant blocked in exopolysaccharide production. However, in Figure S2B of Kolodkin-Gal et al. (2012), the provision of 25 mM exogenous nor- spermidine in the growth medium appears to have signiﬁcantly reduced the amount of residual wispy pellicle material in the exopolysaccharide mutant, a result not commented upon by the authors. We found that the wispy ﬂoating fragments of the exopolysaccharide mutant did not develop when grown in con- centrations of norspermidine that fully inhibited pellicle formation in the wild-type strain (Figure 3B). When B. subtilis NCIB3610 wild-type cells were grown planktonically by Kolodkin-Gal et al. (2012) (see Figure S4 in this reference) in liquid MSgg with exogenous norspermidine present at a concentration 4-fold above the minimum inhibitory concentration (MIC) for pellicle formation determined by the authors (4 3 25 mM), they observed no inhibition of growth. In contrast, when we grew planktonic wild-type cells in a concentration of norspermidine 4-fold above our observed MIC for pellicle formation (4 3 250 mM), planktonic growth was abolished for both wild-type (Figure 3C) and exopolysaccharide mutant (Figure 3D) cells. DISCUSSION Furthermore, we found that norspermidine detection from cells of V. parahaemolyticus, which possesses the norspermidine biosynthetic pathway, was facile (Figure 4B). Figure 6. GabT Is Involved in GABA Catabolism and Not Biosyn- We determined that the concentration of norspermidine that inhibited pellicle formation (250 mM) was ten times higher than that reported by Kolodkin-Gal et al. (2012). Those authors suggested that norspermidine triggers bioﬁlm disassembly by GabT Is Involved in GABA Catabolism and Not Biosyn Figure 6. GabT Is Involved in GABA Catabolism and Not Biosyn- thesis of Norspermidine (A) Schematic of the B. subtilis GABA catabolic operon. The transcription factor-encoding gabR ORF is transcribed divergently from the gabT and gabD ORFs. Arrows and ﬁlled circles represent transcriptional promoters and terminators, respectively. (B) Schematic of the arrangement of B. subtilis strains grown on solid MSgg growth medium plates. Top-left view shows the wild-type B. subtilis NCIB3610, top-right view shows B. subtilis gabT mutant (NRS4007), and bottom view (B) Schematic of the arrangement of B. subtilis strains grown on solid MSgg growth medium plates. Top-left view shows the wild-type B. subtilis NCIB3610, top-right view shows B. subtilis gabT mutant (NRS4007), and bottom view (F) The DABA AT and GabT reactions. 850 Cell 156, 844–854, February 13, 2014 ª2014 The Authors 850 Cell 156, 844–854, February 13, 2014 ª2014 The Authors Figure 7. YaaO Is Not a Homolog of CANSDC Alignment (CLUSTALW) is shown for the amino acid sequences of the C. jejuni CANSDC (GenBank WP_002855819 for Cjej CASDC), B. halodurans CANSDC (GenBank NP_244826) homolog (Bhal CASDC), V. cholerae CANSDC (GenBank NP_231262 for Vcho CANSDC), and the B. subtilis YaaO protein (GenBank NP_387908 for Bsub YaaO). Gaps in the sequence alignment are highlighted in gray, red highlights the identity within the CANSDC/CASDC family of proteins, blue highlights the identity in all four sequences, and black highlights the identity in three out of four sequences regardless of family. See also Figure S1. Figure 7. YaaO Is Not a Homolog of CANSDC g g Alignment (CLUSTALW) is shown for the amino acid sequences of the C. jejuni CANSDC (GenBank WP_002855819 for Cjej CASDC), B. halodurans CANSDC (GenBank NP_244826) homolog (Bhal CASDC), V. cholerae CANSDC (GenBank NP_231262 for Vcho CANSDC), and the B. subtilis YaaO protein (GenBank NP_387908 for Bsub YaaO). DISCUSSION We found that planktonic growth of both wild-type and exopoly- saccharide mutant cells is equally sensitive to lower concentra- tions of exogenous norspermidine (Figures 3A and 3B). The in vitro effects of norspermidine on the hydrodynamic radii of exopolysaccharide reported by Kolodkin-Gal et al. (2012) (see Figure 4 in this reference) were observed using 0.75 mM norsper- In conclusion, we ﬁnd that B. subtilis NCIB3610 does not encode orthologs of the V. cholerae norspermidine biosynthetic pathway. Pellicle bioﬁlms of B. subtilis do not contain natively produced norspermidine at any stage of development, and exogenous norspermidine is not inhibitory to pellicle develop- ment until a concentration 10-fold higher than reported by Kolodkin-Gal et al. (2012). We ﬁnd that exogenous norspermi- dine inhibits planktonic growth and pellicle development in an exopolysaccharide-independent manner. Our data demonstrate that norspermidine is not a self-produced trigger for bioﬁlm disassembly in B. subtilis and reconﬁrm the essential role of spermidine for formation of robust bioﬁlms, a role that can be replaced more effectively at the same concentration by the nonnative polyamine norspermidine. Material Eight-day-old pellicle material grown in 10 ml of MSgg medium in 6-well plates was centrifuged and resuspended in H2O to a volume of 350 ml. One of several norspermidine stock solutions of different concentrations was added (9.6 ml), along with 15 ml of 3 mM 1,7-diaminoheptane to the resuspended pellicle ma- terial, which included cells and pellicle matrix, mixed and then extracted with 117 ml of trichloroacetic acid and made up to a ﬁnal volume of 500 ml. The apparent concentration of the norspermidine spike in the sample was taken as the concentration of norspermidine in the 500 ml ﬁnal volume of extracted pellicle suspension. After three cycles of freeze/thawing, the suspension was centrifuged and then 5 ml of the supernatant was added to 75 ml of borate buffer and 20 ml of AccQ-Fluor reagent. After the labeling reaction, 20 ml of this was used for HPLC analysis. Bacterial Strains and Growth Conditions Strains of B. subtilis used in this study are described in Table S2. All E. coli and B. subtilis strains were routinely grown in LB liquid medium (10 g NaCl, 5 g yeast extract, and 10 g tryptone/l) or on solid medium plates containing 1.5% Select Agar (Invitrogen) at 37C. V. parahaemolyticus RIMD 2210633 (a kind gift of K. Orth, University of Texas Southwestern Medical Center) was grown in modiﬁed LB medium (MLB; tryptone 10 g, yeast extract 5 g, and Cell 156, 844–854, February 13, 2014 ª2014 The Authors 851 and 2 mM thiamine) with 0.5% glycerol as a carbon source, and with either 0.5% glutamic acid or 0.5% 4-aminobutyric acid (GABA) or both as a nitrogen source. For induction of the gabT-complemented strain, 50 mM IPTG was added to the growth medium. Plates were grown again at 37C overnight before being imaged. NaCl 30 g in 1 liter) at 30C. For growth in polyamine-free liquid medium, strains were grown in MSgg medium (5 mM potassium phosphate and 100 mM MOPS at pH 7.0 supplemented with 2 mM MgCl2, 700 mM CaCl2, 50 mM MnCl2, 50 mM FeCl3, 1 mM ZnCl2, 2 mM thiamine, 0.5% glycerol, and 0.5% glutamate) (Branda et al., 2001) or on solid MSgg plates (solidiﬁed with 1.5% Select Agar). When appropriate, antibiotics were used at the following concentrations: 100 mg/ml ampicillin, 100 mg/ml spectinomycin, 12.5 mg/ml tetracycline, 0.5 mg/ml erythromycin, and 12.5 mg/ml lincomycin. Spermidine and sym-norspermidine were obtained from Sigma-Aldrich, and sym-homo- spermidine was a kind gift from Patrick Woster (Medical University of South Carolina). NaCl 30 g in 1 liter) at 30C. For growth in polyamine-free liquid medium, strains were grown in MSgg medium (5 mM potassium phosphate and 100 mM MOPS at pH 7.0 supplemented with 2 mM MgCl2, 700 mM CaCl2, 50 mM MnCl2, 50 mM FeCl3, 1 mM ZnCl2, 2 mM thiamine, 0.5% glycerol, and 0.5% glutamate) (Branda et al., 2001) or on solid MSgg plates (solidiﬁed with 1.5% Select Agar). When appropriate, antibiotics were used at the following concentrations: 100 mg/ml ampicillin, 100 mg/ml spectinomycin, 12.5 mg/ml tetracycline, 0.5 mg/ml erythromycin, and 12.5 mg/ml lincomycin. Spermidine and sym-norspermidine were obtained from Sigma-Aldrich, and sym-homo- spermidine was a kind gift from Patrick Woster (Medical University of South Carolina). p AccQ-Fluor Reagent Pellicles of B. subtilis NCIB3610 and derivative strains were grown in 10 ml of liquid MSgg in 6-well plates (Greiner Bio-One). The 10 ml of pellicle mate- rial and spent medium was transferred to a 15 ml Falcon tube and centri- fuged to pellet the pellicle material including both cells and matrix. The pellet was resuspended with 0.25 ml H20, and then 40% trichloroacetic acid was added to a ﬁnal concentration of 10% and incubated on ice for 30 min. This pellicle suspension was subjected to three cycles of freeze/thawing. After centrifugation (18,000 3 g, 5 min, 4C), the resulting supernatant was used for immediate analysis or stored at 20C until needed. The 1,7-diami- noheptane internal standard and pure polyamine standards were added to the pellicles immediately before extraction of the pellicle material by tri- chloroacetic acid. Colony and Pellicle Bioﬁlm Formation Strains of B. subtilis were grown overnight on MSgg plates at 37C. Single colonies were inoculated into 3 ml MSgg and grown with shaking at 37C for 6 hr. To set up complex colonies, 10 ml of cells was spotted on MSgg plates and incubated for 48 hr at 30C before imaging. For pellicle analysis, pellicles were formed in either 2 or 10 ml of MSgg broth (with 2 or 10 ml of cells as inoc- ulum, respectively) and were grown at 25C for the stated length of time. Preparation of Pellicle Material for Dansylation of Polyamines Preparation of Pellicle Material for Dansylation of Polyamines Pellicles formed in 10 ml of liquid MSgg were set up as described above. Sam- ples were taken for analysis at various days after inoculation. The pellicle ma- terial and liquid medium were both transferred to a 15 ml Falcon tube and centrifuged at 3,750 rpm for 10 min. Supernatant was removed, and the pellicle pellet was resuspended in 0.25 ml H2O for 1-day and 8-day pellicles and 0.5 ml for 3-day pellicles. After resuspension, 15 ml of 0.4 mM 1,7-diaminoheptane Strain Construction All B. subtilis strains, plasmids, and primers used and constructed in this study are listed in Tables S2, S3, and S4. For the construction and maintenance of plasmids, E. coli strain MC1061 (F’lacIQ lacZM15 Tn10 (tet)) was used. Deriv- atives of B. subtilis 168 were generated by transformation of competent cells with plasmids using standard protocols of Harwood and Cutting (1990). For introduction of DNA into B. subtilis strain NCIB3610, SPP1 phage transduc- tions were conducted as described previously by Verhamme et al. (2007). Strains containing in-frame gene deletions of speD and gabT were con- structed using a modiﬁed version of a previously published method (Kiley and Stanley-Wall, 2010). The upstream region of speD was PCR ampliﬁed with primers NSW1500 and NSW1501, and the downstream region with 1502 and 1503. The two puriﬁed products were linked by further PCR using the external primers 1500 and 1503. This product was cut with BamHI and Hin- dIII and ligated into pUC19 cut with the same restriction enzymes, creating the vector pNW1106, and the insert was then released by digestion with BamHI and BglII and ligated into pMAD, creating vector pNW1111. The construct for deletion of gabT was created in a similar way: upstream DNA was ampliﬁed with NSW1508 and NSW1509 and downstream with NSW1510 and NSW1511; the two products were then joined by PCR. Puriﬁed PCR product was cut with XmaI and HindIII and ligated into pUC19, creating pNW1107, and the insert was then released with XmaI and BglII and ligated into pMAD, creating the vector pNW1110. Strains NRS4005 (NCIB3610 DspeD) and NRS4007 (NCIB3610 DgabT) were created by integration and curing of pNW1111 and pNW1110, respectively, in NCIB 3610 as previously described (Kiley and Stanley-Wall, 2010). Planktonic Growth Analysis Pellicles formed in 10 ml of liquid MSgg were set up as described above. Samples were taken for analysis at various days after inoculation. The pellicle material and liquid media were both transferred to a centrifuge tube and centri- fuged at 4,000 3 g for 10 min. The pellet representing all pellicular material, both cells and bioﬁlm matrix were analyzed by HPLC as described below. B. subtilis strains were streaked out to single colonies on an MSgg plate and grown overnight at 37C. Single colonies were inoculated into 25 ml of MSgg broth and grown overnight at 37C with shaking. Cells were diluted to an optical density at 600 nm (OD600nm) of 0.01 in 25 ml of fresh MSgg broth and incubated at 37C with shaking at 200 rpm. Polyamines were added to the growth media at the ﬁnal concentrations indicated immediately prior to inoculation. Planktonic growth was measured by OD600nm readings. Analysis Analysis of trichloroacetic acid-extracted polyamines was performed by pre- column derivatization with AccQ-Fluor reagent labeling kit (Waters; catalog No. WAT052880). For each sample (5 ml), derivatization was carried out in a total volume of 100 ml AccQ-ﬂuor borate buffer and heated to 55C for 10 min. Polyamines labeled with the AccQ-Fluor reagent were separated by HPLC with a hydrolysate amino acid analysis column (AccQ-Tag column, 60 A˚ , 4 mm, 3.9 3 150 mm; Waters) on a Beckman Coulter System Gold with ﬂuorescence detection (excitation, 248 nm; emission, 398 nm [Prostar]). The solvent system consisted of solvent A (520 mM sodium acetic acid, 17 mM triethylamine, and 0.01% sodium azide [pH 4.65]) and solvent B (60% acetonitrile, 0.01% acetone at a ﬂow rate of 1.0 ml/min). Elution was performed using a linear gradient of buffer B: 2–6 min, 0%–20%; 6–11 min, 20%; 11–27 min, 20%–50%; 27–34 min, 50%; 34–37 min, 100%; and 40–50 min, 0%. Calculation of the Limit of Detection of Norspermidine in Pellicle Material To introduce gabT under the control of the IPTG-inducible Phy-spank promoter at the nonessential amyE locus, the coding region of gabT with its ribosome-binding site was ampliﬁed from NCIB3610 genomic DNA with the primers NSW1524 and NSW1525. The puriﬁed product was cut with HindIII and SphI, then ligated into pDR111 cut with the same enzymes, creating the vector pNW1114. Derivatization of Samples with Dansyl Chloride and HPLC Analysis Derivatization of Samples with Dansyl Chloride and HPLC Analysis Fresh dansyl chloride (5-dimethylaminonapthalene-1-sulphonyl chloride) solu- tion was prepared as 10 mg/ml in redistilled acetone, wrapped in foil, and kept on ice (Kabra et al., 1986). A total of 200 ml saturated Na2CO3 (pH rises to approximately 10) and 200 ml of dansyl chloride solution was added to 50 ml of the pellicle sample. After mixing, the solution was heated to 70C for 10 min, cooled to room temperature, and then 100 ml 25% (w/v) L-proline was added to mop up excess dansyl chloride. The top acetone layer was retained for HPLC analysis using ion-paired reverse-phase HPLC on an Ultra- sphere C18 column using a Dionex Ultimate 3000 instrument ﬁtted with a Dionex RF-2000 ﬂuorometer (set at 340 nm excitation and 515 nm emission). The solvent system consisted of solvent A (10 mM sodium phosphate mono- basic phosphate buffer [NaH2PO4.H2O] [pH 4.2]) and solvent B (acetonitrile). Elution was performed at a ﬂow rate of 2.0 ml/min, the column equilibrated in 40% solvent B for 8 min, then a linear gradient of solvent B: 0–25 min, 40%–65%; 25–35 min, 65%–90%; and 35–42 min, 90%. See the Extended Experimental Procedures for more information. Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Korber, D.R., and Lappin- Scott, H.M. (1995). Microbial bioﬁlms. Annu. Rev. Microbiol. 49, 711–745. 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SUPPLEMENTAL INFORMATION Supplemental Information includes Extended Experimental Procedures, four tables, and one ﬁgure and can be found with this article online at http://dx. doi.org/10.1016/j.cell.2014.01.012. Harwood, C.R., and Cutting, S.M. (1990). Molecular Biological Methods for Bacillus (Chichester: John Wiley & Sons). Hobley, L., Ostrowski, A., Rao, F.V., Bromley, K.M., Porter, M., Prescott, A.R., MacPhee, C.E., van Aalten, D.M., and Stanley-Wall, N.R. (2013). BslA is a self- assembling bacterial hydrophobin that coats the Bacillus subtilis bioﬁlm. Proc. Natl. Acad. Sci. USA 110, 13600–13605. ACKNOWLEDGMENTS Joo, H.S., and Otto, M. (2012). Molecular basis of in vivo bioﬁlm formation by bacterial pathogens. Chem. Biol. 19, 1503–1513. We thank David Mangelsdorf for helpful discussions and http://www. sarahjcgillespie.com for design work on the graphical abstract. This work was supported by grant BB/I019464/1 from the Biotechnology and Biological Sciences Research Council, UK (to N.R.S.-W.), by grant 079838 from the Well- come Trust (to A.H.F.), and by a High Impact/High Risk Award from UT South- western Medical Center (to A.J.M.). Genome sequencing and analysis were performed at the Genomic Sequencing Unit, University of Dundee, which is funded by the Wellcome Trust Strategic Grant (098439/Z/12/(Z)). We thank Prof. Richard Losick for providing strain NCIB3610-H. Kabra, P.M., Lee, H.K., Lubich, W.P., and Marton, L.J. (1986). Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal ﬂuid, urine and tissue. J. Chromatogr. A 380, 19–32. Karatan, E., and Michael, A.J. (2013). A wider role for polyamines in bioﬁlm for- mation. Biotechnol. Lett. 35, 1715–1717. Karatan, E., Duncan, T.R., and Watnick, P.I. (2005). NspS, a predicted poly- amine sensor, mediates activation of Vibrio cholerae bioﬁlm formation by nor- spermidine. J. Bacteriol. 187, 7434–7443. Received: October 16, 2013 Revised: December 19, 2013 Accepted: January 7, 2014 Published: February 13, 2014 Revised: December 19, 2013 Kiley, T.B., and Stanley-Wall, N.R. (2010). Post-translational control of Bacillus subtilis bioﬁlm formation mediated by tyrosine phosphorylation. Mol. Micro- biol. 78, 947–963. Accepted: January 7, 2014 Published: February 13, 2014 Kobayashi, K., and Iwano, M. (2012). BslA(YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis bioﬁlms. Mol. Microbiol. 85, 51–66. AUTHOR CONTRIBUTIONS Experiments were designed, performed, and analyzed by N.R.S.-W., A.H.F., L.H., S.W., S.H.K., Y.M., and A.J.M. L.H. and N.R.S.-W. constructed strains, and L.H., N.R.S.-W., and Y.M. performed bioﬁlm and cell growth assays. S.H.K., S.W., and A.H.F. optimized and performed polyamine analysis on sam- ples prepared by L.H. and S.H.K. L.H. prepared samples for whole-genome SNP analysis. SNP identiﬁcation was by L.H. and N.R.S.-W. with further bioin- formatics analysis by N.R.S.-W., A.H.F., and A.J.M. The paper was written and edited by A.J.M., A.H.F., N.R.S.-W., and L.H. Ikai, H., and Yamamoto, S. (1994). Cloning and expression in Escherichia coli of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii. FEMS Microbiol. Lett. 124, 225–228. Ikai, H., and Yamamoto, S. (1997). Identiﬁcation and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii. J. Bacteriol. 179, 5118–5125. Belitsky, B.R., and Sonenshein, A.L. (2002). GabR, a member of a novel protein family, regulates the utilization of gamma-aminobutyrate in Bacillus subtilis. Mol. Microbiol. 45, 569–583. Growth on 4-Aminobutyric Acid as Sole Nitrogen Source Strains of B. subtilis were streaked out to single colonies on an MSgg plate and grown overnight at 37C. Single colonies were then streaked onto MS plates (5 mM potassium phosphate and 100 mM MOPS at pH 7.0 supplemented with 2 mM MgCl2, 700 mM CaCl2, 50 mM MnCl2, 50 mM FeCl3, 1 mM ZnCl2, 852 Cell 156, 844–854, February 13, 2014 ª2014 The Authors 852 Branda, S.S., Gonza´ lez-Pastor, J.E., Ben-Yehuda, S., Losick, R., and Kolter, R. (2001). Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci. USA 98, 11621–11626. (the internal standard) was added to the samples before addition of the trichloroacetic acid. An equal volume (to the H2O used for resuspension) of tri- chloroacetic acid (made in 10 mM HCl) was added to the resuspended pellicle samples. The solution was kept on ice for 30 min to precipitate protein, which was pelleted by centrifugation, and the supernatant was transferred to another tube. This supernatant was extracted ﬁve times with water-saturated ethyl acetate (polyamines remain in the bottom layer; the top layer was discarded). The liquid was removed by freeze-drying overnight, and the solid material was reconstituted with 50 ml 10 mM HCl. (the internal standard) was added to the samples before addition of the trichloroacetic acid. An equal volume (to the H2O used for resuspension) of tri- chloroacetic acid (made in 10 mM HCl) was added to the resuspended pellicle samples. The solution was kept on ice for 30 min to precipitate protein, which was pelleted by centrifugation, and the supernatant was transferred to another tube. This supernatant was extracted ﬁve times with water-saturated ethyl acetate (polyamines remain in the bottom layer; the top layer was discarded). The liquid was removed by freeze-drying overnight, and the solid material was reconstituted with 50 ml 10 mM HCl. Branda, S.S., Vik, S., Friedman, L., and Kolter, R. (2005). 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https://openalex.org/W2899422854	https://projecteuclid.org/journals/bayesian-analysis/volume-13/issue-2/Bayesian-Cluster-Analysis--Point-Estimation-and-Credible-Balls-with/10.1214/17-BA1073.pdf	English	null	Contributed comment on article by Wade and Ghahramani	null	2,018	cc-by	28,570	∗University of Warwick, s.wade@warwick.ac.uk †University of Cambridge, zoubin@eng.cam.ac.uk Bayesian Cluster Analysis: Point Estimation and Credible Balls (with Discussion) Sara Wade∗and Zoubin Ghahramani† Abstract. Clustering is widely studied in statistics and machine learning, with applications in a variety of ﬁelds. As opposed to popular algorithms such as ag- glomerative hierarchical clustering or k-means which return a single clustering solution, Bayesian nonparametric models provide a posterior over the entire space of partitions, allowing one to assess statistical properties, such as uncertainty on the number of clusters. However, an important problem is how to summarize the posterior; the huge dimension of partition space and diﬃculties in visualizing it add to this problem. In a Bayesian analysis, the posterior of a real-valued pa- rameter of interest is often summarized by reporting a point estimate such as the posterior mean along with 95% credible intervals to characterize uncertainty. In this paper, we extend these ideas to develop appropriate point estimates and cred- ible sets to summarize the posterior of the clustering structure based on decision and information theoretic techniques. Keywords: mixture model, random partition, variation of information, Binder’s loss. 13, Number 2, pp. 559–626 13, Number 2, pp. 559–626 Bayesian Analysis (2018) c⃝2018 International Society for Bayesian Analysis 1 Introduction Clustering is widely studied in statistics and machine learning, with applications in a variety of ﬁelds. Numerous models and algorithms for clustering exist, and new studies which apply these methods to cluster new datasets or develop novel models or algorithms are constantly being produced. Classical algorithms such as agglomerative hierarchical clustering or the k-means algorithm (Hartigan and Wong (1979)) are popular but only explore a nested subset of partitions or require specifying the number of clusters apriori. Moreover, they are largely heuristic and not based on formal models, prohibiting the use of statistical tools, for example, in determining the number of clusters. Model-based clustering methods utilize ﬁnite mixture models, where each mixture component corresponds to a cluster (Fraley and Raftery (2002)). Problems of determin- ing the number of clusters and the component probability distribution can be dealt with through statistical model selection, for example, through various information criteria. The expectation-maximization (EM) algorithm is typically used for maximum likelihood estimation (MLE) of the mixture model parameters. Given the MLEs of the parameters, the posterior probability that a data point belongs to a class can be computed through Bayes rule. The cluster assignment of the data point corresponds to the class with max- imal posterior probability, with the corresponding posterior probability reported as a https://doi.org/10.1214/17-BA1073 c⃝2018 International Society for Bayesian Analysis 560 Bayesian Cluster Analysis: Point Estimation and Credible Balls Bayesian Cluster Analysis: Point Estimation and Credible Balls measure of uncertainty. Importantly, however, this measure of uncertainty ignores un- certainty in the parameter estimates. As opposed to MLE, Bayesian mixture models incorporate prior information on the parameters and allow one to assess uncertainty in the clustering structure unconditional on the parameter estimates. measure of uncertainty. Importantly, however, this measure of uncertainty ignores un- certainty in the parameter estimates. As opposed to MLE, Bayesian mixture models incorporate prior information on the parameters and allow one to assess uncertainty in the clustering structure unconditional on the parameter estimates. Bayesian nonparametric mixture models assume that the number of components is inﬁnite. As opposed to ﬁnite mixture models, this not only avoids speciﬁcation of the number of components but also allows the number of clusters present in the data to grow unboundedly as more data is collected. 1 Introduction Bayesian nonparametric mixture models induce a random partition model (Quintana (2006)) of the data points into clusters, and the posterior of the random partition reﬂects our belief and uncertainty of the clustering structure given the data. However, an important problem in Bayesian nonparametric cluster analysis is how to summarize this posterior; indeed, often the ﬁrst question one asks is what is an ap- propriate point estimate of the clustering structure based on the posterior. Such a point estimate is useful for concisely representing the posterior and often needed in applica- tions. Moreover, a characterization of the uncertainty around this point estimate would be desirable in many applications. Even in studies of Bayesian nonparametric models where the latent partition is used simply as a tool to construct ﬂexible models, such as in mixture models for density estimation (Lo (1984)), it is important to understand the behavior of the latent partition to improve understanding of the model. To do so, the researcher needs to be equipped with appropriate summary tools for the posterior of the partition. Inference in Bayesian nonparametric partition models usually relies on Markov chain Monte Carlo (MCMC) techniques, which produce a large number of partitions that represent approximate samples from the posterior. Due to the huge dimension of the partition space and the fact that many of these partitions are quite similar diﬀering only in a few data points, the posterior is typically spread out across a large number of partitions. Clearly, describing all the unique partitions sampled would be unfeasi- ble, further emphasizing the need for appropriate summary tools to communicate our ﬁndings. In a typical Bayesian analysis, the posterior of a univariate parameter of interest is often summarized by reporting a point estimate such as the posterior mean, median, or mode, along with a 95% credible interval to characterize uncertainty. In this paper, we aim to extend these ideas to develop summary tools for the posterior on partitions. In particular, we seek to answer the two questions: 1) What is an appropriate point estimate of the partition based on the posterior? 2) Can we construct a 95% credible region around this point estimate to characterize our uncertainty? We ﬁrst focus on the problem of ﬁnding an appropriate point estimate. A simple solution is to use the posterior mode. 1 Introduction If the marginal likelihood of the data given the partition, that is with all mixture component parameters integrated out, and the prior of the partition are available in closed form, the posterior mode can be estimated based on the MCMC output by the sampled partition which maximizes the non-normalized posterior. In practice, a closed form for the marginal likelihood or prior is often unavail- able, speciﬁcally, if conjugate priors for the component speciﬁc parameters do not exist 561 S. Wade and Z. Ghahramani or are not utilized or hyperpriors are assigned to any hyperparameters. More generally, the posterior mode can be found by reporting the partition visited most frequently in the sampler. Yet this approach can be problematic, as producing reliable frequency counts is intractable due to the huge dimension of the partition space. In fact, in many examples, the MCMC chain does not visit a partition more than once. To overcome this, alter- native search techniques have been developed to locate the posterior mode (Heller and Ghahramani (2005), Heard et al. (2006), Dahl (2009), Raykov et al. (2014)). However, it is well-known that the mode can be unrepresentative of the center of a distribution. Alternative methods have been proposed based on the posterior similarity matrix. For a sample size of N, the elements of this N by N matrix represent the probability that two data points are in the same cluster, which can be estimated by the proportion of MCMC samples that cluster the two data points together. Then, classical hierarchical or partitioning algorithms are applied based on the similarity matrix (Medvedovic and Sivaganesan (2002), Medvedovic et al. (2004), Rasmussen et al. (2009), Molitor et al. (2010)). These methods have the disadvantage of being ad-hoc. A more elegant solution is based on decision theory. In this case, one deﬁnes a loss function over clusterings. The optimal point estimate is that which minimizes the posterior expectation of the loss function. For example, for a real-valued parameter θ, the optimal point estimate is the posterior mean under the squared error loss L2(θ, θ) = (θ −θ)2 and the posterior median under the absolute error loss L1(θ, θ) = \|θ −θ\|. The question to answer then becomes what is an appropriate loss function on the space of clusterings. 1 Introduction The 0-1 loss function, a simple choice which leads to the posterior mode as the point estimate, is not ideal as it does not take into account the similarity between two clusterings. More general loss functions were developed by Binder (1978), and the so-called Binder’s loss, which measures the disagreements in all possible pairs of observations between the true and estimated clusterings, was studied in a Bayesian nonparametric setting by Lau and Green (2007). Alternative loss functions considered in Bayesian nonparametrics can be found in Quintana and Iglesias (2003) and Fritsch and Ickstadt (2009). In this paper, we propose to use the variation of information developed by Meil˘a (2007) as a loss function in a Bayesian nonparametric setting. Both the variation of information and Binder’s loss possess the desirable properties of being metrics on the space of partitions and being aligned with the lattice of partitions. We provide a detailed comparison of these two metrics and discuss the advantages of the variation of informa- tion over Binder’s loss as a loss function in Bayesian cluster analysis. Additionally, we propose a novel algorithm to locate the optimal partition, taking advantage of the fact that both metrics are aligned on the space of partitions. Next, to address the problem of characterizing uncertainty around the point es- timate, we propose to construct a credible ball around the point estimate. As both Binder’s loss and the variation of information are metrics on the partition space, we can easily construct such a ball. Interestingly, the two metrics can produce very diﬀerent credible balls, and we discuss this in detail. In existing literature, quantiﬁcations of uncertainty include reporting a heat map of the estimated posterior similarity matrix. 562 Bayesian Cluster Analysis: Point Estimation and Credible Balls Bayesian Cluster Analysis: Point Estimation and Credible Balls However, there is no precise quantiﬁcation of how much uncertainty is represented by the posterior similarity matrix, and in a comparison with the 95% credible balls, we ﬁnd that the uncertainty is under-represented by the posterior similarity matrix. Finally, we provide an algorithm to construct the credible ball and discuss ways to depict or report it. The paper is organized as follows. Section 2 provides a review of Bayesian nonpara- metric clustering and existing point estimates of the clustering structure from a decision theoretic approach. 2 Review This section provides a review of Bayesian nonparametric clustering models and existing point estimates of the clustering in literature. 1 Introduction In Section 3, we give a detailed comparison of two loss functions, Binder’s loss and the variation of information, pointing out advantages of the latter. The optimal point estimate under the variation of information is derived in Section 4 and a novel algorithm to locate the optimal partition is proposed. In Section 5, we con- struct a credible ball around the point estimate to characterize posterior uncertainty and discuss how to compute and depict it. Finally, simulated and real examples are provided in Section 6. 2.1 Bayesian nonparametric clustering Mixture models are one of the most popular modeling tools in Bayesian nonparametrics. The data is assumed conditionally i.i.d. with density f(y\|P) = K(y\|θ)dP(θ), where K(y\|θ) is a speciﬁed parametric density on the sample space with mixing param- eter θ ∈Θ and P is a probability measure on Θ. In a Bayesian setting, the model is completed with a prior on the unknown parameter, which in this case, is the unknown mixing measure. In the most general setting, this parameter P can be any probability measure on Θ, requiring a nonparametric prior. Typically the nonparametric prior has discrete realizations almost surely (a.s.) with P = ∞ j=1 wjδθj a.s., where it is often assumed that the weights (wj) and atoms (θj) are independent and the θj are i.i.d. from some base measure P0. Thus, the density is modeled with a countably inﬁnite mixture model where it is often assumed that the weights (wj) and atoms (θj) are independent and the θj are i.i.d. from some base measure P0. Thus, the density is modeled with a countably inﬁnite mixture model ∞ f(y\|P) = ∞ j=1 wjK(y\|θj). Since P is discrete a.s., this model induces a latent partitioning c of the data where two data points belong to the same cluster if they are generated from the same mixture 563 S. Wade and Z. Ghahramani component. The partition can be represented by c = (C1, . . . , CkN ), where Cj contains the indices of data points in the jth cluster and kN is the number of clusters in the sample of size N. Alternatively, the partition can be represented by c = (c1, . . . , cN), where cn = j if the nth data point is in the jth cluster. A key diﬀerence with ﬁnite mixture models is that the number of mixture compo- nents is inﬁnite; this allows the data to determine the number of clusters kN present in the data, which can grow unboundedly with the data. Letting yj = {yn}n∈Cj, the marginal likelihood for the data y1:N given the partition is f(y1:N\|c) = kN j=1 m(yj) = kN j=1 n∈Cj K(yn\|θ)dP0(θ). 2.1 Bayesian nonparametric clustering The posterior of the partition, which reﬂects our beliefs and uncertainty in the clus- tering given the data, is simply proportional to the prior times the marginal likelihood p(c\|y1:N) ∝p(c) kN j=1 m(yj), (1) (1) where the prior of the partition is obtained from the selected prior on the mixing mea- sure. For example, a Dirichlet process prior (Ferguson (1973)) for P with mass parameter α corresponds to where the prior of the partition is obtained from the selected prior on the mixing mea- sure. For example, a Dirichlet process prior (Ferguson (1973)) for P with mass parameter α corresponds to p(c) = Γ(α) Γ(α + N)αkN kN j=1 Γ(nj), p(c) = Γ(α) Γ(α + N)αkN kN j=1 Γ(nj), where nj = \|Cj\| is the number of data points in cluster j. Various other priors de- veloped in Bayesian nonparametric literature can be considered for the mixing mea- sure P, such as the Pitman–Yor process (Pitman and Yor (1997)), also known as the two-parameter Poisson–Dirichlet process, or the normalized generalized Gamma pro- cess or more generally, a prior within the class of normalized completely random mea- sures, Poisson–Kingman models (Pitman (2003)), or stick-breaking priors (Ishwaran and James (2001)). See Lijoi and Pr¨unster (2011) for an overview. In general, the marginal likelihood of the data given the partition or the prior of the partition used to compute the posterior in (1) may not be available in closed form. Moreover, there are SN,k = 1 k! k j=0 (−1)j k j (k −j)N, a Stirling number of the second kind, ways to partition the N data points into k groups and N a Stirling number of the second kind, ways to partition the N data points into k groups and N BN = N k=1 SN,k, a Bell number, possible partitions of the N data points. Even for small N, this number is very large, which makes computation of the posterior intractable for the simplest choice a Bell number, possible partitions of the N data points. Even for small N, this number is very large, which makes computation of the posterior intractable for the simplest choice 564 Bayesian Cluster Analysis: Point Estimation and Credible Balls of prior and likelihood. 2.1 Bayesian nonparametric clustering Thus, MCMC techniques are typically employed, such as the marginal samplers described by Neal (2000) with extensions in Favaro and Teh (2013) for normalized completely random measures and in Lomell´ı et al. (2016) for σ-stable Poisson–Kingman models; the conditional samplers described in Ishwaran and James (2001), Papaspiliopoulos and Roberts (2008), or Kalli et al. (2011), with extensions in Favaro and Teh (2013) for normalized completely random measures and in Favaro and Walker (2012) for σ-stable Poisson–Kingman models; or the recently introduced class of hybrid samplers for σ-stable Poisson–Kingman models in Lomell´ı et al. (2015). These algorithms produce approximate samples (cm)M m=1 from the posterior (1). Clearly, describing all the posterior samples is infeasible, and our aim is to develop appropriate summary tools to characterize the posterior. Extensions of Bayesian nonparametric mixture models are numerous and allow one to model increasingly complex data. These include extensions for partially exchangeable data (Teh et al. (2006)), inclusion of covariates (MacEachern (2000)), time dependent data (Griﬃn and Steel (2006)), and spatially dependent data (Duan et al. (2007)) to name a few. See M¨uller and Quintana (2004) and Dunson (2010) for an overview. These extensions also induce latent clusterings of the observations, and the summary tools developed here are applicable for these settings as well. 2.2 Point estimation for clustering Firstly, we seek a point estimate of the clustering that is representative of the posterior, which may be of direct interest to the researcher or, more generally, important for understanding the behavior of the posterior. From decision theory, a point estimate is obtained by specifying a loss function L(c,c), which measures the loss of estimating the true clustering c with c. Since the true clustering is unknown, the loss is averaged across all possible true clusterings, where the loss associated to each potential true clustering is weighted by its posterior probability. The point estimate c∗corresponds to the estimate which minimizes the posterior expected loss, c∗= argmin c E[L(c,c)\|y1:N] = argmin c c L(c,c)p(c\|y1:N). A simple choice for the loss function is the 0-1 loss, L0−1(c,c) = 1(c ̸= c), which assumes a loss of 0 if the estimate is equal to the truth and a loss of 1 otherwise. Under the 0-1 loss, the optimal point estimate is the posterior mode. However, this loss function is unsatisfactory because it doesn’t take into account similarity between two clusterings; a partition which diﬀers from the truth in the allocation of only one observation is penalized the same as a partition which diﬀers from the truth in the allocation of many observations. Moreover, it is well-known that the mode can be unrepresentative of the center of a distribution. Thus, more general loss functions are needed. However, constructing a more general loss is not straightforward because, as pointed out by Binder (1978), the loss function should satisfy basic principles such as invariance to permutations of the data point indices and invariance to permutations of the cluster labels for both the true and estimated clusterings. Binder notes that this ﬁrst condition 565 S. Wade and Z. Ghahramani implies that the loss is a function of the counts ni j = \|Ci ∩Cj\|, which is the cardinality of the intersection between Ci, the set of data point indices in cluster i under c, and Cj, the set of data point indices in cluster j under c, for i = 1, . . . , kN and j = 1, . . . , kN; the notation kN and kN represents the number of clusters in c and c, respectively. He explores loss functions satisfying these principles, starting with simple functions of the counts ni j. 2.2 Point estimation for clustering The so-called Binder’s loss is a quadratic function of the counts, which for all possible pairs of observations, penalizes the two errors of allocating two observations to diﬀerent clusters when they should be in the same cluster or allocating them to the same cluster when they should be in diﬀerent clusters: B(c,c) = n<n′ l11(cn = cn′)1(cn ̸= cn′) + l21(cn ̸= cn′)1(cn = cn′). If the two types of errors are penalized equally, l1 = l2 = 1, then B(c,c) = 1 2 ⎛ ⎝ kN i=1 n2 i + + kN j=1 n2 + j −2 kN i=1 kN j=1 n2 i j ⎞ ⎠, where ni + = j ni j and n+ j = i ni j. Under Binder’s loss with l1 = l2, the optimal partition c∗is the partition c which minimizes where ni + = j ni j and n+ j = i ni j. Under Binder’s loss with l1 = l2, the optimal partition c∗is the partition c which minimizes n<n′ \|1(cn = cn′) −pn n′\| , n<n′ \|1(cn = cn′) −pn n′\| , or equivalently, the partition c which minimizes or equivalently, the partition c which minimizes n<n′ (1(cn = cn′) −pn n′)2 , (2) (2) where pn n′ = P(cn = cn′\|y1:N) is the posterior probability that two observations n and n′ are clustered together. This loss function was ﬁrst studied in Bayesian nonparametrics by Lau and Green (2007). We note that in earlier work Dahl (2006) considered mini- mization of (2) but without the connection to Binder’s loss and the decision theoretic approach. Binder’s loss counts the total number of disagreements, D, in the N 2 possible pairs of observations. The Rand index (Rand (1971)), a cluster comparison criterion, is deﬁned as the number of agreements, A, in all possible pairs divided by the total number of possible pairs. Since D + A = N 2 , Binder’s loss and the Rand index, denoted R(c,c), are related: B(c,c) = (1 −R(c,c)) N 2 , B(c,c) = (1 −R(c,c)) N 2 , and the point estimate obtained from minimizing the posterior expected Binder’s loss is equivalent to the point estimate obtained from maximizing the posterior expected Rand’s index. 2.2 Point estimation for clustering Motivated by this connection, Fritsch and Ickstadt (2009) consider max- imizing the adjusted Rand index, introduced by Hubert and Arabie (1985) to correct the Rand index for chance. An alternative loss function is explored by Quintana and Iglesias (2003) speciﬁcally for the problem of outlier detection. 566 Bayesian Cluster Analysis: Point Estimation and Credible Balls 3 A comparison of the variation of information and Binder’s loss Meil˘a (2007) introduces the variation of information (VI) for cluster comparison, which is constructed from information theory and compares the information in two clusterings with the information shared between the two clusterings. More formally, the VI is deﬁned as VI(c,c) = H(c) + H(c) −2I(c,c) = − kN i=1 ni + N log ni + N − kN j=1 n+ j N log n+ j N −2 kN i=1 kN j=1 ni j N log ni jN ni +n+ j , where log denotes log base 2. The ﬁrst two terms represent the entropy of the two clus- terings, which measures the uncertainty in bits of the cluster allocation of an unknown randomly chosen data point given a particular clustering of the data points. The last term is the mutual information between the two clusterings and measures the reduction in the uncertainty of the cluster allocation of a data point in c when we are told its cluster allocation in c. The VI ranges from 0 to log(N). A review of extensions of the VI to normalize or correct for chance are discussed in Vinh et al. (2010). However, some desirable properties of the VI are lost under these extensions. where log denotes log base 2. The ﬁrst two terms represent the entropy of the two clus- terings, which measures the uncertainty in bits of the cluster allocation of an unknown randomly chosen data point given a particular clustering of the data points. The last term is the mutual information between the two clusterings and measures the reduction in the uncertainty of the cluster allocation of a data point in c when we are told its cluster allocation in c. The VI ranges from 0 to log(N). A review of extensions of the VI to normalize or correct for chance are discussed in Vinh et al. (2010). However, some desirable properties of the VI are lost under these extensions. In this paper, we propose to use the VI as a loss function. 3 A comparison of the variation of information and Binder’s loss Note that since I(c,c) = H(c) + H(c) −H(c,c), we can write VI(c,c) = H(c) + H(c) −2H(c) −2H(c) + 2H(c,c), = −H(c) −H(c) + 2H(c,c), = kN i=1 ni + N log ni + N + kN j=1 n+ j N log n+ j N −2 kN i=1 kN j=1 ni j N log ni j N . = kN i=1 ni + N log ni + N + kN j=1 n+ j N log n+ j N −2 kN i=1 kN j=1 ni j N log ni j N . We provide a detailed comparison with an N-invariant version of Binder’s loss, deﬁned as ˜B(c,c) = 2 N 2 B(c,c) = kN i=1 ni + N 2 + kN j=1 n+ j N 2 −2 kN i=1 kN j=1 ni j N 2 . Both loss functions are considered N-invariant as they only depend on N through the proportions ni j/N. We focus on these two loss functions as they satisfy several desirable properties. The ﬁrst important property is that both VI and ˜B are metrics on the space of partitions. Property 1. Both VI and ˜B are metrics on the space of partitions. A proof for VI can be found in Meil˘a (2007). For ˜B, the proof results from the fact that ˜B can be derived as the Hamming distance between the binary representation of the clusterings. S. Wade and Z. Ghahramani 567 Figure 1: Hasse diagram for the lattice of partitions with a sample of size N = 4. A line is drawn from c up to c when c is covered by c. 567 S. Wade and Z. Ghahramani Figure 1: Hasse diagram for the lattice of partitions with a sample of size N = 4. A line is drawn from c up to c when c is covered by c. The next properties involve ﬁrst viewing the space of partitions as a partially ordered set. In particular, consider the space of partitions C and the binary relation ≤on C deﬁned by set containment, i.e. for c,c ∈C, c ≤c if for all i = 1, . . . , kN, Ci ⊆Cj for some j ∈{1, . . . , kN}. The partition space C equipped with ≤is a partially ordered set. 3 A comparison of the variation of information and Binder’s loss Bayesian Cluster Analysis: Point Estimation and Credible Balls 568 568 Bayesian Cluster Analysis: Point Estimation and Credible Balls Figure 2: Hasse diagram stretched by VI with a sample of size N = 4. Note 2−3 4 log(3) ≈ 0.811. From the VI stretched Hasse diagram, we can determine the distance between any two partitions. Example: if c = ({1, 2}, {3, 4}) and c = ({1}, {3}, {2, 4}), then c ∧c = ({1}, {2}, {3}, {4}) and d(c,c) = d(c ∧c, 1) −d(c, 1) + d(c ∧c, 1) −d(c, 1) = 2 −1 + 2 −1.5 = 1.5. Figure 2: Hasse diagram stretched by VI with a sample of size N = 4. Note 2−3 4 log(3) ≈ 0.811. From the VI stretched Hasse diagram, we can determine the distance between any two partitions. Example: if c = ({1, 2}, {3, 4}) and c = ({1}, {3}, {2, 4}), then c ∧c = ({1}, {2}, {3}, {4}) and d(c,c) = d(c ∧c, 1) −d(c, 1) + d(c ∧c, 1) −d(c, 1) = 2 −1 + 2 −1.5 = 1.5. Figure 3: Hasse diagram stretched by ˜B with a sample of size N = 4. From the ˜B stretched Hasse diagram, we can determine the distance between any two partitions. Example: if c = ({1, 2}, {3, 4}) and c = ({1}, {3}, {2, 4}), then c∧c = ({1}, {2}, {3}, {4}) and d(c,c) = d(c∧c, 1)−d(c, 1)+d(c∧c, 1)−d(c, 1) = 0.75−0.5+0.75−0.625 = 0.375. Figure 3: Hasse diagram stretched by ˜B with a sample of size N = 4. From the ˜B stretched Hasse diagram, we can determine the distance between any two partitions. Example: if c = ({1, 2}, {3, 4}) and c = ({1}, {3}, {2, 4}), then c∧c = ({1}, {2}, {3}, {4}) and d(c,c) = d(c∧c, 1)−d(c, 1)+d(c∧c, 1)−d(c, 1) = 0.75−0.5+0.75−0.625 = 0.375. Property 2. For both VI and ˜B, if c ≥c ≥c, then d(c,c) = d(c,c) + d(c,c). Property 3. For both VI and ˜B, d(c,c) = d(c,c ∧c) + d(c,c ∧c). Proofs can be found in the Supplementary Material. These two properties imply that if the Hasse diagram is stretched to reﬂect the distance between any partition and 1, the distance between any two partitions can be easily determined from the stretched Hasse diagram. 3 A comparison of the variation of information and Binder’s loss For any c,c ∈C, c is covered by c, denoted c ≺c, if c < c and there is no c ∈C such that c < c < c. This covering relation is used to deﬁne the Hasse diagram, where the elements of C are represented as nodes of a graph and a line is drawn from c up to c when c ≺c. An example of the Hasse diagram for N = 4 is depicted in Figure 1. The space of partitions possesses an even richer structure; it forms a lattice. This follows from the fact that every pair of partitions has a greatest lower bound and least upper bound; for a subset S ⊆C, an element c ∈C is an upper bound for S if s ≤c for all s ∈S, and c ∈C is the least upper bound for S, denoted c = l.u.b.(S), if c is an upper bound for S and c ≤c′ for all upper bounds c′ of S. A lower bound and the greatest lower bound for a subset S ⊆C are similarly deﬁned, the latter denoted by g.l.b.(S). We deﬁne the operators ∧, called the meet, and ∨, called the join, as c ∧c = g.l.b.(c,c) and c ∨c = l.u.b.(c,c). Following the conventions of lattice theory, we will use 1 to denote the greatest element of the lattice of partitions, i.e. the partition with every observation in one cluster c = ({1, . . . , N}), and 0 to denote the least element of the lattice of partitions, i.e. the partition with every observation in its own cluster c = ({1}, . . . , {N}). See Nation (1991) for more details on lattice theory and the Supplementary Material (Wade and Ghahramani, 2017) for speciﬁc details on the lattice of partitions. A desirable property is that both VI and ˜B are aligned with the lattice of partitions. Speciﬁcally, both metrics are vertically aligned in the Hasse diagram; if c is connected up to c and c is connected up to c, then the distance between c and c is the vertical sum of the distances between c and c and between c and c (see Property 2). And, both metrics are horizontally aligned; the distance between any two partitions is the horizontal sum of the distances between each partition and the meet of the two partitions (see Property 3). 3 A comparison of the variation of information and Binder’s loss Figures 2 and 3 depict the Hasse diagram for N = 4 in Figure 1 stretched according to VI and ˜B respectively. From the stretched Hasse diagram, we gain several insights into the similarities and diﬀerences between the two metrics. An evident diﬀerence is the scale of the two diagrams. 569 S. Wade and Z. Ghahramani S. Wade and Z. Ghahramani S. Wade and Z. Ghahramani Property 4. A distance on partitions satisfying Properties 2 and 3 has the property that for any two partitions c and c, d(c,c) ≤d(1, 0). Thus, Thus, VI(c,c) ≤log(N) and ˜B(c,c) ≤1 −1 N . A proof can be found in the Supplementary Material. In both cases, the bound on the distance between two clusterings depends on the sample size N. However, the behavior of this bound is very diﬀerent; for VI, it approaches inﬁnity as N →∞, and for ˜B, it approaches one as N →∞. As N grows, the number of total partitions BN increases drastically. Thus, it is sensible that the bound on the metric grows as the size of the space grows. In particular, 1 and 0 become more distant as N →∞, as there is an increasing number, BN −2, of partitions between these two extremes; for ˜B, the loss of estimating one of these extremes with the other approaches the ﬁxed number one, while for VI, the loss approaches inﬁnity. From the stretched Hasse diagram in Figures 2 and 3, we can determine the clos- est partitions to any c. For example, the closest partitions to 1 are the partitions which split 1 into two clusters, one singleton and one containing all other observa- tions; and the closest partitions to ({1}, {2}, {3, 4}) are the partition which merges the two smallest clusters ({1, 2}, {3, 4}) and the partition which splits the cluster of size two ({1}, {2}, {3}, {4}). Property 5. For both metrics VI and ˜B, the closest partitions to a partition c are: Property 5. For both metrics VI and ˜B, the closest partitions to a partition c a • if c contains at least two clusters of size one and at least one cluster of size two, the partitions which merge any two clusters of size one and the partitions which split any cluster of size two. Bayesian Cluster Analysis: Point Estimation and Credible Balls Next, we note that the Hasse diagram stretched by ˜B in Figure 3 appears asymmetric, in the sense that 1 is more separated from the others when compared to the Hasse diagram stretched by VI in Figure 2. Property 6. Suppose N is divisible by k, and let ck denote a partition with k clusters of equal size N/k. of equal size N/k. ˜B(1, ck) = 1 −1 k > 1 k −1 N = ˜B(0, ck). VI(1, ck) = log(k) ≤log(N) −log(k) = VI(0, ck), for k ≤ √ N, and VI(1, ck) = log(k) ≥log(N) −log(k) = VI(0, ck), for k ≥ √ N. Property 6 reﬂects the asymmetry apparent in Figure 3. In particular, for ˜B, a partition with two clusters of equal size c2 will always be closer to the extreme 0 of each data point in its own cluster than the extreme 1 of everyone in one cluster. However, as the sample size increases, c2 becomes equally distant between the two extremes. For all other values of k, the extreme 0 will always be closer. This behavior is counter-intuitive for a loss function on clusterings. VI is much more sensible in this regard. If k = √ N, 0 and 1 are equally good estimates of ck. For k < √ N, ck is better estimated by 1 and for k > √ N, ck is better estimated by 0; as the sample size increases, these preferences become stronger. In particular, note that loss of estimating c2 with 1 will always be smaller than estimating it with 0 for N > 4. Additionally, we observe from Figure 3 that the partitions with two clusters of sizes one and three are equally distant between the two extremes under ˜B. The following property generalizes this observation. Property 7. Suppose N is an even and square integer. Then, the partitions with two clusters of sizes n = 1 2(N − √ N) and N −n are equally distant from 1 and 0 under ˜B. Property 7. Suppose N is an even and square integer. Then, the partitions with two clusters of sizes n = 1 2(N − √ N) and N −n are equally distant from 1 and 0 under ˜B. 3 A comparison of the variation of information and Binder’s loss • if c contains at least two clusters of size one and no clusters of size two, the partitions which merge any two clusters of size one. • if c contains at most one cluster of size one, the partitions which split the smallest cluster of size greater than one into a singleton and a cluster with the remaining observations of the original cluster. A proof can be found in the Supplementary Material. This property characterizes the set of estimated partitions which are given the smallest loss. Under both loss functions, the smallest loss of zero occurs when the estimated partition is equal to the truth. Otherwise, the smallest loss occurs when the estimated clustering diﬀers from the truth by merging two singleton clusters or splitting a cluster of size two, or, if neither is possible, splitting the smallest cluster of size n into a singleton and a cluster of size n −1. We further note that the loss of estimating the true clustering with a clustering which merges two singletons or splits a cluster of size two, is 2 N and 2 N 2 for VI and ˜B respectively, which converges to 0 as N →∞for both metrics, but at a faster rate for ˜B. 570 Bayesian Cluster Analysis: Point Estimation and Credible Balls 4 Point estimation via the variation of information As detailed in the previous section, both VI and ˜B share several desirable properties including being aligned with the lattice of partitions and coinciding in the smallest non-trivial ball around any clustering. However, in our comparison, diﬀerences also emerged. Particularly, we ﬁnd that ˜B exhibits some peculiar asymmetries, preferring to split clusters over merging, and we ﬁnd that the VI ball more closely reﬂects our intuition of the neighborhood of a partition. In light of this, we propose to use VI as a loss function in Bayesian cluster analysis. Under the VI, the optimal partition c∗is c∗= argmin c E[VI(c,c)\|D] = argmin c N n=1 log( N n′=1 1(cn′ = cn)) −2 N n=1 E[log( N n′=1 1(cn′ = cn, cn′ = cn))\|D], (3) c∗= argmin c E[VI(c,c)\|D] = argmin c N n=1 log( N n′=1 1(cn′ = cn)) −2 N n=1 E[log( N n′=1 1(cn′ = cn, cn′ = cn))\|D], (3) (3) with D denoting the data. For a given c, the second term in (3) can be approximated based on the MCMC output, and evaluating this term is of order O(MN 2) (recall M is the number of MCMC samples). This may be computationally demanding if the number of MCMC samples is large and if (3) must be evaluated for a large number of c. Alternatively, one can use Jensen’s inequality, swapping the log and expectation, to obtain a lower bound on the expected loss which is computationally more eﬃcient to evaluate: argmin c N n=1 log( N n′=1 1(cn′ = cn)) −2 N n=1 log( N n′=1 P(cn′ = cn\|D)1(cn′ = cn)). (4) (4) Similar to minimization of the posterior expected Binder’s loss, minimization of (4) only depends on the posterior through the posterior similarity matrix, which can be pre-computed based on the MCMC output. In this case, computational complexity for a given c is reduced to O(N 2). Due to the huge dimensions of the partition space, computing the lower bound in (4) for every possible c is practically impossible. A simple technique to ﬁnd the optimal partition c∗restricts the search space to some smaller space of partitions. Bayesian Cluster Analysis: Point Estimation and Credible Balls This property is unappealing for a loss function, as it states that the loss of esti- mating a partition consisting of two clusters of sizes 1 2(N − √ N) and 1 2(N + √ N) with the partition of only one cluster or with the partition of all singletons is the same. In- tuitively, however, 1 is a better estimate. The behavior of VI is much more reasonable, as partitions with two clusters will always be better estimated by 1 than 0 for N > 4 and partitions with √ N clusters of equal size are equally distant from 0 and 1. Finally, we note that as both VI and ˜B are metrics on the space of clusterings, we can construct a ball around c of size ϵ, deﬁned as: Bϵ(c) = {c ∈C : d(c,c) ≤ϵ}. From Property 5, the smallest non-trivial ball will be the same for the two metrics. When considering the next smallest ball, diﬀerences emerge; a detailed example is provide in the Supplementary Material. In the authors’ opinions, the VI ball more closely reﬂects our intuition of the closest set of partitions to c. 571 S. Wade and Z. Ghahramani 4 Point estimation via the variation of information The R package ‘mcclust’ (Fritsch (2012)), which contains tools for point estimation in Bayesian cluster analysis and cluster comparison, includes a function minbinder() that ﬁnds the partition minimizing the poster expected Binder’s loss among the subset of partitions 1) visited in the MCMC chain or 2) explored in a hierarchical clustering algorithm with a distance of 1 −P(cn = cn′\|D) and average or complete linkage. An alternative search algorithm developed in Lau and Green (2007), which is based on binary integer programming, is also implemented. We propose a greedy search algorithm to locate the optimal partition c∗based on the Hasse diagram, which can be used for both VI and ˜B. In particular, given some partition c, we consider the l closest partitions that cover c and the l closest partitions that c covers. Here, the distance used to determine the closest partitions corresponds to the selected loss of VI or ˜B. Next, the posterior expected loss E[L(c,c)\|D] is computed for all proposed partitions c, and we move in the direction of minimum posterior expected loss, that is the partition c′ with minimal E[L(c, c′)\|D] is selected. The algorithm stops when 572 Bayesian Cluster Analysis: Point Estimation and Credible Balls Bayesian Cluster Analysis: Point Estimation and Credible Balls no reduction in the posterior expected loss is obtained or when a maximum number of iterations has been reached. At each iteration, the computational complexity is O(lN 2). We have developed an R package ‘mcclust.ext’ (Wade (2015)), expanding upon the ‘mcclust’ package, that is currently available on the author’s website1 and includes functions minbinder.ext() and minVI() to ﬁnd the partition minimizing the poste- rior expected Binder’s loss and VI, respectively. In addition to implementing the search algorithms of minbinder() in ‘mcclust’ described previously, the greedy search algo- rithm is also included. As is common in greedy search algorithms, results are sensitive to both the starting value of c and the step size l. In practice, we recommend multiple restarts, for example, at diﬀerent MCMC samples or the best partition found by the other search algorithms. A larger value of l will allow more exploration and reduce the need for multiple restarts, and we have chosen a default value of l = 2N as this showed good exploration in the examples considered with little sensitivity to the initial value of c. 4 Point estimation via the variation of information However, for larger datasets, this may be too expensive and multiple restarts with smaller l may be preferred. An advantage of the greedy search algorithm over simply restricting to partitions visited in the chain is that partitions not explored in the MCMC algorithm can be considered; in fact, in almost all simulated and real examples, the clustering estimate is not among the sampled partitions and results in a lower expected loss than any sampled partition. S. Wade and Z. Ghahramani To characterize the credible ball, we deﬁne the vertical and horizontal bounds of the credible ball. The vertical upper bounds consist of the partitions in the credible ball with the smallest number of clusters that are most distant from c∗. The vertical lower bounds consist of the partitions in the credible ball with the largest number of clusters that are most distant from c∗. The horizontal bounds consist of the partitions in the credible ball that are most distant from c∗. The bounds are deﬁned more formally below, where the notation k(c) is used for the number of clusters in c. Deﬁnition 1 (Vertical upper bounds). The vertical upper bounds of the credible ball Bϵ∗(c∗), denoted vu ϵ∗(c∗), are deﬁned as vu ϵ∗(c∗) = {c ∈Bϵ∗(c∗) : k(c) ≤k(c′) ∀c′ ∈Bϵ∗(c∗) and d(c, c∗) ≥d(c′′, c∗) ∀c′′ ∈Bϵ∗(c∗) with k(c) = k(c′′)}. Deﬁnition 2 (Vertical lower bounds). The vertical lower bounds of the credible ball Bϵ∗(c∗), denoted vl ϵ∗(c∗), are deﬁned as vl ϵ∗(c∗) = {c ∈Bϵ∗(c∗) : k(c) ≥k(c′) ∀c′ ∈Bϵ∗(c∗) and d(c, c∗) ≥d(c′′, c∗) ∀c′′ ∈Bϵ∗(c∗) with k(c) = k(c′′)}. Deﬁnition 3 (Horizontal bounds). The horizontal bounds of the credible ball Bϵ∗(c∗), denoted hϵ∗(c∗), are deﬁned as Deﬁnition 3 (Horizontal bounds). The horizontal bounds of the credible ball Bϵ∗(c∗), denoted hϵ∗(c∗), are deﬁned as hϵ∗(c∗) = {c ∈Bϵ∗(c∗) : d(c, c∗) ≥d(c′, c∗) ∀c′ ∈Bϵ∗(c∗)}. These bounds describe the extremes of the credible ball and with 1 −α posterior probability, how diﬀerent we believe the partition may be from c∗. An example is pro- vided in the Supplementary Material. In practice, we deﬁne the vertical and horizontal bounds based on the partitions in the credible ball with positive estimated posterior probability. In existing literature, quantiﬁcation of uncertainty in the clustering structure is typically described through a heat map of the estimated posterior similarity matrix. However, as opposed to the credible ball of Bayesian conﬁdence level 1 −α, there is no precise quantiﬁcation of how much uncertainty is represented by the posterior similar- ity matrix. Moreover, in the examples of Section 6, we ﬁnd that in a comparison with the 95% credible balls, the uncertainty is under-represented by the posterior similar- ity matrix. Additionally, the credible balls have the added desirable interpretation of characterizing the uncertainty around the point estimate c∗. 5 Credible balls of partitions To characterize the uncertainty in the point estimate c∗, we propose to construct a credible ball of a given credible level 1 −α, α ∈[0, 1], deﬁned as Bϵ∗(c∗) = {c : d(c∗, c) ≤ϵ∗}, where ϵ∗is the smallest ϵ ≥0 such that P(Bϵ(c∗)\|D) ≥1 −α. The credible ball is the smallest ball around c∗with posterior probability at least 1 −α. It reﬂects the posterior uncertainty in the point estimate c∗; with probability 1 −α, we believe that the clustering is within a distance of ϵ∗from the point estimate c∗given the data. It can be deﬁned based on any metric on the space of partitions, such as VI and ˜B. If the smallest non-trivial ball under VI or ˜B has posterior probability of at least 1 −α, the credible balls under the two metrics will coincide (see Property 5). Typically, however, they will be diﬀerent. From the MCMC output, we can obtain an estimate of ϵ∗, and thus the credible ball of level 1−α. First, the distance between all MCMC samples {cm} and c∗is computed. For any ϵ ≥0, P(Bϵ(c∗)\|D) = E[1(d(c∗, c) ≤ϵ)\|D] ≈1 M M m=1 1(d(c∗, cm) ≤ϵ), and ϵ∗is the smallest ϵ ≥0 such that 1 M M m=1 1(d(c∗, cm) ≤ϵ) ≥1 −α. and ϵ∗is the smallest ϵ ≥0 such that 1 M M m=1 1(d(c∗, cm) ≤ϵ) ≥1 −α. 1https://www2.warwick.ac.uk/fac/sci/statistics/staff/academic-research/wade/. 1https://www2.warwick.ac.uk/fac/sci/statistics/staff/academic-research/wade/. 1https://www2.warwick.ac.uk/fac/sci/statistics/staff/academic-research/wade/. 573 6 Examples We provide both simulated and real examples to compare the point estimates from VI and Binder’s loss and describe the credible ball representing uncertainty in the clustering estimate. Bayesian Cluster Analysis: Point Estimation and Credible Balls 574 Figure 4: The data is simulated from a mixture of four normals with locations (±2, ±2)′ and colored by cluster membership. In (b) components having varying standard devia- tions. Figure 4: The data is simulated from a mixture of four normals with locations (±2, ±2)′ and colored by cluster membership. In (b) components having varying standard devia- tions. 6.1 Simulated examples Two datasets of size n = 200 are simulated from: Xi iid ∼ 4 j=1 1 4N (−1)⌊(j−1) 2 ⌋2 (−1)j−12 , σ2 j 0 0 σ2 j . In the ﬁrst example, σj = 1 for all components, while in the second example, components have varying standard deviations; σj = 1 for the two components located in the ﬁrst and third quadrants, σj = 0.5 in the second quadrant, and σj = 1.5 in the fourth quadrant. The datasets for both examples are depicted in Figure 4 and colored by cluster membership. We consider a Dirichlet process (DP) mixture model: We consider a Dirichlet process (DP) mixture model: Xi\|P iid ∼ N μ1 μ2 , σ2 1 0 0 σ2 2 dP(μ, Σ) and P ∼DP(αP0), (5) (5) where μ = (μ1, μ2)′ and Σ is a diagonal matrix with diagonal elements (σ2 1, σ2 2). The base measure of the DP is the conjugate product of normal inverse gamma priors with parameters (μ0,i, ci, ai, bi) for i = 1, 2, i.e. P0 has density p0(μ1, μ2, σ2 1, σ2 2) ∝ 2 i=1 ci σ2 i exp −ci 2σ2 i (μi −μ0,i)2 (σ2 i )−ai−1 exp −bi σ2 i . The parameters were ﬁxed to μ0,i = 0, ci = 1/2, ai = 2, bi = 1 for i = 1, 2. The mass parameter α is given a Gam(1, 1) hyperprior. A marginal Gibbs sampler is used for inference (Neal (2000)) with 10,000 iterations after a burn in period of 1,000 iterations. Trace plots and autocorrelation plots (not 575 S. Wade and Z. Ghahramani Figure 5: Clustering estimate with color representing cluster membership for Binder’s loss (ﬁrst row) and VI (second row) with columns corresponding to examples. Figure 5: Clustering estimate with color representing cluster membership for Binder’s loss (ﬁrst row) and VI (second row) with columns corresponding to examples. shown) suggest convergence. Among partitions sampled in the MCMC, only one is visited twice and all others are visited once in the ﬁrst example, while no partitions are visited more than once in the second example. 6.1 Simulated examples Ex 1 Loss k∗ N NI E[˜B\|D] ˜B(ct, c∗) E[VILB\|D] E[VI\|D] VI(ct, c∗) N = 200: ˜B 9 13 0.062 0.045 0.545 0.816 0.643 VI 4 9 0.064 0.044 0.426 0.77 0.569 N = 400: ˜B 17 31 0.068 0.052 0.674 1.0 0.769 VI 4 18 0.073 0.044 0.505 0.933 0.54 N = 800: ˜B 24 62 0.068 0.061 0.615 1.016 0.903 VI 4 47 0.069 0.056 0.477 0.943 0.742 N = 1600: ˜B 41 93 0.058 0.044 0.551 0.898 0.719 VI 4 49 0.0596 0.045 0.403 0.814 0.629 Table 2: Example 1 with increasing sample size: a comparison of the clustering estimate with ˜B or VI in terms of 1) number of clusters k∗ N; 2) number of data points incorrectly classiﬁed, denoted NI; 3) expected ˜B; 4) ˜B between the optimal and true clusterings; 5) expected lower bound of VI; 6) expected VI; and 7) VI between the optimal and true clusterings. VI estimate achieve the lowest posterior expected loss for ˜B and VI, respectively, but interestingly, the VI estimate has the smallest distance from the truth for both ˜B and VI in both examples, with the greatest improvement in the second example. Furthermore, the number of incorrectly classiﬁed data points is greater for the ˜B estimate than the VI estimate. Additional simulated experiments were performed to analyze the eﬀect of increasing the sample size in the ﬁrst example. The results are succinctly summarized in Table 2. As the sample size increases, more points are located on the border where cluster mem- bership is uncertain. This results in an increasing number of clusters in the ˜B estimate (up to 41 clusters for N = 1600), while the VI estimate contains only four clusters for all sample sizes. In both estimates, the number of incorrectly classiﬁed data points increases with the sample size, however this number is smaller for the VI estimate in all sample sizes, with the diﬀerence between this number for Binder’s and VI growing with the sample size. Furthermore, the VI estimate has improved VI distance with truth and improved or comparable ˜B distance with truth when compared with the ˜B estimate. Further experiments were carried out to consider highly unbalanced clusters. 6.1 Simulated examples Figure 5 depicts the partition estimate found by the greedy search algorithm for Binder’s loss and VI and for both examples (with multiple restarts and the default value of l = 2N); colors represent cluster membership with the posterior expected cluster-speciﬁc mean and variance represented through stars and ellipses, respectively. Tables in the Supplementary Material provide a comparison of the true partition with the estimates through a cross tabulation of cluster labels. In all examples, the four true clusters are visible; however, Binder’s loss creates new small clusters for observations on the border between clusters where cluster membership is uncertain, overestimating the number of clusters. This eﬀect is most extreme for the second example, where the fourth cluster (blue in Figure 4b) has increased overlap with the second and third clusters (red and green in Figure 4b), while the ﬁrst cluster (black in Figure 4b) with decreased variance is well separated from the other clusters and identiﬁed in both estimates. A further comparison of the true partition with the estimates under Binder’s loss and VI, for both examples, is provided in Table 1. As expected, the ˜B estimate and Bayesian Cluster Analysis: Point Estimation and Credible Balls Bayesian Cluster Analysis: Point Estimation and Credible Ba 576 Loss k∗ N NI E[˜B\|D] ˜B(ct, c∗) E[VILB\|D] E[VI\|D] VI(ct, c∗) Ex 1: ˜B 9 13 0.062 0.045 0.545 0.816 0.643 VI 4 9 0.064 0.044 0.426 0.77 0.569 Ex 2: ˜B 12 18 0.088 0.056 0.846 1.068 0.764 VI 4 10 0.093 0.049 0.668 0.99 0.561 Table 1: A comparison of the clustering estimate with ˜B or VI in terms of 1) number of clusters k∗ N; 2) number of data points incorrectly classiﬁed, denoted NI; 3) expected ˜B; 4) ˜B between the optimal and true clusterings; 5) expected lower bound of VI; 6) expected VI; and 7) VI between the optimal and true clusterings for both examples. Table 1: A comparison of the clustering estimate with ˜B or VI in terms of 1) number of clusters k∗ N; 2) number of data points incorrectly classiﬁed, denoted NI; 3) expected ˜B; 4) ˜B between the optimal and true clusterings; 5) expected lower bound of VI; 6) expected VI; and 7) VI between the optimal and true clusterings for both examples. 6.1 Simulated examples In this case, the conclusions continue to hold; Binder’s loss overestimates the number of clusters present, placing uncertain observations in new small clusters, and this eﬀect becomes more pronounced with increased overlap between clusters (results not shown). 577 S. Wade and Z. Ghahramani Figure 6: Example 1: 95% credible ball with Binder’s loss around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). Figure 6: Example 1: 95% credible ball with Binder’s loss around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). For the ﬁrst example, Figures 6 and 7 represent the 95% credible ball around the opti- mal partition for ˜B and VI, respectively, through the upper vertical bound, lower vertical bound, and horizontal bound, with data points colored according to cluster membership. Analogous plots for the second example are found in Figures 8 and 9. The Supplemen- tary Material provides tables comparing the bounds with the true clustering through a cross tabulation of the true cluster labels with the cluster labels for each bound. In the ﬁrst example, we observe that elements of the 95% credible ball with positive estimated posterior probability have at least four clusters for both metrics and at most 18 clusters for ˜B or 16 clusters for VI, while the most distant elements contain 11 clusters for ˜B and VI (Table 3). For both metrics, these bounds reallocate uncertain data points on the border with these points either added to one of the four main clusters or to new small to medium-sized clusters. For example, in the ˜B upper bound, 19 elements of the third cluster (green in Figure 4a) are added to the fourth cluster (blue in Figure 4a) and in the ˜B lower bound, the fourth cluster (blue in Figure 4a) is split in two medium-sized clusters and several small clusters. 6.1 Simulated examples In the second example, the ﬁrst cluster (black in Figure 4b) is stable in all bounds, while the 95% credible ball reﬂects posterior uncertainty on whether to divide the re- 578 Bayesian Cluster Analysis: Point Estimation and Credible Balls Figure 7: Example 1: 95% credible ball with VI around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). Figure 7: Example 1: 95% credible ball with VI around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). Figure 7: Example 1: 95% credible ball with VI around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). Loss Upper Lower Horizontal ku N d(c∗, cu) kl N d(c∗, cl) kh N d(c∗, ch) Ex 1: ˜B 4 0.097 18 0.097 11 0.097 VI 4 1.02 16 1.152 11 1.213 Ex 2: ˜B 4 0.137 19 0.131 10 0.137 VI 3 1.043 16 1.342 6 1.403 Table 3: A summary of the credible bounds with ˜B or VI in terms of the number of clusters and distance to the clustering estimate for the upper vertical, lower vertical, and horizontal bounds and for both examples. maining data points into 3 to 18 clusters for ˜B and 2 to 15 clusters for VI (Table 3). Notice the high uncertainty in the fourth cluster with increased variance (blue in Fig- ure 4b). Additionally, note the greater uncertainty around the optimal estimate in Ex- ample 2, as the horizontal distance in Table 3 is greater for Example 2 for both metrics. maining data points into 3 to 18 clusters for ˜B and 2 to 15 clusters for VI (Table 3). Notice the high uncertainty in the fourth cluster with increased variance (blue in Fig- ure 4b). Additionally, note the greater uncertainty around the optimal estimate in Ex- ample 2, as the horizontal distance in Table 3 is greater for Example 2 for both metrics. Figures 6–9 also present heat maps of the posterior similarity matrix for both ex- amples. 6.1 Simulated examples In general, the posterior similarity matrix appears to under-represent the un- certainty; indeed, one would conclude from the similarity matrix that there is only 579 S. Wade and Z. Ghahramani Figure 8: Example 2: 95% credible ball with Binder’s loss around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bounds (only one of two shown for conciseness), and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). Figure 8: Example 2: 95% credible ball with Binder’s loss around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bounds (only one of two shown for conciseness), and (d) lower vertical bound, where color denotes cluster membership, and a heat map of the posterior similarity matrix (e). uncertainty in allocation of a few data points in Example 1. Moreover, the 95% credible ball gives a precise quantiﬁcation of the uncertainty. uncertainty in allocation of a few data points in Example 1. Moreover, the 95% credible ball gives a precise quantiﬁcation of the uncertainty. 6.2 Galaxy example We consider an analysis of the galaxy data (Roeder (1990)), available in the MASS package of R, which contains measurements of velocities in km/sec of 82 galaxies from a survey of the Corona Borealis region. The presence of clusters provides evidence for voids and superclusters in the far universe. The data is modeled with a DP mixture (5). The parameters were selected empirically with μ0 = ¯x, c = 1/2, a = 2, b = s2, where ¯x represents the sample mean and s2 represents the sample variance. The mass parameter α is given a Gam(1, 1) hyperprior. With 10,000 samples after 1,000 burn in, the posterior mass is spread out over 9,636 partitions, emphasizing the need for appropriate summary tools. Figure 10 plots the point estimate of the partition found by the greedy search algorithm for Binder’s loss and VI (with multiple restarts and the default value of l = 2N). The data values are Bayesian Cluster Analysis: Point Estimation and Credible Balls 580 Figure 9: Example 2: 95% credible ball with VI around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership and a heat map of the posterior similarity matrix (e). Figure 9: Example 2: 95% credible ball with VI around c∗(a) represented by the (b) horizontal bound, (c) upper vertical bound, and (d) lower vertical bound, where color denotes cluster membership and a heat map of the posterior similarity matrix (e). plotted against the estimated density values from the DP mixture model and colored according to cluster membership, with correspondingly colored stars and bars along the x-axis representing the posterior mean and variance within cluster. Again, we observe that Binder’s loss places observations with uncertain allocation into singleton clusters, with a total of 7 clusters, 4 of which are singletons, while the VI solution contains 3 clusters. Table 4 compares the point estimates in terms of the posterior expected ˜B, lower bound of VI, and VI; as anticipated, the ˜B solution has the smallest posterior expected ˜B and the VI solution has the smallest posterior expected VI. Loss k∗ N E[˜B\|D] E[VILB\|D] E[VI\|D] ˜B 7 0.218 0.746 1.014 VI 3 0.237 0.573 0.939 Table 4: Galaxy example: a comparison of the optimal partition with Binder’s loss and VI in terms of posterior expected ˜B, lower bound to VI, and VI. 6.2 Galaxy example Table 5: Galaxy example: a summary of the credible bounds with VI in terms of the number of clusters and distance to the clustering estimate for the upper vertical, lower vertical, and horizontal bounds. in cross tabulation tables in the Supplementary Material. We observe a large amount of variability around the optimal partition. With 95% posterior probability, we believe that, on one extreme, the data could be modeled using only 2 components, one with a large variance to account for outliers (black cluster in Figure (11a)). On the other extreme, the data could be further split into one medium sized cluster and many, 14 to be precise, smaller clusters. The horizontal bound, the most extreme partition in the credible ball, splits the largest cluster (red in Figure 10b) into two medium sized clusters and four small clusters and reallocates some of its data points to the ﬁrst cluster (black in Figure 10b). Figure 11d emphasizes that the posterior similarity ma- trix under-represents the uncertainty around the point estimate in comparison to the credible ball. 6.2 Galaxy example Loss k∗ N E[˜B\|D] E[VILB\|D] E[VI\|D] ˜B 7 0.218 0.746 1.014 VI 3 0.237 0.573 0.939 Table 4: Galaxy example: a comparison of the optimal partition with Binder’s loss and VI in terms of posterior expected ˜B, lower bound to VI, and VI. Table 4: Galaxy example: a comparison of the optimal partition with Binder’s loss and VI in terms of posterior expected ˜B, lower bound to VI, and VI. Table 4: Galaxy example: a comparison of the optimal partition with Binder’s loss and VI in terms of posterior expected ˜B, lower bound to VI, and VI. Table 4: Galaxy example: a comparison of the optimal partition with Binder’s loss and VI in terms of posterior expected ˜B, lower bound to VI, and VI. The 95% VI credible ball contains all partitions with a VI distance less than 1.832. Figure 11 depicts the 95% credible ball through the upper vertical, lower vertical, and horizontal bounds, which are further described and summarized in Table 5 and 581 S. Wade and Z. Ghahramani Figure 10: Galaxy example: optimal clustering estimate with color representing cluster membership for Binder’s loss and VI, with correspondingly colored stars and bars along the x-axis representing the posterior mean and variance within cluster. Figure 10: Galaxy example: optimal clustering estimate with color representing cluster membership for Binder’s loss and VI, with correspondingly colored stars and bars along the x-axis representing the posterior mean and variance within cluster. Upper Lower Horizontal ku N d(c∗, cu) kl N d(c∗, cl) kh N d(c∗, ch) Galaxy 2 1.364 15 1.669 8 1.832 Table 5: Galaxy example: a summary of the credible bounds with VI in terms of the number of clusters and distance to the clustering estimate for the upper vertical, lower vertical, and horizontal bounds. Upper Lower Horizontal ku N d(c∗, cu) kl N d(c∗, cl) kh N d(c∗, ch) Galaxy 2 1.364 15 1.669 8 1.832 Table 5: Galaxy example: a summary of the credible bounds with VI in terms of the number of clusters and distance to the clustering estimate for the upper vertical, lower vertical, and horizontal bounds. Table 5: Galaxy example: a summary of the credible bounds with VI in terms of the number of clusters and distance to the clustering estimate for the upper vertical, lower vertical, and horizontal bounds. 7 Discusssion Bayesian cluster analysis provides an advantage over classical cluster analysis, in that the Bayesian procedure returns a posterior distribution over the entire partition space, reﬂecting uncertainty in the clustering structure given the data, as opposed to returning a single solution or conditioning on the parameter estimates and number of clusters. This allows one to assess statistical properties of the clustering given the data. However, due to the huge dimension of the partition space, an important problem in Bayesian cluster analysis is how to appropriately summarize the posterior. To address this problem, we Bayesian Cluster Analysis: Point Estimation and Credible Balls 582 Figure 11: Galaxy example: 95% credible ball with VI represented by the (a) upper vertical bound, (b) lower vertical bound, and (c) horizontal bound, where color denotes cluster membership, with correspondingly colored stars and bars along the x-axis rep- resenting the posterior mean and variance within cluster, and (d) a heat map of the posterior similarity matrix. Figure 11: Galaxy example: 95% credible ball with VI represented by the (a) upper vertical bound, (b) lower vertical bound, and (c) horizontal bound, where color denotes cluster membership, with correspondingly colored stars and bars along the x-axis rep- resenting the posterior mean and variance within cluster, and (d) a heat map of the posterior similarity matrix. have developed tools to obtain a point estimate of clustering based on the posterior and describe uncertainty around this estimate via the 95% credible ball. Obtaining a point estimate through a formal decision theory framework requires the speciﬁcation of a loss function. Previous literature focused on Binder’s loss. In this work, we propose to use an information theoretic measure, the variation of information, and provide a detailed comparison of the two metrics. We ﬁnd that Binder’s loss ex- hibits peculiar asymmetries, preferring to split over merge clusters, and the variation of information is more symmetric in this regard. This behavior of Binder’s loss causes the optimal partition to overestimate the number of clusters, allocating uncertain data points to small additional clusters. In addition, we have developed a novel greedy search algorithm to locate the optimal partition, allowing one to explore beyond the space of partitions visited in the MCMC chain. To represent uncertainty around the point estimate, we construct 95% credible balls around the point estimate and depict the credible ball through the upper vertical, lower 583 S. Wade and Z. 7 Discusssion Ghahramani vertical, and horizontal bounds. In addition to a heat map of the posterior similarity matrix, which is often reported in literature, the 95% credible ball enriches our un- derstanding of the uncertainty present. Indeed, it provides a precise quantiﬁcation of the uncertainty present around the point estimate, and in examples, we ﬁnd that an analysis based on the posterior similarity matrix leads one to be over certain in the clus- tering structure. The developed posterior summary tools for Bayesian cluster analysis are available2 through an R package ‘mcclust.ext’ (Wade (2015)), expanding upon the existing R package ‘mcclust’ (Fritsch (2012)). In future work, we aim to extend these ideas to Bayesian feature allocation analy- sis, an extension of clustering which allows observations to belong to multiple clusters (Griﬃths and Ghahramani (2011)). A further direction of research will be to explore posterior consistency for the number of clusters based on the VI estimate for Bayesian nonparametric mixture models; this is of particular interest in light of the negative re- sults of Miller and Harrison (2013) and Miller and Harrison (2014) and the positive results in our simulation studies (Table 2). Finally, scalability issues of Bayesian non- parametric mixture models are an important concern for very large datasets. To scale with large sample sizes, a number of papers have avoided exploration of the posterior on partitions through MCMC and focused on ﬁnding a point estimate of the partition, of- ten through MAP inference (Heller and Ghahramani (2005), Dahl (2009), Raykov et al. (2014)) or the DP-means algorithm and its extensions (Kulis and Jordan (2012), Jiang et al. (2012), Broderick et al. (2013)). One direction of future research is to develop an algorithm to ﬁnd the point estimate which minimizes the posterior expected VI that avoids MCMC. Of course, while gaining in scalability, we lose the uncertainty in the clustering structure. Supplementary Material Supplementary material for Bayesian cluster analysis: Point estimation and credible balls (DOI: 10.1214/17-BA1073SUPP; .pdf). 2Through the author’s website https://www2.warwick.ac.uk/fac/sci/statistics/staff/ academic-research/wade/. References Binder, D. (1978). “Bayesian Cluster Analysis.” Biometrika, 65: 31–38. MR0501592. doi: https://doi.org/10.1093/biomet/65.1.31. 561, 564 Broderick, T., Kulis, B., and Jordan, M. (2013). “MAD-Bayes: MAP-based asymptotic derivations from Bayes.” In Proceedings of the 30th International Conference on Ma- chine Learning, 226–234. 583 Dahl, D. (2006). “Model-based clustering for expression data via a Dirichlet process mixture model.” In Do, K., M¨uller, P., and Vannucci, M. 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(2014). “Simple approximate MAP In- ference for Dirichlet processes.” Available at https://arxiv.org/abs/1411.0939. MR3572859. doi: https://doi.org/10.1214/16-EJS1196. 561, 583 Roeder, K. (1990). “Density estimation with conﬁdence sets exempliﬁed by superclusters and voids in galaxies.” Journal of the American Statistical Association, 85: 617–624. 579 Teh, Y., Jordan, M., Beal, M., and Blei, D. (2006). “Hierarchical Dirichlet pro- cess.” Journal of the American Statistical Association, 101: 1566–1581. References MR2279480. doi: https://doi.org/10.1198/016214506000000302. 564 Vinh, N., Epps, J., and Bailey, J. (2010). “Information theoretic measures for clusterings comparison: Variants, properties, normalization and correction for chance.” Journal of Machine Learning Research, 11: 2837–2854. MR2738784. 566 Wade, S. (2015). mcclust.ext: Point estimation and credible balls for Bayesian cluster analysis. URL https://www.researchgate.net/publication/279848500 mcclustext-manual. 572, 583 Wade, S. and Ghahramani, Z. (2017). “Supplementary material for Bayesian cluster analysis: Point estimation and credible balls.” Bayesian Analysis. doi: https://doi.org/10.1214/17-BA1073SUPP. 567 his work was supported by the Engineering and Physical Sciences Research Council [grant mber EP/I036575/1]. Invited comment on Article by Wade and Ghahramani Stefano Monni∗ Professor Wade and Professor Ghahramani have written an interesting paper that deals with the very important question of how to summarize the posterior distribution of partitions in nonparametric models. The summary of the posterior they propose is in the form of a point estimate and an associated credible ball, which quantiﬁes the uncertainty of the estimate, using a decision-theoretic approach. I enjoyed reading the paper and would like to make two comments. The ﬁrst one is a clariﬁcation. The second is concerned with the graphical representation of the credible ball. The authors consider the variation of information (VI) and Binder’s loss; show that these criteria for comparison of clusters are metrics/distances; give proofs of some of their properties, such as vertical and horizontal collinearity; determine their bounds and their scales; obtain the closest cluster to a given cluster according to those distances. All these properties (and a few more others) are already described and proven in the paper of Meil˘a (2007), where the VI distance is introduced. Indeed, the authors explicitly refer to that paper for some proofs. However, they do so only for the VI. Because of this, some readers may be left with the impression that the properties of the Binder’s loss and some of the advantages of the VI over the Binder’s loss when comparing clusters were never described in detail before this paper. Thus, I think it is important to stress that Meil˘a does give details of the properties of the Binder’s loss. To be precise, Meil˘a considers a number of criteria useful to compare clusters, among which the Mirkin metric (Mirkin and Chernyi, 1970). The latter metric is equal to twice the Binder’s loss B(c, ˆc), and its N-invariant version is equal to the N-invariant Binder’s loss ˜B(c, ˆc). In fact Meil˘a provides a quite explicit comparison of the VI with the Mirkin metric. With this clariﬁed, the entire Section 3 of the paper under discussion should only be seen as a review of the comparison of Binder’s loss with the VI as criteria for comparison of clusters. To be fair, in other sections of the paper, the contrasts between these two distances are analyzed further (for example in the description of the credible balls) although not at the same level of formality. Acknowledgments This work was supported by the Engineering and Physical Sciences Research Council [grant number EP/I036575/1]. 588 Contributed comment on Article by Wade and Ghahramani ∗Department of Mathematics, American University of Beirut, sm150@aub.edu.lb Invited comment on Article by Wade and Ghahramani The fact that the Binder’s loss and the VI are distances is brought to bear in the deﬁnition of the credible ball, which is the most interesting part of the paper. The credible ball is a very useful concept and I agree with the authors that it allows a characterization of uncertainty of the point estimate. Naturally, since the ball is a subset of the partition space, one is faced, yet again, with the problem of summarizing a subset of partitions. It is perhaps for this reason that the authors introduce the concept of vertical and horizontal bounds. If one looks at the graphical representations of the credible balls presented in the paper (Figures 7 and 8 for instance), one will undoubtedly 589 S. Monni ﬁnd them pleasing and informative. However, some questions should be asked about such plots. Namely, I’m concerned about graphically representing the credible ball when the vertical and horizontal bounds consist of more than one partition. Indeed, the deﬁnitions of the bounds do not prevent such occurrences and, in fact, examples of such bounds are explicitly given in section 2 of the supplementary material. If the number of objects to cluster is large, it is quite plausible that the horizontal and vertical bounds too are sets of large size. The problem of representing a credible ball has turned into the problem of representing the bounds, which appears to be just as intricate, if not identical. In the paper it is stated that what is used in practice to deﬁne the bounds is the subset of partitions in the credible ball that have positive estimated posterior probabilities, but, even so, the bounds will hardly contain one partition. I would be very interested in knowing what the authors suggest should be done when the sets of the bounds are large. One can perhaps just depict one representative partition for each of the bounds that is selected on the basis of additional considerations. As a selection criterion one could employ the value of the posterior probability or of the expected posterior loss, but it is diﬃcult to see whether this could really work well. To put it another way, I’m suggesting the authors should think of a reﬁnement of the deﬁnition of bounds. Wade and Ghahramani propose the credible ball as an elegant alternative to the posterior similarity matrix in assessing the uncertainty of a cluster estimate. Invited comment on Article by Wade and Ghahramani They state that the posterior similarity matrix under-represents this uncertainty, when compared with the credible ball. While I’m sure that they will agree with me that much more evidence is necessary to conclude whether this is true in general, I suspect that the diﬃculty I see in representing the credible ball may limit its success. The heatmap of the posterior similarity matrix continues to be in my view a very valid (if not irreplaceable) tool for assessing the uncertainty of cluster estimates. However, I hope to hear from Wade and Ghahramani that my concerns about the graphical representation of the credible ball are misplaced. Invited comment on Article by Wade and Ghahramani Giorgio Paulon∗, Lorenzo Trippa†, and Peter M¨uller ‡ We thank the authors for an interesting discussion of estimates and uncertainty sum- maries for random partitions. A coherent description of uncertainties is one of the strengths of the Bayesian approach, but it is diﬃcult to summarize and report it in the case of a random partition. The clever and elegant approach of Wade and Ghahra- mani addresses this critical gap in the literature. However, the approach relies on loss functions that ignore the underlying inference problem that gave rise to the random partition. In other words, the loss functions are generic inference losses that ignore the context of the scientiﬁc question that the investigators are trying to address. In this discussion we would like to elaborate on the authors’ related comment that alternative loss functions could be tailored to speciﬁc problems. We assume that the inference problem and sampling model include cluster-speciﬁc parameters, θ⋆ j , j = 1, . . . , kN. For example, if θ⋆ j were the mean times to progres- sion for patients in a clinical trial, the clusters would describe patient subpopulations with diﬀerent mean time to progression. A summary of the random partition should then focus on partitions with meaningfully diﬀerent θ⋆ j ’s. Similarly, in some contexts, one might prefer avoiding inclusion and reporting of small clusters. Inspired by Xu et al. (2016) who use a determinantal point process to favor conﬁgurations with diverse cluster-speciﬁc parameters, we propose the following loss function. The loss function formalizes a tradeoﬀbetween reporting clusters that are representative of the posterior and, with the second term, favoring partitions with clusters Cj that are diverse: Lrep(c, ˆc, θ⋆, ˆθ⋆) = 1 N N n=1 θ⋆ cn −ˆθ⋆ ˆcn 2 −λ det(Φ), where [Φij]i,j = φτ(ˆθ⋆ i , ˆθ⋆ j ) for some kernel φτ(x, y), e.g. the squared exponential φτ(x, y) = exp{−0.5[(x −y)/τ]2}. That is, det(Φ) is the volume of a parallelotope spanned by the columns of Φ, which is zero when θ⋆ i = θ⋆ j for any i ̸= j, and maximized when they are very distinct. Of course the squared distance in the loss can be replaced by a diﬀerent distance, e.g. one that allows for asymmetric costs of misﬁt. ∗Department of Statistics & Data Science, University of Texas, Austin, TX, giorgio.paulon@utexas.edu †Dana-Faber Cancer Institute, Boston, MA, ltrippa@jimmy.harvard.edu ‡Department of Statistics & Data Science, University of Texas, Austin, TX, pmueller@math.utexas.edu References Meil˘a, M. (2007). “Comparing clusterings-an information based distance.” Journal of Multivariate Analysis, 98(5): 873–895. MR2325412. doi: https://doi.org/10.1016/ j.jmva.2006.11.013. 588 Mirkin, B. G. and Chernyi, L. B. (1970). “Measurement of the distance between distinct partitions of a ﬁnite set of objects.” Automation and Remote Control, 31(5): 786–792. MR0300907. 588 590 Invited comment on Article by Wade and Ghahramani Invited comment on Article by Wade and Ghahramani The second component of the loss function could also be modiﬁed to mirror speciﬁc goals, for ex- ample penalizing conﬁgurations that include small clusters. The point here is that, in general, the particular application should drive the choice of the loss function. G. Paulon, L. Trippa, and P. M¨uller 591 Figure 1: Optimal ˆc for the normal mixture example. The histogram shows the data; the black curve shows the estimated posterior mean of the random probability mea- sure, along with pointwise 95% credible intervals. Color shows the estimated cluster membership for xi. Figure 1: Optimal ˆc for the normal mixture example. The histogram shows the data; the black curve shows the estimated posterior mean of the random probability mea- sure, along with pointwise 95% credible intervals. Color shows the estimated cluster membership for xi. We compared Lrep with the VI loss and also with the squared loss (Dahl, 2006) in the following example. Let N(x; m, s) denote a normal p.d.f. with location m and scale s evaluated at x, and let μ = (−3, −3.5, −2.6, 0, 1.8, 2.4, 7.1). We simulated N = 1000 observations from a mixture of 7 normals, p(xi \| μ) ∝7 j=1 N(xi; μj, 1). We ﬁt the data using a Dirichlet process mixture of normals model. In this case, only four com- ponents of the mixture are likely to be practically meaningful. The three values around -3 and the two around 2 are not meaningfully diﬀerent (relative to the variances in the normal kernels). Inference summaries under Lrep and VI loss are shown in Figure 1. In this example the posterior mode for kN is ˆkN = 7. But both loss functions penalize excessive complexity and shrink the reported partition to the 4 groups shown in the ﬁgure. Although the VI loss does not explicitly favor easy interpretation, it does sur- prisingly well in this example. We used an implementation that restricted the search for the Bayes estimate of the partition under Lrep to the simulated partitions only, which might explain the counter-intuitive lack of monotonicity in the cluster membership in Figure 1a. One could alternatively use better search algorithms such as, for example, the sequentially-allocated latent structure optimization (SALSO) in the sdols R package (Dahl and M¨uller, 2017). Invited comment on Article by Wade and Ghahramani using a Dirichlet process mixture of probit models. Inference under VI and squared loss reports 10 singleton clusters, a partition which is diﬃcult to interpret, also because of the negligible diﬀerences between estimated cluster-speciﬁc response rates. See Figure 2 for a summary of the posterior estimated response rates πi. In contrast, the desired preference for interpretable structure is explicitly included in Lrep, leading us to report ˆc = (1, 1, 1, 1, 1, 1, 1, 1, 2, 1), which appears more plausible in the light of the estimated response probabilities (the singleton cluster is Ewings’ sarcoma). Figure 2: 90% posterior credible intervals of the Binomial success probabilities πi for each sarcoma. For reference the dashed vertical line marks 0.1. Figure 2: 90% posterior credible intervals of the Binomial success probabilities πi for each sarcoma. For reference the dashed vertical line marks 0.1. There are two more aspects of inference for random partitions that we would like to brieﬂy discuss. Both are related to the underlying data analysis problem. In many applications the main inference target is not the entire partition, but only a special subset. Assume, for example, that in an analysis of clinical trial data cluster-speciﬁc parameters θ⋆ j are interpreted as treatment eﬀects. An important problem is to ﬁnd the subset of patients who most beneﬁt from the treatment under consideration, that is, the subset with the largest θ⋆ j . This is known as subgroup analysis. Let B = Cj⋆denote the subset Cj with the largest θ⋆ j . Characterizing uncertainty on a random partition now reduces to reporting uncertainty on B. Schnell et al. (2016) develop a clever approach to determine a pair of subsets (D, S) such that p(D ⊆B ⊆S \| data) > 1−α. Subgroup analysis is in general not necessarily linked with random partitions and involves several other issues. The point here is to emphasize that relevant uncertainty on a random partition need not treat all subsets symmetrically. Investigators might only be concerned about a particular subset. Finally, we would like to bring up one more aspect about summaries of clustering uncertainty, related to reproducibility. Above, we used a decision theoretic framework to summarize a random partition with a good estimate that is constructed to be represen- tative of the posterior distribution. Additionally, we report uncertainty measures that mirror the distance between the selected conﬁguration and a ﬁctitious latent partition. Invited comment on Article by Wade and Ghahramani We do not show the results obtained under squared loss or Binder’s loss, since both clearly overﬁt the data reporting kN = 53 components. Next we investigate a scenario with a small number of observations. We compare the same two loss functions with a dataset from a clinical trial for sarcoma patients with binary endpoints (tumor response) (Le´on-Novelo et al., 2013). The goal of the study is to cluster N = 10 diﬀerent sarcomas subtypes. That is, the experimental units for the random partition are the disease subtypes. The sampling model is binomial sampling, xi\|πi ∼Bin(Mi, πi) for the number of tumor responses xi for a given number of patients Mi under each sarcoma subtype, i = 1, . . . , N. The number of patients, Mi for each subtype are moderately small, between 2 and 29. We implement inference 592 Invited comment on Article by Wade and Ghahramani Although primarily meant to summarize the posterior distribution, these uncertainty measures are also vaguely related to the (frequentist) variability of the estimate ˆc. In- deed, consider repeating the entire experiment de novo, including both, data generation and analysis. It remains unclear how diﬀerent the estimated conﬁguration ˆc might turn 593 G. Paulon, L. Trippa, and P. M¨uller out. In most Bayesian estimation problems of key parameters, including means or me- dians, estimating this variability is unnecessary to express uncertainty, and the focus is exclusively on the posterior distribution of the parameter of interest. But clustering is an attempt to organize data points into conveniently created categories. An under- lying true unknown partition might be useless or not exist at all. These considerations lead us to suggest the report of replicability measures that could contrast ˆc and esti- mates under independent replicates, possibly including variations in the sample size. An extended set of uncertainty metrics could scrutinize the main drivers of variability, in- cluding limitations in the measurement of the statistical units (low sample size for each sarcoma subtype, in the previous application), data preprocessing, clustering methods, and experimental designs. References Dahl, D. B. (2006). Model-based clustering for expression data via a Dirichlet process mixture model. In M. Vannucci, K.-A. Do, and P. M¨uller, editors, Bayesian Inference for Gene Expression and Proteomics. Cambridge University Press. MR2706330. 591 Dahl, D. B. and M¨uller, P. (2017). sdols: Summarizing Distributions of Latent Struc- tures. R package version 1.4. 591 Le´on-Novelo, L. G., M¨uller, P., Arap, W., Kolonin, M., Sun, J., Pasqualini, R., and Do, K.-A. (2013). Semiparametric Bayesian inference for phage display data. Biometrics, 69(1), 174–183. MR3058064. doi: https://doi.org/10.1111/j.1541- 0420.2012.01817.x. 591 Schnell, P. M., Tang, Q., Oﬀen, W. W., and Carlin, B. P. (2016). A Bayesian cred- ible subgroups approach to identifying patient subgroups with positive treatment eﬀects. Biometrics, 72, 1026–1036. MR3591587. doi: https://doi.org/10.1111/ biom.12522. 592 Xu, Y., M¨uller, P., and Telesca, D. (2016). Bayesian inference for latent biologic structure with determinantal point processes (DPP). Biometrics, 72, 955–964. MR3545688. doi: https://doi.org/10.1111/biom.12482. 590 594 Invited comment on Article by Wade and Ghahramani ∗School of Mathematics and Statistics and Insight Centre for Data Analytics, University College Dublin, Ireland, nial.friel@ucd.ie †Institute for Statistics and Mathematics, WU Vienna University of Economics and Business, Aus- tria, riccardo.rastelli@wu.ac.at 1 Choice of loss function and computational eﬃciency As W&G clearly point out in their paper, commonly used loss functions such as the 0−1 loss or the squared error loss are not ideally suited to compare partitions, due to the discrete nature of the variables and because of the lack of total order in the space. This leads to the important issue of ﬁnding an appropriate and reasonable loss function to compare partitions. A popular choice in this context is Binder’s loss, primarily for two main reasons: its close connection to the Rand index; but also since the corresponding optimal partition can be estimated via the posterior similarity matrix, which itself can be routinely estimated by Markov chain Monte Carlo, for example. The posterior similarity matrix is an N × N matrix with element n, n′ (denoted pn,n′ in W&G) equal to the posterior probability that observations n and n′ are allocated to the same cluster and where N denotes the size of the dataset. The Variation of Information (VI) loss does not possess such a representation in terms of the posterior similarity matrix and as such it turns out that this brings with it an increased computational overhead. However, W&G neatly sidestep this problem by exploiting Jensen’s inequality to obtain a lower bound for the VI loss which relies only on the posterior similarity matrix. This input is interesting, though we note that the eﬀect that this approximation has on the estimated optimal partition is not clear. In R&F, the approach we advocate does not rely on the posterior similarity matrix representation and does not involve any approximation. In fact, our method may be used with any loss function, L(a, z) that depends on the two partitions, a and z through the counts nij, denoting the number of data points allocated to group i in partition a and to group j in partition z, which can conceptually be considered as depending on the contingency table deﬁned by both partitions. Binders’ loss and VI loss are included in this family, along with other known losses such as the normalised VI and the normalised information distance. Moreover, since our approach does not require the posterior sim- ilarity matrix, its computational complexity in N is decreased to a linear order (See Figure 1 of Rastelli and Friel (2017)). However, the computational cost of our approach also becomes increasingly costly as sample size of partitions drawn from the posterior increases. Invited comment on Article by Wade and Ghahramani Friel and R. Rastelli the following aspects: the choice of loss function used and the ensuing computational complexity; alternatives to the credible balls approach; the wider applicability of the methods proposed. Invited comment on Article by Wade and Ghahramani Nial Friel∗and Riccardo Rastelli† Abstract. We present a discussion of the paper “Bayesian cluster analysis: point estimation and credible balls” by Sara Wade and Zoubin Ghahramani. We believe that this paper contributes substantially to the literature on Bayesian clustering by ﬁlling in an important methodological gap, by providing a means to assess the uncertainty around a point estimate of the optimal clustering solution based on a given loss function. In our discussion we reﬂect on the characterisation of uncertainty around the Bayesian optimal partition, revealing other possible alter- natives that may be viable. In addition, we suggest other important extensions of the approach proposed which may lead to wider applicability. Keywords: Bayesian clustering, greedy optimisation, latent variable models, Markov chain Monte Carlo. We congratulate the authors, Wade and Ghahramani (W&G hereafter), on a wonderful article which is an excellent contribution to the area of Bayesian cluster analysis. Here the authors address the problem of appropriately summarising a partition based on a posterior. This is a crucial issue arising in a variety of clustering contexts. While Markov chain Monte Carlo techniques, for example, can be used to eﬃciently sample the cluster membership variables from the posterior distribution of a variety of mixture models, it is not immediately clear then how one can reasonably summarise such information. Similarly to other previous papers, notably Lau and Green (2007), the authors deﬁne the optimal partition as the one minimising the posterior expectation of a suitable loss function, and propose a greedy algorithm to estimate such an optimal solution. Somewhat surprisingly, there has been very little in the literature around how one might assess the uncertainty in this point estimate. W&G address this crucial gap by introducing a strategy to characterise the uncertainty around the optimal partition using an adaptation of the credible intervals approach. We consider this to be a major contribution and expect it stimulate future developments in this ﬁeld. We have recently worked on the same problem and published our ﬁndings in Rastelli and Friel (2017) (hereafter referred to as R&F). Similarly to W&G, we rely on a decision theoretic framework to summarise a collection of partitions, however, diﬀerently from their approach, our contribution is primarily focused on the computational aspects of the problem. Our method is implemented in the R package GreedyEPL available on CRAN. In this discussion we compare our ﬁndings to those of W&G mainly focusing on 595 N. 2 Quantifying the uncertainty around the estimated Bayes partition In our experience, the marginal posteriors for the cluster membership variables generally exhibit some degree of multimodality, even after labeling issues have been taken into account. This is one important reason why often Markov chain Monte Carlo sampling methods generally struggle to explore the discrete search space eﬃciently. We believe that the same multimodality may also have non-negligible eﬀects on the characterisation of the uncertainty around the estimated optimal Bayes partition. In a nutshell: if, by deﬁnition, the credible ball has to include 95% of the posterior mass, it will contain most of the relevant modes, but it may also include many of irrelevant partitions “between” them, in the sense of the loss considered. This would result in a quite heterogeneous set which may be hard to characterise, and where the horizontal and vertical bounds may not be so relevant to the clustering problem. We present a small experiment here to illustrate this point. Here we simulated a data set by sampling from a uniform distribution in the square [−1, 1]×[−1, 1]. We assumed the data followed a Gaussian mixture model and then obtained a posterior sample of partitions using the R package bayesm. We then applied the methodology proposed by W&G to assess the uncertainty in the estimated Bayes partition and present the output of this experiment in Figure 1. In this case, while the optimal Bayes partition seems very reasonable, having found three contiguous group, the bounds of the credible ball appear quite diverse and “distant” from the actual optimal solution (particularly the horizontal one). We feel that these bounds do not necessarily convey much information regarding which partitions are inside the ball and which are not. Of course, this is a situation where the model is mis-speciﬁed, as is the usual case in practice, and this may partially explain the results in Figure 1. Alternatively, one may instead consider an approach based on the idea of high poste- rior density regions, and simply list all of the partitions that have posterior probability above a certain threshold. This method would include all of the relevant partitions re- gardless of their distance from the Bayes partition (in the sense of the distance induced by the Hasse diagram), providing a good representation of what the possible optimal alternatives look like. 1 Choice of loss function and computational eﬃciency Additionally, R&F empirically assess the eﬀect of the various loss functions on simu- lated data and in particular we refer the reader to Figure 3 of Rastelli and Friel (2017). The main take home message is that the VI loss typically achieved the best results in terms of the number of estimated groups, while the other loss functions, including Binders loss, the normalised VI loss and the normalised information distance often ex- hibit unreasonable behaviour and overestimation of the number of groups. However, our ﬁndings also reveal that the VI loss tends to be biased towards an overestimation of the number of groups. This seems not to be case with the results presented in W&G. We wonder if the approximation the authors introduce may have an impact on the estima- 596 Invited comment on Article by Wade and Ghahramani tion of the number of groups? All things considered, we deem the research question of ﬁnding an optimal loss function and associated computational strategy still very open. 2 Quantifying the uncertainty around the estimated Bayes partition From a computational perspective, both methods are straightfor- ward to implement once the posterior values and the distances to the Bayes solution are available for all of the partitions sampled. 3 Wider application of mixture models W&G propose applications of their methodology to Gaussian mixture models. We would like to conclude our discussion by remarking that the method they proposed may be applied in more general mixture modelling contexts, thereby widening their applicability. N. Friel and R. Rastelli 597 N. Friel and R. Rastelli 597 Figure 1: VI loss optimal clustering and credible ball bounds for the simulated uniform data proposed. Figure 1: VI loss optimal clustering and credible ball bounds for the simulated uniform data proposed. For instance, recent research has focused much on mixture models for network data (Daudin et al., 2008). Computationally eﬃcient Markov chain Monte Carlo sampling strategies for network clustering models have been proposed by McDaid et al. (2013) and Wyse and Friel (2012). In R&F, we propose several applications of the decision theoretic framework to Gaussian mixture models, but also to stochastic block models for networks, and to latent block models for bipartite networks. Furthermore, mixed- membership models (Airoldi et al., 2008) extend the basic clustering structures to partial memberships, where nodes of the network may distribute their aﬃliation among the groups. Extending the decision theoretic framework proposed by W&G to these contexts would be a great next step forward. For instance, recent research has focused much on mixture models for network data (Daudin et al., 2008). Computationally eﬃcient Markov chain Monte Carlo sampling strategies for network clustering models have been proposed by McDaid et al. (2013) and Wyse and Friel (2012). In R&F, we propose several applications of the decision theoretic framework to Gaussian mixture models, but also to stochastic block models for networks, and to latent block models for bipartite networks. Furthermore, mixed- membership models (Airoldi et al., 2008) extend the basic clustering structures to partial memberships, where nodes of the network may distribute their aﬃliation among the groups. Extending the decision theoretic framework proposed by W&G to these contexts would be a great next step forward. Invited comment on Article by Wade and Ghahramani 598 Contributed comment on Article by Wade and Ghahramani William Weimin Yoo∗ Abstract. I begin my discussion by giving an overview of the main results. Then I proceed to touch upon issues about whether the credible ball constructed can be interpreted as a conﬁdence ball, suggestions on reducing computational costs, and posterior consistency or contraction rates. Keywords: Bayesian clustering, variation of information, Binder’s loss, credible ball, overﬁtted mixtures, Bayes Lepski. The authors should be congratulated for producing such an interesting and important work. In the present paper, Wade and Ghahramani (2017) investigated the issues of point estimation and uncertainty quantiﬁcation for Bayesian clustering analysis. Here, the data density is modelled as a countably inﬁnite mixture and latent variables attach- ing to each observation are introduced to represent cluster membership. A common prior for the mixing distribution is the Dirichlet process, and they used this as the default prior in the simulations and real data analysis. They derived point estimators through decision theory by considering two diﬀerent clustering losses/metrics, i.e., Binder’s loss (N-invariant version) and variation of information (VI). They endowed the space of partitions with a lattice by including partial order and the covering relation, and this enables them to compare properties of these two metrics and deﬁne a consistent no- tion of closeness between partitions. This latter notion was further used to develop a method to construct credible ball over partitions using the aforementioned metrics. The optimization problem needed to ﬁnd the point estimate (for VI) is computational de- manding and the search space is very high-dimensional. To scale up computations, the authors proposed a greedy search algorithm. I start my discussion by asking the question whether the credible balls constructed can be interpreted as conﬁdence balls in the frequentist sense? Speciﬁcally, do the 95% credible balls based on Binder’s loss or VI with their vertical and horizontal bounds, have also approximate 95% frequentist coverage probability (contains the true clustering 95% of the time)? For ﬁnite dimensional parameters, we have the Bernstein-von Mises theorem to ensure this equivalence; however in the nonparametric setting as in this paper, this equivalence breaks down and it is in general not true that Bayesian credible ball is also a frequentist conﬁdence ball. References Airoldi, E. M., Blei, D. M., Fienberg, S. E., and Xing, E. P. (2008). “Mixed membership stochastic blockmodels.” Journal of Machine Learning Research, 9(Sep): 1981–2014. 597 Daudin, J. J., Picard, F., and Robin, S. (2008). “A mixture model for random graphs.” Statistics and Computing, 18(2): 173–183. MR2390817. doi: https://doi.org/ 10.1007/s11222-007-9046-7. 597 Lau, J. W. and Green, P. J. (2007). “Bayesian model-based clustering procedures.” Journal of Computational and Graphical Statistics, 16(3): 526–558. MR2351079. doi: https://doi.org/10.1198/106186007X238855. 594 McDaid, A. F., Murphy, T. B., Friel, N., and Hurley, N. J. (2013). “Improved Bayesian inference for the stochastic block model with application to large networks.” Compu- tational Statistics & Data Analysis, 60: 12–31. MR3007016. doi: https://doi.org/ 10.1016/j.csda.2012.10.021. 597 Rastelli, R. and Friel, N. (2017). “Optimal Bayesian estimators for latent variable cluster models.” Statistics and Computing. doi: https://doi.org/10.1007/s11222-017- 9786-y. 594, 595 Wyse, J. and Friel, N. (2012). “Block clustering with collapsed latent block mod- els.” Statistics and Computing, 22(2): 415–428. MR2865026. doi: https://doi.org/ 10.1007/s11222-011-9233-4. 597 599 W. W. Yoo W. W. Yoo ∗Mathematical Institute, Leiden University, The Netherlands, yooweimin0203@gmail.com Contributed comment on Article by Wade and Ghahramani It would be very interesting if we can give some theoretical guarantees on coverage for the VI credible ball, or maybe compare the extent of its uncertainty in a simulation with a conﬁdence ball over partitions constructed based on non-Bayesian methods (if there are any). In complex models, it is straightforward to use Markov Chain Monte Carlo (MCMC) samples to construct credible balls, as compared to frequentist methods which rely on complicated asymptotic normality analysis or bootstrap, and hence such comparisons and coverage guarantees 600 Contributed comment on Article by Wade and Ghahramani Contributed comment on Article by Wade and Ghahramani 600 Contributed comment on Article by Wade and Ghahramani will provide good incentives for statisticians (particularly non-Bayesians) to use the methods proposed in this paper to do clustering in their own work. will provide good incentives for statisticians (particularly non-Bayesians) to use the methods proposed in this paper to do clustering in their own work. A recurring theme that came up when designing algorithms in the paper is the abil- ity to scale to massive datasets and to speed up computations. Instead of using inﬁnite mixtures which entails searching over the entire partition space, one can use overﬁtted mixtures as investigated in Rousseau and Mengersen (2011), where one intentionally overﬁt the model by choosing a larger but ﬁnite number of components than necessary and use some sparsity-inducing priors to zero out the unnecessary components. Alterna- tively, by observing in Table 2 that the number of clusters for the VI credible ball stays constant for the diﬀerent sample sizes considered, its robust property suggests that we could ﬁrst try to estimate the correct number of clusters, through MAP (Maximum a posteriori) or the recently proposed Bayes Lepski’s method (Yoo and van der Vaart (2018)), and only explore the part of the partition space corresponding to this estimated number of clusters. I totally agree with the authors that we need results on posterior consistency and contraction rates, in order to fully resolve the ambiguity caused by the positive results of the present paper and the negative results of Miller and Harrison (2014). Question of interests include characterizing the rate at which the number of clusters estimated under the VI posterior approaches the true number, and whether this rate is optimal. Contributed comment on Article by Wade and Ghahramani In addition, it would also be interesting to study miss-classiﬁcation errors and how they grow with sample size or depend on the chosen loss function. A deeper understanding of these issues will help statisticians choose the right priors and design algorithms to control these errors. The present paper proposes a very promising method to obtain point estimate and uncertainty quantiﬁcation for Bayesian cluster analysis, which is a great improvement in terms of interpretability over posterior similarity matrices commonly considered in the literature. I envision that the lattice-based framework introduced here can be extended to other settings as well, e.g., multiple membership clusters, and I am certain this work will further spur research in these areas. Contributed comment on Article by Wade and Ghahramani∗ Sylvia Fr¨uhwirth-Schnatter†, Bettina Gr¨un‡, and Gertraud Malsiner-Walli§ We would like to congratulate the authors on addressing the diﬃcult problem of summa- rizing the posterior distribution of partitions. The high dimensionality of the partition space and the low support for any single partition make this problem very challeng- ing. To our knowledge, their approach is the ﬁrst one, which tries to systematically estimate bounds for conﬁdence regions of the partition posterior. In this comment, we would like to emphasize that their proposed procedure is not only useful for Bayesian nonparametric mixture models, but can also prove very useful for ﬁnite mixture models. 1 Sparse ﬁnite mixture models As opposed to common belief which is also expressed in the introduction of the paper, the number of clusters in the data is not necessarily ﬁxed a priori for ﬁnite mixtures and can be estimated from the data, in particular when using sparse ﬁnite mixture models (Malsiner-Walli et al., 2016, 2017; Fr¨uhwirth-Schnatter and Malsiner-Walli, 2018). The authors’ procedure for summarizing uncertainty in the posterior of the partitions is particularly appealing for such sparse ﬁnite mixture models where the number of data clusters is random. Data clusters in this context refer to clusters of data points induced by the partitions. Sparse ﬁnite mixture models are based on an overﬁtting ﬁnite mixture distribution with the number K of components exceeding the number of data clusters, in combination with a very small value for the hyperparameter e0 of the Dirichlet prior on the mixture weights. Such a setting encourages partitions with less clusters than there are components, implying that during Markov chain Monte Carlo (MCMC) sampling data points are only assigned to a subset of the components and some components are left empty. Sampling from the posterior of the partitions for sparse ﬁnite mixture models is straightforward as standard MCMC sampling schemes developed for ﬁnite mixtures can be used. ∗This research was funded by the Austrian Science Fund (FWF): P28740. †Institute for Statistics and Mathematics, Wirtschaftsuniversit¨at Wien, Austria, sylvia.fruehwirth-schnatter@wu.ac.at ‡Department of Applied Statistics, Johannes Kepler University Linz, Austria, Bettina.Gruen@jku.at §Institute for Statistics and Mathematics, Wirtschaftsuniversit¨at Wien, Austria, gertraud.malsiner-walli@wu.ac.at References Miller, J. and Harrison, M. (2014). “Inconsistency of Pitman-Yor process mixtures for the number of components.” Journal of Machine Learning Research, 15: 3333–3370. MR3277163. 600 Rousseau, J. and Mengersen, K. (2011). “Asymptotic behaviour of the posterior dis- tribution in overﬁtted mixture models.” Journal of the Royal Statistical Society. Se- ries B (Statistical Methodology), 73(5): 689–710. MR2867454. doi: https://doi.org/ 10.1111/j.1467-9868.2011.00781.x. 600 Wade, S. and Ghahramani, Z. (2017). “Bayesian Cluster Analysis: Point Estimation and Credible Balls.” Bayesian Analysis, 1–29. Advance publication. 599 Yoo, W. W. and van der Vaart, A. W. (2018). “The Bayes Lepski’s Method and Credi- ble Bands through Volume of Tubular Neighborhoods.” arXiv:1711.06926 [math.ST]. 601 S. Fr¨uhwirth-Schnatter, B. Gr¨un, and G. Malsiner-Walli ‡Department of Applied Statistics, Johannes Kepler University Linz, Austria, Bettina.Gruen@jku.at §Institute for Statistics and Mathematics, Wirtschaftsuniversit¨at Wien, Austria, gertraud.malsiner-walli@wu.ac.at ∗This research was funded by the Austrian Science Fund (FWF): P28740. †Institute for Statistics and Mathematics, Wirtschaftsuniversit¨at Wien, Austria, sylvia.fruehwirth-schnatter@wu.ac.at 2 Illustration using example 1 of the paper To illustrate how the proposed inference tools can be used for post-processing the par- titions sampled from a sparse ﬁnite mixture model, we ﬁt a sparse ﬁnite mixture model Contributed comment on Article by Wade and Ghahramani 602 Figure 1: Left: trace plot of the number of data clusters K+ including burn-in. Middle and right: scatter plots of the data indicating the ﬁnal partition c⋆using four diﬀerent colors except for the data points marked with black stars which belong to diﬀerent clusters in the upper and lower bounds using α = 0.50 (middle) and α = 0.05 (right). Figure 1: Left: trace plot of the number of data clusters K+ including burn-in. Middle and right: scatter plots of the data indicating the ﬁnal partition c⋆using four diﬀerent colors except for the data points marked with black stars which belong to diﬀerent clusters in the upper and lower bounds using α = 0.50 (middle) and α = 0.05 (right). Partition k∗ N NI Cluster sizes ARI Upper bound α = 0.05 4 17 65, 50, 45, 40 0.78 Upper bound α = 0.50 4 16 60, 59, 42, 39 0.80 c⋆ 4 6 56, 54, 47, 43 0.92 Lower bound α = 0.50 5 13 55, 51, 48, 45, 1 0.84 Lower bound α = 0.05 5 30 50, 48, 47, 41, 14 0.71 Table 1: The ﬁnal partition c⋆and boundary partitions of the credible balls. For each partition, the number of clusters k∗ N, the number of misclassiﬁed data points NI, the cluster sizes (in decreasing order) and the adjusted Rand indices (ARI) are reported. Table 1: The ﬁnal partition c⋆and boundary partitions of the credible balls. For each partition, the number of clusters k∗ N, the number of misclassiﬁed data points NI, the cluster sizes (in decreasing order) and the adjusted Rand indices (ARI) are reported. Table 1: The ﬁnal partition c⋆and boundary partitions of the credible balls. For each partition, the number of clusters k∗ N, the number of misclassiﬁed data points NI, the cluster sizes (in decreasing order) and the adjusted Rand indices (ARI) are reported. with K = 10 in combination with e0 = 0.01 to the data set of their example 1. The priors on the component means and variances follow Fr¨uhwirth-Schnatter (2006). 2 Illustration using example 1 of the paper Gibbs sampling with data augmentation is initialized by assigning data points to all available components and 10,000 posterior samples are drawn after a burn-in of 1,000 iterations. For each partition drawn during MCMC sampling, the number of data clusters K+ induced by the non-empty components is determined and the corresponding trace plot is shown in Figure 1. During burn-in, most components become empty and the sampler iterates between partitions with 4 and 5 data clusters. These partitions (excluding the burn-in) are summarized based on the VI loss using the R package mcclust.exe. Table 1 shows characteristics of the estimated ﬁnal partition c⋆and reports the upper and lower bounds for α = 0.50 and α = 0.05. If α decreases, the cluster size of the largest cluster increases for the upper bounds and the cluster size of the smallest data cluster increases for the lower bounds. This behavior might be expected from the order relation discussed in Property 5 of the paper. The adjusted Rand index (ARI) measures the correspondence between the true clustering and each of the partitions. The partition which minimizes the expected VI loss has the highest ARI. The ARI decreases with decreasing α for both, the lower as well as the upper bounds. Figure 1 illustrates the ﬁnal partition in a scatter plot of the data using diﬀerent colors for the data clusters identiﬁed. In addition, data points which are not consistently allocated to 603 S. Fr¨uhwirth-Schnatter, B. Gr¨un, and G. Malsiner-Walli the same clusters in the ﬁnal and boundary partitions using either α = 0.50 or α = 0.05 are marked with black stars. These data points could be regarded as “uncertain” in their cluster membership. 3 Final remarks The close relationship between Bayesian cluster analysis based on ﬁnite and inﬁnite mixtures is again demonstrated by indicating how inference tools developed for the inﬁnite case also prove useful in the ﬁnite case. For ﬁnite mixtures, the proposed infer- ence tools have a number of advantages for post-processing samples from the partition posterior: (1) no model selection needs to be performed, (2) no identiﬁed model where label-switching is resolved is required and (3) uncertainty estimates for the partition posterior are readily available based on the credible balls. We hope that future work on Bayesian cluster analysis follows our example and develops and demonstrates inference tools not only for the inﬁnite mixture case, but also considers the ﬁnite case. MSC 2010 subject classiﬁcations: Primary 62G05, 62F15, 60G57, 60G09. MSC 2010 subject classiﬁcations: Primary 62G05, 62F15, 60G57, 60G09. Keywords: Bayesian nonparametrics, Dirichlet process prior, model selection, variation of information criterion. Contributed comment on Article by Wade and Ghahramani Roberto Casarin∗and Stefano Tonellato† Abstract. This article discusses the Wade and Ghahramani’s (2017) paper on a new estimator for clustering structures based on the variation of information (VI) metric. The present discussion focuses on the estimation of concentration parameter of the Dirichlet process. In estimating the clustering structure, the concentration parameter is integrated out and the marginal posterior distribution of the random partition is used to evaluate the posterior loss. Here we propose to use the optimal VI for model selection. SC 2010 subject classiﬁcations: Primary 62G05, 62F15, 60G57, 60G09. ∗Department of Economics, University Ca’ Foscari of Venice, Cannaregio 873, 30121, Venezia, Italy, r.casarin@unive.it †Department of Economics, University Ca’ Foscari of Venice, Cannaregio 873, 30121, Venezia, Italy, stone@unive.it †Department of Economics, University Ca’ Foscari of Venice, Cannaregio 873, 30121, Venezia, Italy, stone@unive.it References Fr¨uhwirth-Schnatter, S. (2006). Finite Mixture and Markov Switching Models. Springer Series in Statistics. New York: Springer. MR2265601. 602 Fr¨uhwirth-Schnatter, S. and Malsiner-Walli, G. (2018). “From Here to Inﬁnity – Sparse Finite versus Dirichlet Process Mixtures in Model-Based Clustering.” URL http://arxiv.org/pdf/1706.07194.pdf. 601 Malsiner-Walli, G., Fr¨uhwirth-Schnatter, S., and Gr¨un, B. (2016). “Model-Based Clus- tering based on Sparse Finite Gaussian Mixtures.” Statistics and Computing, 26(1): 303–324. MR3439375. doi: https://doi.org/10.1007/s11222-014-9500-2. 601 Malsiner-Walli, G., Fr¨uhwirth-Schnatter, S., and Gr¨un, B. (2017). “Identifying Mix- tures of Mixtures Using Bayesian Estimation.” Journal of Computational and Graphical Statistics, 26(2): 285–295. MR3640186. doi: https://doi.org/10.1080/ 10618600.2016.1200472. 601 604 Contributed comment on Article by Wade and Ghahramani ∗Department of Economics, University Ca’ Foscari of Venice, Cannaregio 873, 30121, Venezia, Italy, r.casarin@unive.it References Dahl, D. B. (2006). “Model-based clustering for expression data via a Dirichlet process mixture model.” In Do, K.-A., P. Muller, P., and Vannucci, M. (eds.), Bayesian In- ference for Gene Expression and Proteomics, 201–218. Cambridge University Press. MR2706330. 604 Meilˇa, M. (2007). “Comparing clusterings—an information based distance.” Journal of Multivariate Analysis, 98(5): 873–895. MR2325412. doi: https://doi.org/10.1016/ j.jmva.2006.11.013. 604 Neal, R.M. (2000). “Markov Chain sampling methods for Dirichlet process mixture models.” Journal of Computational and Graphical Statistics, 9:249–265. 604 Wade, S. and Ghahramani, Z. (2017). “Bayesian Cluster Analysis: Point Estimation and Credible Balls.” Bayesian Analysis. 604 2 Conclusion In their paper, the authors sketch a number of possible extensions. We would suggest as further research line also the combination of posterior clustering probabilities obtained from the diﬀerent models. It is clear that this is an exciting and stimulating work. We are therefore very pleased to be able to propose the vote of thanks to the authors for their work. 1 Introduction The authors are to be congratulated on their excellent intuition, which has culminated in the development of a new Bayesian point estimator for clustering structure which can ﬁnd applications in many Bayesian nonparametrics studies. Their Bayesian ap- proach to clustering estimation is inspired by the paper of Meilˇa (2007). The proposed model provides an alternative to the Dahl (2006) method widely used in the Bayesian nonparametric literature. In the application to the galaxy data we assume the same DP mixture model as in equation (5) of the paper and the same prior setting μ0 = ¯x, c = 1/2, a = 2 and b = s2. Instead of estimating α we assume the concentration parameter α takes values in the ﬁnite set A = {α1, . . . , αn} and for each element αj of this regular grid we evaluate the partition posterior distribution p(c\|y1:N, αj) given by p(c\|y1:N, αj) ∝ Γ(αj) Γ(αj + N)αkN j kN j=1 Γ(nnj)m(yj), where m(yj) is the marginal likelihood of the observations in the j-th partition. For each value of α ∈A we run the Gibbs sampler as in the algorithm 8 of Neal (2000) and ﬁnd the optimal value of the VI criterion at α (VICα) as where m(yj) is the marginal likelihood of the observations in the j-th partition. For each value of α ∈A we run the Gibbs sampler as in the algorithm 8 of Neal (2000) and ﬁnd the optimal value of the VI criterion at α (VICα) as VICα = min ˆc L(c, ˆc)p(c\|y1:N, α)dc. 605 R. Casarin and S. Tonellato c VICα VI α = 0.5 α = 1 α = 1.5 1/2 0.61 0.72 0.83 0.74 1/10 0.23 0.32 0.41 0.29 Table 1: Optimal VICα for α ∈{0.5, 1, 1.5} and diﬀerent values of c. The VICα obtained are given in Table 1 for α ∈{0.5, 1, 1.5}. The optimal VI value with α integrated out, using a Ga(1, 1) prior, is reported in the last column. The minimumVICα is attained for α = 0.5 and it is always smaller than the integrated VI. The result suggests the VICα is favoring smaller values of the concentration parameter. This research used the multiprocessor cluster system at University Ca’ Foscari of Venice (SCSCF). Acknowledgments This research used the multiprocessor cluster system at University Ca’ Foscari of Venice (SCSCF). 606 Contributed comment on Article by Wade and Ghahramani Contributed comment on Article by Wade and Ghahramani Eduard Belitser∗and Nurzhan Nurushev†‡ We would like to congratulate the authors on an impressive paper that solves an open problem on uncertainty quantiﬁcation in cluster analysis from a practical point of view. Let us ﬁrst summarize some key ides of the present paper. Due to the huge dimension of the partition space in Bayesian nonparametric cluster analysis, one of the main prob- lems in Bayesian cluster analysis is how to appropriately summarize the posterior. This problem in the present paper is addressed by providing tools to obtain a point estimate of clustering based on the posterior and describe uncertainty around this estimate via the 95% credible ball. The computation of the point estimate c∗is based on the greedy search algorithm and Hasse diagram, which can be used for both the variation of infor- mation and Binder’s loss. In simulation study the authors construct a credible ball of a given credible level 1 −α, α ∈[0, 1], deﬁned as Bϵ∗(c∗) = {c : d(c∗, c) ≤ϵ∗}, where ϵ∗ is the smallest ϵ such that P(Bϵ(c∗)\|D) ≥1 −α. However, the practical results of the present paper leads to the question of whether the credible ball Bϵ∗(c∗) is “optimal”. Namely, does the credible ball Bϵ∗(c∗) lead to conﬁdence? The point estimate c∗can be very close to, or far away from the true clustering c, without us knowing the actual distance. One would like to have some sort of quantiﬁcation for the reliability of the estimator c∗, which can be seen as the problem of constructing conﬁdence balls for c∗. Conﬁdence balls are a type of set estimates intended to quantify the accuracy of the estimator. The size of the ball quantiﬁes the level of uncertainty of the estimator c∗. Let us specify the optimality framework for conﬁdence balls. Assume that any par- tition c belongs to some functional class Cβ indexed by unknown structural parameter β ∈B (e.g., number of clusters). Denote the probability measure of the data D by Pc = P(N) c , the minimax concentration rate over Cβ by rN,β. The goal is to construct such a conﬁdence ball BCϵ∗(c∗) = {c : d(c∗, c) ≤Cϵ∗} that for any α1, α2 ∈(0, 1] there exist C, c > 0 such that sup c∈C0 Pc c /∈BCϵ∗(c∗) ≤α1, sup c∈C1 Pc ϵ∗≥crN,β ≤α2, (1) (1) for some C0, C1 ⊆Cβ and all β ∈B. ‡Research funded by the Netherlands Organisation for Scientiﬁc Research NWO. Department of Mathematics, VU Amsterdam, e.n.belitser@vu.nl †Korteweg-de Vries Institute for Mathematics, University of Amsterdam, n.nurushev@uva.nl ‡Research funded by the Netherlands Organisation for Scientiﬁc Research NWO. ∗Department of Mathematics, VU Amsterdam, e.n.belitser@vu.nl †Korteweg-de Vries Institute for Mathematics, University of Amsterdam, n.nurushev@uva.nl ‡Research funded by the Netherlands Organisation for Scientiﬁc Research NWO. p , , †Korteweg-de Vries Institute for Mathematics, University of Amsterdam, n.nurushev@uv ‡ ∗Department of Mathematics, VU Amsterdam, e.n.belitser@vu.nl †Korteweg-de Vries Institute for Mathematics, University of Amsterdam, n.nurushev@uva.nl ‡Research funded by the Netherlands Organisation for Scientiﬁc Research NWO p , , †Korteweg-de Vries Institute for Mathematics, University of Amsterdam, n.nurushev@uva.nl ‡Research funded by the Netherlands Organisation for Scientiﬁc Research NWO ∗Department of Mathematics, VU Amsterdam, e.n.belitser@vu.nl † d f h f A d h @ l ∗Department of Mathematics, VU Amsterdam, e.n.belitser@vu.nl Contributed comment on Article by Wade and Ghahramani The minimax concentration rate rN,β is a bench- mark for the eﬀective radius of the conﬁdence ball BCϵ∗(c∗). The ﬁrst expression in (1) is called coverage relation and the second size relation. It is desirable to have the coverage and size relations to be hold for the biggest C0, C1. For example, if we insist on overall uniformity C0 = C1 = Cβ, then the results in Li (1989) and Cai and Low 607 E. Belitser and N. Nurushev (2004) (more reﬁned versions are in Baraud (2004) say basically that the radius of con- ﬁdence ball cannot be of a bigger order than N 1/4. Many good conﬁdence balls cannot be optimal in this sense (called “honest” in some papers), e.g., in sparse normal means model. Instead, it makes sense to sacriﬁce in the set C0 = Cβ\C′, by removing a prefer- ably small portion of “deceptive parameters” C′ from C so that the optimal minimax rate becomes attainable in the size relation with interesting (preferably “massive”) sets C1 (see Belitser and Nurushev (2017) for details). To the best of our knowledge, it is not known whether it is possible to construct a conﬁdence ball simultaneously with a good coverage and optimal size adaptively to some scale Cβ in the studied model. This is a challenging problem and of great importance to our understanding of uncovering partitions c. Admittedly, the above optimality framework is formulated from the frequentists perspective whereas the authors pursue a purely Bayesian approach. An advantage is that such a framework allows to compare diﬀerent procedures. We wonder whether the authors could come up with a general idea of how to compare diﬀerent Bayesian procedures from the purely Bayesian perspective. We understand that the authors were mainly focused on the practical results related to the construction of credible balls, but we hope this comment will inspire the authors and other people to work on this interesting problem in the future. We would like to ﬁnish with the question to the authors whether the point estimates studied in the present paper can be used for the Hamming loss function. If it was possible then it might be interesting to create a new simulation study in the stochastic block model and compare the radius ϵ∗of credible ball BCϵ∗(c∗) (based on the Hamming loss function) with the minimax rate for community detection problem studied in Zhang and Zhou (2016). Contributed comment on Article by Wade and Ghahramani Then one could answer the question whether the radius ϵ∗of credible ball BCϵ∗(c∗) is optimal in this sense or not. ∗Dept. of Operation Management, University of Amsterdam, Netherlands, a.mohammadi@uva.nl; url: http://www.uva.nl/proﬁle/a.mohammadi References Baraud, Y. (2004). “Conﬁdence balls in Gaussian regression.” Annals of Statistics, 32(2): 528–551. MR2060168. doi: https://doi.org/10.1214/009053604000000085. 607 Belitser, E. and Nurushev, N. (2017). “Needles and straw in a haystack: robust conﬁ- dence for possibly sparse sequences.” https://arxiv.org/abs/1511.01803. 607 Cai, T. T. and Low, M. G. (2004). “An adaptation theory for nonparamet- ric conﬁdence intervals.” Annals of Statistics, 32(5): 1805–1840. MR2102494. doi: https://doi.org/10.1214/009053604000000049. 606 Li, K.-C. (1989). “Honest Conﬁdence Regions for Nonparametric Regression.” Annals of Statistics, 17(3): 1001–1008. MR1015135. doi: https://doi.org/10.1214/aos/ 1176347253. 606 Zhang, A. Y. and Zhou, H. H. (2016). “Minimax rates of community detection in stochastic block models.” Annals of Statistics, 44(5): 2252–2280. MR3546450. doi: https://doi.org/10.1214/15-AOS1428. 607 608 Contributed comment on Article by Wade and Ghahramani Contributed comment on Article by Wade and Ghahramani Reza Mohammadi∗ I would ﬁrst like to congratulate Dr Wade and Professor Ghahramani for their excellent exposition of the Bayesian nonparametric cluster analysis by developing point estimates and credible sets to summarize the posterior of the clustering structure. Their method is based on a greedy search algorithm to locate the optimal partition based on Hasse diagram, which can be used for both the variation of information and the Binder’s loss. Here, I would like to contribute to the discussion by suggesting a comparison with the Bayesian parametric methods of ﬁnite mixture distributions based on the trans- dimensional Markov chain Monte Carlo (MCMC) algorithms. Comparison with ﬁnite mixture distributions This paper illustrates the high potential of the Bayesian nonparametric cluster analysis. Here, I focus on the Bayesian parametric approaches for ﬁnite mixture distributions based on trans-dimensional MCMC sampling algorithms. In the Bayesian analysis of ﬁnite mixture distributions with an unknown number of components, the main problem is sampling from the posterior distributions. Since the number of components is unknown, it requires advanced search algorithms which can potentially move in the model space. Transdimensional search algorithms explore the model space when the model does not have a ﬁxed dimension; common ones are the reversible jump MCMC by Green (1995) and birth-death MCMC Stephens (2000). In the context of ﬁnite mixture distributions, these methods have been used by Green (1995); Stephens (2000); Mohammadi et al. (2013) and in the case of graphical models Mohammadi and Wit (2015). To compare the performance of the Bayesian nonparametric method, proposed in the paper, with the Bayesian parametric method based on ﬁnite mixture distributions, we apply the ﬁnite mixture of normal distribution for galaxy data with the same sce- nario as in subsection 6.2 of the paper. We run the birth-death MCMC algorithm pro- posed by Stephens (2000) using the R-package bmixture (Mohammadi, 2018), function bmixnorm(), with 10K samples with 10K burnin. Figure 1(b) shows that the data came from 5 or 6 clusters. The sum of the estimated posterior probability of the number of clusters for the case of 4 up to 8 clusters is 0.95, which can be considered as the 95 percent conﬁdent interval for the number of clusters; The results are comparable with the results in Table 5 of this paper. 609 609 R. Mohammadi R. Mohammadi R. Mohammadi 609 Figure 1: (a) Histogram of galaxy data and estimated density based on the ﬁnite mixture of Normal distribution and (b) the estimation of the posterior distribution of the number of clusters. Figure 1: (a) Histogram of galaxy data and estimated density based on the ﬁnite mixture of Normal distribution and (b) the estimation of the posterior distribution of the number of clusters. It would be quite useful if the authors could comment on the comparison of their approach to ﬁnite mixture distributions as Bayesian parametric approaches and the pos- sibility of replacing greedy search algorithm in their Bayesian nonparametric framework with the trans-dimensional MCMC algorithms. Contributed comment on Article by Wade and Ghahramani Stephens, M. (2000). “Bayesian analysis of mixture models with an unknown number of components-an alternative to reversible jump methods.” Annals of Statistics, 40–74. MR1762903. doi: https://doi.org/10.1214/aos/1016120364. 608 Mohammadi, R. (2018). bmixture: Bayesian Estimation for Finite Mixture of Distribu- tions. R package version 0.6. 608 ∗Univ. Grenoble Alpes, Inria, CNRS, LJK, 38000 Grenoble, France, julyan.arbel@inria.fr; michal.lewandowski@inria.fr †DISMEQ, University of Milano Bicocca, 20126 Milano MI, Italy, riccardo.corradin@unimib.it 1Package available at https://github.com/rcorradin/BNPmix, can be installed via devtools. 2Code of the simulation study available at https://github.com/rcorradin/WGdiscussion. Contributed comment on Article by Wade and Ghahramani Julyan Arbel∗, Riccardo Corradin†, and Michal Lewandowski∗ Abstract. We propose a simulation study to emphasise the diﬀerence between Variation of Information and Binder’s loss functions in terms of number of clusters estimated by means of (1) the use of the Markov chain Monte Carlo (MCMC) output only and (2) a “greedy” method. Wade and Ghahramani’s paper is a very neat contribution to Bayesian cluster analysis in at least two respects: (i) by formalizing cluster credible coverage via Hasse diagrams, and (ii) by recasting the problem in a decision theory framework, with tangible improvements brought by the Variation of Information (VI) loss function (Meil˘a, 2007) over Binder’s (Binder, 1978; Dahl, 2006). We propose a simulation study implementing two algorithms provided by Wade and Ghahramani’s package mcclust.ext for ﬁnding the argument minimizing the posterior expected loss: (1) the draw algorithm, which restricts the minimization problem to the MCMC output, and (2) the greedy algorithm, which is more reliable as it also scans the neighbouring clusters of the MCMC output, but with a larger computational cost. While increasing the sample size, we point out the radically diﬀerent behavior of the number of clusters estimated under VI and Binder, especially with the greedy algorithm. Our simulation study is based on the same data generation as in the ﬁrst example of Section 6.1 in Wade and Ghahramani (2017): a mixture of four Gaussian distributions equally weighted with means (±2, ±2) and identity covariance matrix. We estimated the model using a marginal approach provided by BNPmix1 R package. We synthesised the output with mcclust.ext package.2 The Dirichlet process mixture model was estimated with mass parameter ﬁxed to 1, and by specifying an independent base measure on locations and scales, with a 0-vector prior mean for the location component and an identity matrix prior mean for the scale component (25 000 iterations with 5 000 burn- in period). We considered four diﬀerent sample sizes n = {20, 40, 100, 300}. The results are shown in Figure 1. With the draw algorithm, the cluster estimates under both losses are quite close in terms of number of clusters. In contrast, the greedy algorithm leads to cluster estimates obtained via Binder’s loss function with excessive size, while that obtained via VI remains coherent with the number of components of the model (four). References Green, P. J. (1995). “Reversible jump Markov chain Monte Carlo computation and Bayesian model determination.” Biometrika, 82(4): 711–732. MR1380810. doi: https://doi.org/10.1093/biomet/82.4.711. 608 Mohammadi, A., Salehi-Rad, M., and Wit, E. C. (2013). “Using mixture of Gamma distributions for Bayesian analysis in an M/G/1 queue with optional second service.” Computational Statistics, 28: 683–700. MR3064474. doi: https://doi.org/10.1007/ s00180-012-0323-3. 608 Mohammadi, A. and Wit, E. C. (2015). “Bayesian Structure Learning in Sparse Gaussian Graphical Models.” Bayesian Analysis, 10(1): 109–138. MR3420899. doi: https://doi.org/10.1214/14-BA889. 608 Contributed comment on Article by Wade and Ghahramani 610 Mohammadi, R. (2018). bmixture: Bayesian Estimation for Finite Mixture of Distribu- tions. R package version 0.6. 608 Mohammadi, R. (2018). bmixture: Bayesian Estimation for Finite Mixture of Distribu- tions. R package version 0.6. 608 Stephens, M. (2000). “Bayesian analysis of mixture models with an unknown number of components-an alternative to reversible jump methods.” Annals of Statistics, 40–74. MR1762903. doi: https://doi.org/10.1214/aos/1016120364. 608 611 J. Arbel, R. Corradin, and M. Lewandowski Contributed comment on Article by Wade and Ghahramani Similarly to the authors’ ﬁnding, ours’ indicates that Binder’s loss function exhibits an undesirable property of overestimating the number of clusters (Miller and Harrison, Contributed comment on Article by Wade and Ghahramani 612 Figure 1: Size of the cluster estimate under VI (yellow line) and Binder (green light). Left: draw algorithm. Right: greedy algorithm. Figure 1: Size of the cluster estimate under VI (yellow line) and Binder (green light). Left: draw algorithm. Right: greedy algorithm. 2013, 2014). Variation of Information tends to lessen this problem. As alluded to by the authors, a theoretical study of the asymptotic behavior of the VI estimator would be very timely. Especially in light of the recent contribution by Rajkowski (2016) about the asymptotic behavior of the cluster estimator under the 0 −1 loss (MAP estimator). Contributed comment on Article by Wade and Ghahramani Bernardo Nipoti∗and Weining Shen† We vividly congratulate the authors for providing very interesting insight on how to summarize posterior belief on the space of partitions, and characterize its uncertainty. In this comment we focus on the latter point and we build upon the authors’ deﬁnition and characterization of credible balls of partitions. Speciﬁcally, we would like to suggest that an alternative formulation of such quantities might be considered and might provide additional insight, useful to the diﬃcult task of summarizing a posterior belief on the space of partitions. We articulate our comment in two points. 1. Where to center? Commonly adopted measures of posterior uncertainty, such as highest posterior density or quantile-based posterior intervals, are deﬁned independently of any posterior estimator, which might vary based on the choice of the loss function. Similarly, while the authors deﬁne a credible ball of partitions Bϵ∗(c∗) as centered around a point estimate c∗, we would like to observe that such deﬁnition could be tweaked so to make it independent of any point estimator. This could be achieved by centering the credible ball around the true partition c0 and by averaging across all possible true partitions. That is, following the same idea used in Section 2.2 of Wade and Ghahramani (2017), we can deﬁne a credible ball Bϵ∗of credible level 1 −α as Bϵ∗= {c : Ec0[d(c, c0) \| D] ≤ϵ∗} ≈ c : 1 M M m=1 d(c, cm) ≤ϵ∗ , where {cm}M m=1 is the set of partitions visited by the Markov chain Monte Carlo (MCMC) algorithm. An estimate of ϵ∗in this case is obtained from the MCMC output by choosing ϵ∗as the smallest ϵ ≥0 such that the average of pairwise distances satisﬁes 2 M(M−1) M n=1 M m>n 1(d(cn, cm) ≤ϵ) ≥1 −α. Compared with the authors’ credible ball deﬁnition, the alternative construction is computationally more expensive, but it does not rely on the correct estimation of a posterior point estimator. It would be inter- esting to investigate the theoretical properties (e.g. frequentist coverage) of these two diﬀerent deﬁnitions. where {cm}M m=1 is the set of partitions visited by the Markov chain Monte Carlo (MCMC) algorithm. An estimate of ϵ∗in this case is obtained from the MCMC output by choosing ϵ∗as the smallest ϵ ≥0 such that the average of pairwise distances satisﬁes 2 M(M−1) M n=1 M m>n 1(d(cn, cm) ≤ϵ) ≥1 −α. References Binder, D. A. (1978). Bayesian cluster analysis. Biometrika, 65(1):31–38. MR0501592. doi: https://doi.org/10.1093/biomet/65.1.31. 611 Dahl, D. B. (2006). Model-based clustering for expression data via a dirichlet process mixture model. Bayesian inference for gene expression and proteomics, pages 201– 218. MR2706330. 611 Meil˘a, M. (2007). Comparing clusterings—an information based distance. Journal of Multivariate Analysis, 98(5):873–895. MR2325412. doi: https://doi.org/10.1016/ j.jmva.2006.11.013. 611 Miller, J. W. and Harrison, M. T. (2013). A simple example of Dirichlet process mix- ture inconsistency for the number of components. In Advances in neural information processing systems, pages 199–206. 611 Miller, J. W. and Harrison, M. T. (2014). Inconsistency of Pitman-Yor process mix- tures for the number of components. The Journal of Machine Learning Research, 15(1):3333–3370. MR3277163. 611 Rajkowski, L. (2016). Analysis of MAP in CRP Normal-Normal model. arXiv preprint arXiv:1606.03275. 612 Wade, S. and Ghahramani, Z. (2017). Bayesian cluster analysis: Point estimation and credible balls. Bayesian Analysis. 611 613 d W. Shen Contributed comment on Article by Wade and Ghahramani B. Nipoti and W. Shen ∗School of Computer Science and Statistics, Trinity College Dublin, Ireland, nipotib@tcd.ie †Department of Statistics, University of Califorinia, Irvine, United States, weinings@uci.edu School of Computer Science and Statistics, Trinity College Dublin, Ireland, nipotib@tcd.ie †Department of Statistics, University of Califorinia, Irvine, United States, weinings@uci.edu ∗School of Computer Science and Statistics, Trinity College Dublin, Ireland, nipotib@tcd.ie † Contributed comment on Article by Wade and Ghahramani Compared with the authors’ credible ball deﬁnition, the alternative construction is computationally more expensive, but it does not rely on the correct estimation of a posterior point estimator. It would be inter- esting to investigate the theoretical properties (e.g. frequentist coverage) of these two diﬀerent deﬁnitions. 2. To center or not to center? Alternatively, credible balls could be deﬁned without resorting to the idea of centering them at any given partition, being it a point estimate c∗or the true c0. A simple approach consists in sorting the MCMC samples via a certain measure, such as entropy. More speciﬁcally, if we call H(c) the entropy of a partition c = (C1, . . . , CkN ), deﬁned as H(c) = log(N) −1/N kN j=1 nj log(nj), where nj = \|Cj\| is the number of data points in cluster j, then a entropy-based credible ball B(H) ϵ∗ can 614 Contributed comment on Article by Wade and Ghahramani Contributed comment on Article by Wade and Ghahramani be deﬁned as be deﬁned as B(H) ϵ∗ = {c : HL ≤H(c) ≤HU}, where HL := H(c(l) H ) and HU := H(c(u) H ), and c(l) H and c(u) H are empirical quantiles of the sorted set of observed partitions, such that M −1 M m=1 1{HL ≤H(c(m)) ≤ HU} ≥1 −α. As a by-product a point estimate c∗ H can be obtained by considering the posterior median partition. Following Example 2 in Section 6.1 of the main paper, we considered the simulated data from a mixture of four bivariate normal distributions with unequal covariance matrices, used the posterior samples output from the R package “mcclust.ext”, and obtained a quantile-based 95% credible ball B(H) ϵ∗. The plots for the posterior median c∗ H, upper bound c(u) H and lower bound c(l) H are presented in Figure 1. Compared with Figure 9 in the main paper, the clustering results look quite similar, with a lower bound c(l) H showing a moderately smaller number of clusters than the VI lower vertical bound. For both methods, the eﬀect of outliers and over-estimation of the number of clusters in the lower bound is apparent. This is expected since Dirichlet mixture priors are known to provide consistent density estimation (Shen et al., 2013), while overestimating the number of clusters (Miller and Harrison, 2013). Contributed comment on Article by Wade and Ghahramani It would be interesting to investigate and compare entropy-based and VI vertical bounds, using other prior distributions known to have better inferential properties in terms of clustering, such as normalized random measures (Barrios et al., 2013). Figure 1: Posterior median c∗ H (4 clusters, left), upper bound c(u) H (5 clusters, middle) and lower bound c(l) H (12 clusters, right) of a quantile-based 95% credible ball B(H) ϵ∗. Figure 1: Posterior median c∗ H (4 clusters, left), upper bound c(u) H (5 clusters, middle) and lower bound c(l) H (12 clusters, right) of a quantile-based 95% credible ball B(H) ϵ∗. Miller, J. W. and Harrison, M. T. (2013). “A simple example of Dirichlet process mixture inconsistency for the number of components.” In Advances in neural information processing systems, 199–206. 614 Barrios, E., Lijoi, A., Nieto-Barajas, L. E., and Pr¨unster, I. (2013). “Modeling with nor- malized random measure mixture models.” Statistical Science, 313–334. MR3135535. doi: https://doi.org/10.1214/13-STS416. 614 Shen, W., Tokdar, S. T., and Ghosal, S. (2013). “Adaptive Bayesian multivariate den- sity estimation with Dirichlet mixtures.” Biometrika, 100(3): 623–640. MR3094441. doi: https://doi.org/10.1093/biomet/ast015. 614 Wade, S. and Ghahramani, Z. (2017). “Bayesian cluster analysis: Point estimation and credible balls.” Bayesian Analysis. 613 Contributed comment on Article by Wade and Ghahramani Federico Castelletti∗and Stefano Peluso† We congratulate the authors for the insightful paper. We ﬁnd particularly interesting the adoption of the two ball sizes as concise and easily interpretable measure of uncer- tainty associated to point estimates on non-standard and large supports. In the present discussion we want to highlight the applicability of the method proposed by the authors beyond the space of random partitions, to Directed Acyclic Graphs (DAGs) and to their Markov equivalence classes, namely to Essential Graphs (EGs). In Consonni and La Rocca (2012) an Objective Bayes model selection procedure is proposed for DAGs, later extended in Consonni et al. (2017) to covariate-adjusted model selection, and to EGs in Castelletti et al. (2018). Similarly to the space of random partitions, exhaustive enumeration of DAGs and EGs is not feasible (Madigan et al., 1996), since the number of DAGs and EGs grows super-exponentially in the number of nodes (Gillispie and Perlman, 2002). Therefore the posterior probability associated to DAGs and EGs for non-trivial dimensions is only available up to a normalizing constant, requiring appropriate Markov Chain Monte Carlo (MCMC) procedures to perform posterior inference. The need of summarizing MCMC visits on the space of DAGs and EGs through a point estimate and an uncertainty measure raises, among others, diﬃculties similar to those in the paper under discussion: the 0-1 loss function adopted in Consonni et al. (2017) resulted in the choice of the modal DAG as point estimate, whilst the point estimator of the EG in Castelletti et al. (2018) is based on the inclusion of edges with marginal posterior inclusion probability higher than 50%. Also, in both cases no mea- sure of uncertainty on the whole structure has been provided, but only MCMC-based uncertainty measures on graph features (as on the number of chain components and v-structures), or rough uncertainty measures based on variation of the edge inclusion probability threshold. Potentially, both estimates can be improved and the uncertainty around the esti- mated graph can be measured following the lines suggested in Wade et al. (2017). First, the 0-1 loss function can be replaced by a graph-equivalent of the ﬁrst metric proposed in the current paper: the error in the classiﬁcation to the correct cluster becomes the error in the inclusion of an edge. ∗Universit`a Cattolica del Sacro Cuore, Milan, Italy, federico.castelletti@unicatt.it †Universit`a Cattolica del Sacro Cuore, Milan, Italy, stefano.peluso@unicatt.it References Barrios, E., Lijoi, A., Nieto-Barajas, L. E., and Pr¨unster, I. (2013). “Modeling with nor- malized random measure mixture models.” Statistical Science, 313–334. MR3135535. doi: https://doi.org/10.1214/13-STS416. 614 Miller, J. W. and Harrison, M. T. (2013). “A simple example of Dirichlet process mixture inconsistency for the number of components.” In Advances in neural information processing systems, 199–206. 614 615 B. Nipoti and W. Shen B. Nipoti and W. Shen 616 Contributed comment on Article by Wade and Ghahramani F. Castelletti and S. Peluso Second, the information-based metric from Meil˘a (2007) is strictly related to the Hamming distance between the binary representations of the clusterings. It could be interesting to ﬁnd in the graphical context a metric related to the Structural Hamming Distance (the number of edge insertions, deletions or ﬂips needed to transform one graph into another) among (classes of) graphs. Finally, metrics alternative to the 0-1 loss introduce an additional layer of complexity for DAGs and EGs: the estimated graph might lie outside of its support, and therefore some projection onto the proper space could be necessary (Castelletti et al., 2018). Third, the adoption of the VI metric as a measure of distance between clusterings can be also interesting when model selection of DAG models can be performed in an interventional setting (Hauser and B¨uhlmann, 2015). In general, the size of a Markov equivalence class of DAGs is used as a measure of complexity of causal learning (He et al., 2013). Assuming faithfulness of the observed data to some true DAG model, interventions on variables from randomized experiments can be used to improve the identiﬁability of such a model and then reduce the size of the estimated equivalence class. The problem of optimal choice of intervention targets is carried out in He and Geng (2008) from a design of experiments perspective. Speciﬁcally, each Markov equivalence class can be partitioned into DAG sub-classes, each with common edges orientations on the intervened nodes. A diﬀerent intervention target induces a diﬀerent partition of DAGs, and the VI metric among DAG partitions suggests maximum-entropy-based criteria for optimized choices of targets. Contributed comment on Article by Wade and Ghahramani A ball at level (1 −α) would be represented by the set of visited EGs whose distance with respect to the point-estimated graph ˆG is less than the threshold ϵ∗. Then, vertical and horizontal bounds, as deﬁned in the paper, can be also introduced. Coherently, one can for instance consider the graph with the smallest (largest) number of edges that are most distant from ˆG as the vertical lower (upper) bound. Similarly for the horizontal bound. 617 References Castelletti, F., Consonni, G., Della Vedova, M., and Peluso, S. (2018). “Learning Markov Equivalence Classes of Directed Acyclic Graphs: an Objective Bayes Approach.” Bayesian Analysis. 616, 617 Consonni, G. and La Rocca, L. (2012). “Objective Bayes Factors for Gaussian Di- rected Acyclic Graphical Models.” Scandinavian Journal of Statistics, 39: 743–756. MR3000846. doi: https://doi.org/10.1111/j.1467-9469.2011.00785.x. 616 Consonni, G., La Rocca, L., and Peluso, S. (2017). “Objective Bayes Covariate-Adjusted Sparse Graphical Model Selection.” Scandinavian Journal of Statistics, 44(3): 741– 764. MR3687971. doi: https://doi.org/10.1111/sjos.12273. 616 Gillispie, S. B. and Perlman, M. D. (2002). “The size distribution for Markov equivalence classes of acyclic digraph models.” Artiﬁcial Intelligence, 141: 137–155. MR1935281. doi: https://doi.org/10.1016/S0004-3702(02)00264-3. 616 Hauser, A. and B¨uhlmann, P. (2015). “Jointly interventional and observational data: estimation of interventional Markov equivalence classes of directed acyclic graphs.” Journal of the Royal Statistical Society. Series B (Methodology), 77: 291–318. MR3299409. doi: https://doi.org/10.1111/rssb.12071. 617 He, Y. and Geng, Z. (2008). “Active learning of causal networks with intervention experiments and optimal designs.” Journal of Machine Learning Research, 9: 2523– 2547. MR2460892. 617 618 Wade, S., Ghahramani, Z., et al. (2017). “Bayesian Cluster Analysis: Point Estimation and Credible Balls.” Bayesian Analysis. 616 Contributed comment on Article by Wade and Ghahramani Bent Natvig∗ In Wade et al. (2018), considering Bayesian cluster analysis, it is stated in the intro- duction that a more elegant solution, than the one presented in some papers, is based on decision theory. Furthermore, it is stated that the question to answer then becomes what is an appropriate loss function over clusterings? This leads my mind to a paper, Natvig and Tvete (2007), on Bayesian hierarchical space-time modeling of earthquake data. Our aim was to get some insight into where and when large earthquakes occur, or otherwise stated, we were interested in the clustering of large earthquakes. We would like to judge our model more speciﬁcally, based upon its ability to avoid two errors by not predicting the large earthquakes and signal false alarms. In that paper we took a coarse point of view considering grid cells of 50×50 km and time periods of 4 months, which seems suitable for predictions. We discussed diﬀerent alternatives of a Bayesian hierarchical space-time model inspired by the paper Wikle et al. (2001). For each time period the observations were the magnitudes of the largest observed earthquake within each grid cell. As data we applied parts of an earthquake catalogue provided by The Northern California Earthquake Center where we limited ourselves to the area 32–37 degrees N and 115–120 degrees W, and for the time period January 1981 through December 1999 containing the Landers and Hector Mine earth- quakes, respectively measuring 7.3 and 7.1 on the Richter scale. Based on space-time model alternatives one step earthquake predictions for the time periods containing these two events for all grid cells are arrived at. We constructed a specially designed loss function, weighted over the X spatial cells, that penalizes both these errors. The weight for a given spatial cell, x, for a given prediction period,t, is dependent both upon the magnitude of the observed earthquake, M(x,t), and the distance between the observed earthquake and the predicted value ˆ M(x, t, j) for sample j. To take signalized earthquakes in neighbouring cells into account we let, suppressing the time notation t, ˆ M(x, j) be a spatially weighted average of the predictions, {M ∗(x, j)}X x=1, where each of the two, three, or four predictions at a neighbouring cell has half the weight of the one at x and the rest given weight zero. Contributed comment on Article by Wade and Ghahramani Contributed comment on Article by Wade and Ghahramani He, Y., Jia, J., and Yu, B. (2013). “Reversible MCMC on Markov equivalence classes of sparse directed acyclic graphs.” The Annals of Statistics, 41: 1742–1779. MR3127848. doi: https://doi.org/10.1214/13-AOS1125. 617 Madigan, D., Andersson, S., Perlman, M., and Volinsky, C. (1996). “Bayesian Model Averaging and Model Selection for Markov Equivalence Classes of Acyclic Di- graphs.” Communications in Statistics: Theory and Methods, 2493–2519. MR1439312. doi: https://doi.org/10.1214/aos/1031833662. 616 Meil˘a, M. (2007). “Comparing clusterings – an information based distance.” Journal of Multivariate Analysis, 98: 873–895. MR2325412. doi: https://doi.org/10.1016/ j.jmva.2006.11.013. 617 Wade, S., Ghahramani, Z., et al. (2017). “Bayesian Cluster Analysis: Point Estimation and Credible Balls.” Bayesian Analysis. 616 619 B. Natvig B. Natvig ∗Department of Mathematics, University of Oslo, bent@math.uio.no Contributed comment on Article by Wade and Ghahramani The specially designed loss function, for a given predicted period, is given by: L2(M, ˆ M) = 1 X X x=1 1 N N j=1 w( ˆ M(x, j), M(x))( ˆ M(x, j) −M(x))2 1/2 , 620 Contributed comment on Article by Wade and Ghahramani Contributed comment on Article by Wade and Ghahramani w( ˆ M(x, j), M(x)) = ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ 0.2 if M(x) ∈[0, 1) and \| ˆ M(x, j) −M(x)\| ≥3, 0.24 if M(x) ∈[1, 2) and \| ˆ M(x, j) −M(x)\| ≥2.3, 0.28 if M(x) ∈[2, 3) and \| ˆ M(x, j) −M(x)\| ≥1.6, 0.36 if M(x) ∈[3, 4) and \| ˆ M(x, j) −M(x)\| ≥1.0, 0.48 if M(x) ∈[4, 5) and \| ˆ M(x, j) −M(x)\| ≥0.8, 0.64 if M(x) ∈[5, 6) and \| ˆ M(x, j) −M(x)\| ≥0.7, 0.80 if M(x) ∈[6, 7) and \| ˆ M(x, j) −M(x)\| ≥0.6, 1.00 if M(x) ≥7 and \| ˆ M(x, j) −M(x)\| ≥0.5, 0 otherwise. The weights w( ˆ M(x, j), M(x))) are subjectively designed to punish a lack of ability to predict large earthquakes. This is reﬂected in the equation above by giving a weight equal to 0.2 when M(x) ∈[0, 1) increasing to 1, in nondecreasing steps, when M(x) ≥7. The weights are also designed to punish a lack of ability to predict the large earthquakes more accurately than the small ones. Hence, to have a loss when M(x) ∈[0, 1) the absolute diﬀerence between the predicted and the maximal earthquake must be greater or equal to 3. This threshold decreases, in nonincreasing steps, to 0.5 when M(x) ≥ 7. The basic idea behind the equation above should be reﬂected in any modiﬁcation of it. Let ˆ M p be the spatial average of the pth percentiles in the simulated prediction distribution {M ∗(x, j)}N j=1. We then also compute another specially designed loss func- tion: L3(M, ˆ M p) = 1 X X x=1 w( ˆ M p(x), M(x))( ˆ M p(x) −M(x))2 1/2 . L3 is diﬀerent from L2 in that we consider the pth percentile rather than all the sampled predictions. Contributed comment on Article by Wade and Ghahramani This is a sensible loss function to consider in the search of predictions giving the smallest overall loss. It can be shown that the losses L2 and L3 are quite small for the less extreme period, somewhat larger for the Hector Mine period and again larger for the Landers period. This is as expected due to the earthquake activity in the various periods. Due to the fact that all weights are less than or equal to 1, the L2 values are much smaller than the L1 values given in Natvig and Tvete (2007). Obviously, one will obtain large losses when predicting periods where there is a high earthquake activity, and small losses in less extreme periods. W feel that the estimated loss values are not alarmingly high. Natvig, B. and Tvete, I. F. (2007). “Bayesian Hierarchical Space-time Modeling of Earthquake Data.” Methodology and Computing in Applied Probability, 9: 89–114. MR2364983. doi: https://doi.org/10.1007/s11009-006-9008-0. 619, 620 References Natvig, B. and Tvete, I. F. (2007). “Bayesian Hierarchical Space-time Modeling of Earthquake Data.” Methodology and Computing in Applied Probability, 9: 89–114. MR2364983. doi: https://doi.org/10.1007/s11009-006-9008-0. 619, 620 621 B. Natvig Wikle, C., Milliﬀ, R., Nychka, D., and Berliner, L. (2001). “Spatio-temporal hierar- chial Bayesian modeling: tropical occean surface winds.” Journal of the American Statistical Association, 96: 382–397. MR1939342. doi: https://doi.org/10.1198/ 016214501753168109. 619 Sara Wade∗and Zoubin Ghahramani† Sara Wade∗and Zoubin Ghahramani† We sincerely thank the discussants for the interesting and insightful comments. Our paper investigates approaches to summarize the posterior distribution over the space of partitions. The massive dimension of the partition space and its categorical nature com- bined with the low posterior probability of any single partition make this a challenging problem. As highlighted by the discussants, there are a number of relevant extensions and open problems. Properties. As noted by Monni, Meil˘a (2007) details various properties of the VI and other cluster comparison measures, including an equivalent version of Binder’s loss. The aim of Section 3 was to highlight the asymmetry of Binder’s loss compared to VI, accumulating in Properties 6 and 7. This was not discussed in Meil˘a (2007), and for completeness, we include a through review of some relevant properties described by Meil˘a (2007), such as vertical and horizontal collinearity. Credible balls. To characterize uncertainty in the point estimate, we deﬁned credible balls around the point estimate based on a chosen metric, e.g. VI or Binder’s loss. We agree with Monni that the posterior similarity matrix is an important tool for assessing uncertainty but emphasize that the credible ball provides additional information that enriches our understanding of uncertainty around the point estimate. We proposed to summarize these balls based on the vertical and horizontal bounds, and as noted by Monni (and in the paper), these bounds may consist of more than one partition. In theory, this could be a large number, but in practice, we restrict the bounds to partitions with positive posterior probability, which in our experience provides at most a handful of partitions for each bound. This restriction is for computational purposes, but also serves as a reﬁnement of the bound. We encourage further research into the construction of credible balls of partitions. Friel and Rastelli propose an interesting idea based on highest posterior density (HPD) regions. In practice, the MCMC may not visit any partition more than once; thus, the suggested HPD region, which considers partitions with posterior probability over a threshold, would contain all or no partitions in this setting. Moreover, relevant summaries of the HPD regions may be needed in practice. Nipoti and Shen outline some alternative ideas for deﬁning credible balls. ∗University of Warwick, s.wade@warwick.ac.uk †University of Cambridge, zoubin@eng.cam.ac.uk B. Natvig B. Natvig Wade, S. and Ghahramani, Z. (2018). “Bayesian Cluster Analysis: Point Estimation and Credible Balls.” Bayesian Analysis, 13. 619 Wade, S. and Ghahramani, Z. (2018). “Bayesian Cluster Analysis: Point Estimation and Credible Balls.” Bayesian Analysis, 13. 619 Wikle, C., Milliﬀ, R., Nychka, D., and Berliner, L. (2001). “Spatio-temporal hierar- chial Bayesian modeling: tropical occean surface winds.” Journal of the American Statistical Association, 96: 382–397. MR1939342. doi: https://doi.org/10.1198/ 016214501753168109. 619 622 Rejoinder Sara Wade∗and Zoubin Ghahramani† Sara Wade∗and Zoubin Ghahramani† First, they consider centering the ball at the true partition, not the point estimate; this however comes at a computational cost, as it requires evaluating pairwise distances between all MCMC samples. Second, they propose a construction of entropy-based credible balls. These ideas are certainly interesting and more work is needed to investigate and compare the credible balls in theory and simulations. We suspect that the entropy-based credible balls could be quite diﬀerent from VI credible balls. For example, consider the case with N = 4 depicted in Figure 1. We note that the Hasse diagram stretched by the 623 S. Wade and Z. Ghahramani S. Wade and Z. Ghahramani 623 Figure 1: Example of the VI ball around c = ({1, 2}, {3, 4}), with rainbow color indi- cating increasing distance from c. The smallest non-trivial credible ball contains all the red clusterings, the next smallest contains the red and orange clusterings, and so on. Figure 1: Example of the VI ball around c = ({1, 2}, {3, 4}), with rainbow color indi- cating increasing distance from c. The smallest non-trivial credible ball contains all the red clusterings, the next smallest contains the red and orange clusterings, and so on. Figure 1: Example of the VI ball around c = ({1, 2}, {3, 4}), with rainbow color indi- cating increasing distance from c. The smallest non-trivial credible ball contains all the red clusterings, the next smallest contains the red and orange clusterings, and so on. entropy results in the same relative positions of the partitions, with the values on the y-axis simply rescaled to be between 0 and log(4). Suppose that the VI point estimate of c = ({1, 2}, {3, 4}) has posterior probability of 0.5, then the 50% VI credible ball would contain only that partition. On the other hand, the 50% entropy credible ball would contain the black and blue partitions, that is, all partitions with two clusters of equal size; the blue partitions, however, have the greatest VI distance from c = ({1, 2}, {3, 4}), and the VI credible ball would have to be extended to 100% credibility to contain these partitions. Another interesting direction is discussed by Paulon, Trippa, and M¨uller, where it may be relevant to understand uncertainty of a subset of the partition. Computations. Sara Wade∗and Zoubin Ghahramani† Friel and Rastelli in Rastelli and Friel (2017) expand upon our work and develop an alternative technique for optimizing the posterior expected loss, which is linear in N but increasingly expensive in the number of MCMC samples. Also, their approach does not require the approximation of the posterior expected loss through Jensen’s inequality. As they highlight, more work is needed to understand the impact of this approximation, particularly, as their studies suggest that optimizing the lower bound to the posterior expected VI may actually perform better at recovering the true number of clusters. Arbel, Corradin, and Lewandowski point out the improvements in optimization of the greedy search algorithm over restricting to the MCMC samples (at a computational cost). Related to this, we note that the simple proposed approach to locate the credible bounds restricts to the MCMC samples. Similar improvements could be expected here by searching outside of the MCMC samples; this would however require a novel algorithm to locate the bounds as well as an appropriate reﬁnement of the bounds (because, as highlighted by Monni, without restriction to the MCMC samples the bounds could contain a large number of partitions). One simple approach could be to restrict the search to partitions that are vertically aligned with the point estimate; this, however, also has its limitations. Asymptotics. A study of the asymptotic properties of the proposed estimators and credible balls is an important and timely research direction, especially in light of the negative results of Miller and Harrison (2014) on posterior inconsistency for the number 624 Rejoinder Figure 2: Credible probability against estimated coverage probability from 50 replicated experiments of Example 1 with N = 200; the solid represents the optimal setting when the 1 −α credible ball achieves 1 −α coverage. 624 Rejoinder Rejoinder Figure 2: Credible probability against estimated coverage probability from 50 replicated experiments of Example 1 with N = 200; the solid represents the optimal setting when the 1 −α credible ball achieves 1 −α coverage. of clusters and the positive results of the experiments in Table 2, as well as the recent results for the MAP estimator Rajkowski (2016), pointed out by Arbel, Corradin, and Lewandowski. Belitser and Nurushev and Yoo raise an interesting question on optimality of the proposed (1 −α) ∗100% credible balls; speciﬁcally, do they also have (1−α)∗100% frequentist coverage probability? Sara Wade∗and Zoubin Ghahramani† We carried out a small experiment by simulating 50 datasets with the same data-generating mechanism as described in Example 1. We ﬁrst note that kN = 4 for the VI estimate in 90% of the experiments (although only one starting point was considered for the greedy search, and this may be improve with multiple restarts). Figure 2 depicts the estimated coverage probabilities as a function of the credible probability. This suggests that, in this setting, VI credible balls can be interpreted as conﬁdence balls, although they are not optimal and have quite large coverage probabilities. The reasons for this could be a combination of the nonparametric model and the data-generating mechanism, as well as the credible ball deﬁnition, and it would be interesting to extend this simulation study for parametric models, large sample sizes, and other data-generating mechanisms. Overall, a deeper understanding of the frequentist coverage of the credible balls and other asymptotic properties is needed. Belitser and Nurushev: to the best of our knowledge, we do not know of results on minimax rates in the community detection problem for Binder’s loss or VI, but note that Binder’s loss can be viewed as the Hamming distance between the binary representation of clusterings. And, we would be intrigued to understand if the work of Zhang and Zhou (2016) could help to shed light on the coverage of the proposed credible balls for the community detection problem. Applications. Our paper focused on Bayesian nonparametric mixture models and exper- iments considered Gaussian mixtures, but as highlighted by the discussants, the pro- posed tools have applications beyond this. Friel and Rastelli and Belitser and Nu- rushev discuss stochastic block models for networks. Fr¨uhwirth–Schnatter, Gr¨un, and Malsiner–Walli and Yoo discuss sparse ﬁnite mixture models and Fr¨uhwirth– 625 S. Wade and Z. Ghahramani Schnatter, Gr¨un, and Malsiner–Walli extend Example 1 by using the proposed summary tools for sparse ﬁnite mixtures. Mohammadi considers ﬁnite mixture mod- els and trans-dimensional MCMC, which allows exploration of the space of partitions. The proposed summary tools are relevant in this case as well, and instead of “replacing” the trans-dimensional MCMC, the greedy search algorithm would be used to ﬁnd a point estimate of the partition based on those explored in the trans-dimensional MCMC. In addition to considering marginal properties, such as the posterior on the number of clusters, we have developed tools to further understand the posterior on the clustering structure. Sara Wade∗and Zoubin Ghahramani† Castelletti and Peluso describe an interesting application and extension to DAGs and EGs. Loss functions. We have proposed and motivated the use of VI as a general loss func- tions for partitions, and developed tools for summarizing MCMC samples of partitions. However, in some applications one may be interested in more problem-speciﬁc loss func- tions. An interesting example for clinical trials is provided by Paulon, Trippa, and M¨uller, where the loss function is a combination of the squared error loss for the true and estimated parameters of each patient and a penalization term that encourages clus- ter parameters to be distinct. Another interesting example is provided by Natvig and Tvete, where a problem-speciﬁc loss function is designed for earthquake data. Model selection. Casarin and Tonellato propose an interesting use of the posterior expected VI as a model selection tool to identify hyperparameters. Although this re- quires ﬁtting several models, it can result in a lower posterior expected VI, compared with the hierarchical model with hyperpriors on the hyperparameters. Casarin and Tonellato have outlined a promising research direction, and we would, for example, be interested in the use of the posterior expected VI as a model selection tool to com- pare nonparametric priors on partitions, beyond the the Dirichlet process (Lijoi and Pr¨unster (2011), Barrios et al. (2013)) or the sparse ﬁnite mixture models investigated by Fr¨uhwirth–Schnatter, Gr¨un, and Malsiner–Walli. References Barrios, E., Lijoi, A., Nieto-Barajas, L., and Pr¨unster, I. (2013). “Modeling with nor- malized random measure mixture models.” Statistical Science, 313–334. MR3135535. doi: https://doi.org/10.1214/13-STS416. 625 Lijoi, A. and Pr¨unster, I. (2011). “Models beyond the Dirichlet process.” In Hjort, N., Holmes, C., M¨uller, P., and Walker, S. (eds.), Bayesian Nonparametrics, 80–136. Cambridge, UK: Cambridge University Press. MR2730661. 625 Meil˘a, M. (2007). “Comparing clusterings – an information based distance.” Journal of Multivariate Analysis, 98: 873–895. MR2325412. doi: https://doi.org/10.1016/ j.jmva.2006.11.013. 622 Miller, J. and Harrison, M. (2014). “Inconsistency of Pitman-Yor process mixtures for the number of components.” Journal of Machine Learning Research, 15: 3333–3370. MR3277163. 623 626 Rejoinder Rejoinder Rajkowski, L. (2016). “Analysis of MAP in CRP normal-normal model.” ArXiv preprint arXiv:1606.03275. 624 Rastelli, R. and Friel, N. (2017). “Modeling with normalized random measure mix- ture models.” Statistics and Computing. doi: https://doi.org/10.1007/s11222- 017-9786-y. 623 Zhang, A. and Zhou, H. (2016). “Minimax rates of community detection in stochastic block models.” Annals of Statistics, 44: 2252–2280. MR3546450. doi: https://doi.org/10.1214/15-AOS1428. 624
https://openalex.org/W3116168092	https://bearworks.missouristate.edu/cgi/viewcontent.cgi?article=2408&context=articles-chhs	English	null	Simple, Reliable Isolation, Purification and Cultivation of Murine Skeletal Muscle Microvascular Endothelial Cells	Journal of biomedical science and engineering	2,020	cc-by	4,896	Follow this and additional works at: https://bearworks.missouristate.edu/articles-chhs Follow this and additional works at: https://bearworks.missouristate.edu/articles-chhs Simple, Reliable Isolation, Purification and Cultivation of Murine Simple, Reliable Isolation, Purification and Cultivation of Murine Skeletal Muscle Microvascular Endothelial Cells Skeletal Muscle Microvascular Endothelial Cells Jianjie Wang Missouri State University BearWorks BearWorks College of Health and Human Services 12-31-2020 Simple, Reliable Isolation, Purification and Cultivation of Murine Simple, Reliable Isolation, Purification and Cultivation of Murine Skeletal Muscle Microvascular Endothelial Cells Skeletal Muscle Microvascular Endothelial Cells Jianjie Wang Missouri State University Joseph Harvey Richard C. Garrad Missouri State University Virginia H. Huxley Laurie Erb See next page for additional authors BearWorks BearWorks Recommended Citation Recommended Citation Wang, Jianjie, Joseph Harvey, Richard Garrad, Virginia Huxley, Laurie Erb, and Gary Weisman. "Simple, Reliable Isolation, Purification and Cultivation of Murine Skeletal Muscle Microvascular Endothelial Cells." Journal of Biomedical Science and Engineering 13, no. 12 (2020): 290. This article or document was made available through BearWorks, the institutional repository of Missouri State University. The work contained in it may be protected by copyright and require permission of the copyright holder for reuse or redistribution. For more information, please contact bearworks@missouristate.edu. Authors Authors Jianjie Wang, Joseph Harvey, Richard C. Garrad, Virginia H. Huxley, Laurie Erb, and Gary Weisman Wang, Joseph Harvey, Richard C. Garrad, Virginia H. Huxley, Laurie Erb, and Gary Weisman Authors Authors Jianjie Wang, Joseph Harvey, Richard C. Garrad, Virginia H. Huxley, Laurie Erb, and Gary Weisman ABSTRACT https://doi.org/10.4236/jbise.2020.1312026 290 J. Biomedical Science and Eng ABSTRACT Objectives: Microvascular dysfunction in skeletal muscle is involved in metabolic and cular diseases. Microvascular endothelial cells (MEC) are poorly characterized in the gression of associated diseases in part due to lack of availability of MEC from various an models. The objective was to provide a fast, simple, and efficient method to isolate mu MEC derived from skeletal muscle. Methods: Dissected abdominal skeletal muscles C57BL/6J mice at 8 - 12 weeks of age were enzymatically dissociated. MEC were iso using a modified two-step Dynabeads™-based purification method. With a combinatio Dynabeads™ - Griffonia simplicifolia lectin-I and Dynabeads™ - monoclonal anti against CD31/PECAM-1, MEC were isolated and purified twice followed by cultiva Results: Isolated and purified cells were viable and cultured. MEC were characterize using immunofluorescence to identify CD31/PECAM-1, an EC marker, and two spe functional assays, which include a capillary-like tube formation and the uptak Dil-Ac-LDL. The purity of isolated cell populations from skeletal muscle microve which was assessed by flow cytometry, was 88.02% ± 2.99% (n = 6). Conclusions: This thod is simple, fast, and highly reproducible for isolating MEC from murine skeletal mu The method will enable us to obtain primary cultured MEC from various genetic or dise murine models, contributing to insightful knowledge of diseases associated with the function of microvessels. 1. INTRODUCTION Microvascular endothelial cells (MEC) constitute the inner lining of tiny vessels and play piv Objectives: Microvascular dysfunction in skeletal muscle is involved in metabolic and vas- cular diseases. Microvascular endothelial cells (MEC) are poorly characterized in the pro- gression of associated diseases in part due to lack of availability of MEC from various animal models. The objective was to provide a fast, simple, and efficient method to isolate murine MEC derived from skeletal muscle. Methods: Dissected abdominal skeletal muscles from C57BL/6J mice at 8 - 12 weeks of age were enzymatically dissociated. MEC were isolated using a modified two-step Dynabeads™-based purification method. With a combination of Dynabeads™ - Griffonia simplicifolia lectin-I and Dynabeads™ - monoclonal antibody against CD31/PECAM-1, MEC were isolated and purified twice followed by cultivation. Results: Isolated and purified cells were viable and cultured. MEC were characterized by using immunofluorescence to identify CD31/PECAM-1, an EC marker, and two specific functional assays, which include a capillary-like tube formation and the uptake of Dil-Ac-LDL. Authors Authors J. Biomedical Science and Engineering, 2020, Vol. 13, (No. 12), pp: 290-299 https://www.scirp.org/journal/jbise Jianjie Wang1, Joseph Harvey2, Richard Garrad1, Virginia Huxley3, Laurie Erb4, Gary Weisman4 1Department of Biomedical Sciences, Missouri State University, Springfield, USA; 2Department of Cell Biology and Physiology, Washington University, St. Louis, USA; 3Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri, Columbia, USA; 4Department of Biochemistry, Life Sciences Center, University of Missouri, Columbia, USA Correspondence to: Jianjie Wang, Keywords: Microvascular Endothelial Cells, Isolation, Primary Cultured, Skeletal Muscle, Mouse Received: December 1, 2020 Accepted: December 28, 2020 Published: December 31, 2020 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. py g y g This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ This work is licensed under the Creative Commons Attribution International License (CC BY htt // ti /li /b /4 0/ p Open Access ABSTRACT The purity of isolated cell populations from skeletal muscle microvessels, which was assessed by flow cytometry, was 88.02% ± 2.99% (n = 6). Conclusions: This me- thod is simple, fast, and highly reproducible for isolating MEC from murine skeletal muscle. The method will enable us to obtain primary cultured MEC from various genetic or diseased murine models, contributing to insightful knowledge of diseases associated with the dys- function of microvessels. 1. INTRODUCTION 1. INTRODUCTION Microvascular endothelial cells (MEC) constitute the inner lining of tiny vessels and play pivotal roles https://doi.org/10.4236/jbise.2020.1312026 290 J. Biomedical Sc 290 in blood coagulation, angiogenesis, blood perfusion to tissue, and vascular exchange [1, 2]. Dysfunction of MEC is associated with a wide spectrum of disorders and diseases, such as coronary microvascular disease [3], tumor angiogenesis [4], sepsis, and inflammation [5, 6]. However, the role of microvessels, and partic- ularly that of MEC, in the progression of those diseases remains to be investigated. Given the heterogenic properties of vascular endothelial cells, physiological and pathophysiological studies using species- and origin-specific cultured MEC are critical for gaining novel insights into molecu- lar mechanisms underlying diseases associated with dysfunction of MEC. Although all vascular EC share some common properties, heterogeneity between microvascular and macrovascular EC have been well documented [7]. Furthermore, a large degree of functional heterogeneity was also found among MEC de- rived from different tissues [8]. It would be ideal for insightful molecular and functional studies to utilize MEC derived from the same tissue in disease models or genetically engineered mice, and with sex- and age-matched control groups. To that end, the challenge is to obtain large numbers of pure primary cul- tured MEC. The purpose of this study is to develop a simple and reliable method for isolation and culture of MEC derived from murine abdominal skeletal muscle. The skeletal muscle is the largest component of body mass in human and the microvessels of skeletal muscles are important constituents of the microcircula- tion. Microvascular dysfunction of skeletal muscle is involved in the pathogenesis of obesity, diabetes, hypertension, atherosclerosis, and peripheral vascular disease. Endothelial dysfunction in the skeletal muscles associated with these diseases is poorly characterized. Such studies are restricted, in part, by the availability of MEC derived from sex-, age-, and tissue-matched disease models vs. controls. Although a method using multicolor fluorescent-activated cell sorting (FACS) was reported to im- prove the purity of isolated cells derived from murine skeletal muscle [9], the sorting is expensive and complicated with technical concerns [10]. Our previous work has demonstrated successful isolation and culture of MEC from rat skeletal muscle. However, it is not simple to directly apply the method used in rats to another rodent species. 1. INTRODUCTION The specific differences in MEC properties among species, such as prolife- ration, metabolism, molecular signaling pathways, and responses to various environments, have been re- ported in stroke [11]. For isolation of mouse MEC, the primary challenges include, but are not limited to, low yield and purity. We proposed to isolate MEC derived from murine skeletal muscle via modification and refinement of our previous method [10, 12] to meet the following criteria: 1) fast and simple prepara- tion; 2) high yield; 3) high purity; and 4) high reproducibility. Dynabeads™-based two-step purification scheme was employed Griffonia simplicifolia lectin-I (GS-I), to recognize the carbohydrate moiety, which is abundantly expressed on the cell surface, followed by mo- noclonal antibody against CD31/PECAM-1, an endothelial marker. The isolated cells were characterized by using immunofluorescence and functional assays to validate properties of MEC. The purity of isolated cell populations from skeletal muscle microvessels was about 88%, assessed by using flow cytometry. 2.1. Experimental Animals and Reagents All animal care and experimental protocols on adult male (8 - 12 weeks of age) C57BL/6J mice were conducted in accordance with the “Care of Human Use of Laboratory Animals” under the supervision of Office of Research Administration at Missouri State University. Unless otherwise noted, reagents were purchased from Fisher Scientific (Hampton, NH). 2.4. Flow Cytometry The cell pellet was suspended in 2% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS) for 10 min at 37˚C for fixation. The cells were blocked using 5% (w/v) BSA for 1 hr at RT followed by incubation with mouse monoclonal antibody against CD31/PECAM-1 (ABD Serotec, catalogue MCA1334G, 1:30 dilution) in 0.5% BSA/DPBS overnight at 4˚C with constant rotation. After washing, the cells were labeled with secondary antibody, which was goat anti-mouse IgG conjugated with Alexa Fluor®-488 (1:333 dilution), for 30 min with constant rotation. The negative control was prepared in the same way as described above except the omission of primary antibody labeling. Subsequently, all the sam- ples were assessed by flow cytometry (AccuriTM 6C, BD Bioscience, Franklin Lake, NJ). The cells labeled with primary-secondary antibody-Alexa Fluro®-488 were detected and the data were analyzed by its soft- ware. 2.2. Isolation and Culture of Skeletal Muscle Microvascular Endothelial Cells The procedure of MEC isolation was modified from our previous work for rats [12]. The abdominal wall muscles were excised carefully using sterile procedures after the mice were anesthetized with 150 mg/kg ketamine and 7.5 mg/kg xylazine. The excised muscles pooled from four male C57BL/6J mice were cut into ~ 0.5 mm2 pieces, and then digested in an enzyme solution consisting of dispase (0.12 mg/ml; https://doi.org/10.4236/jbise.2020.1312026 291 J. Biomedical Science and Engineering https://doi.org/10.4236/jbise.2020.1312026 291 J. Biomedical Science and Engineering 291 J. Biomedical Science and Engineering 291 Worthington, Lakewood, NJ), trypsin (0.12 mg/ml; Invitrogen, Carlsbad, CA), collagenase type II (0.84 mg/ml; Sigma Aldrich, St. Louis, MO), and bovine serum albumin (BSA, 1.62 mg/ml) in DMEM-F12 (In- vitrogen, Carlsbad, CA) at 37˚C until the solution became cloudy (40 - 50 min). The cellular suspension was separated from tissue by filtration through sterile gauze and then a cell strainer (40 µm in pore size; BD Falcon, San Jose, CA). For the first step of the isolation, MEC were isolated using Dynabeads™ coated with GS-I (catalogue: L-2380, Sigma Aldrich, St. Louis, MO) for 10 - 15 min at room temperature and followed by collection of cells that bound to GS-I using a magnet. The cells isolated by the first step were cultured with DMEM-F12 containing 20% fetal bovine serum (FBS; vol/vol %), endothelial cell growth supplement (50 µg/ml), hepa- rin (5 U/ml), antimycotic-antibiotic solution (10 µl/ml), and L-glutamine (0.1 mg/ml). A second isolation was then performed using Dynabeads™ coated with monoclonal antibody against CD-31/PECAM-1 (BD Biosciences, San Jose, CA) for 30 min at 4˚C. The cells were collected using a mag- net and cultured with medium containing 10%, instead of 20% FBS. All subcultures (>2 passage) were grown in 10% of FBS medium. 2.3. Identification of Isolated Cells Immunofluorescence assay. Cells were seeded and grown on a Lab-Tek® II Chamber Slide™. MEC were fixed in 4% (w/v) paraformaldehyde at room temperature (RT) for 10 min and then permeabilized with 0.1% (v/v) Triton-X-100 for 5 min at RT. After blocking with 5% (w/v) IgG-free BSA (Jackson Im- munoresearch, West Grove, PA) for 30 min at RT, the cells were incubated overnight with specific mouse monoclonal antibody anti-CD31/PECAM-1 (1:50 dilution; Serotec Immunological Excellence) at 4˚C. The cells were incubated with goat anti-mouse IgG conjugated with Alexa Fluor®-488 or 568 (1:333 dilution, Molecular Probes), for 1 hr at RT followed by nucleic acid staining with 0.1% DAPI. Cultured HEK-293 cells were used as a negative control (Data not shown). The images were taken by Q-image camera coupled with BX60 Olympus microscope. p y p p To identify the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) by MEC, the fixed and permeabilized cells were incu- bated with Dil-Ac-LDL for (1:100; Invitrogen, Carlsbad, CA) 4 hr followed by acquisition of imaging. g y q g g Capillary-like tube formation. The cells isolated using the two-step scheme were seeded in 6-well plates coated with MatrigelTM (BD Biosciences, Franklin Lake, NJ) in accordance with the manufacturer’s instructions for EC functional characterization. Tube formation was observed and images were acquired by phase contrast microscopy (IX71 Olympus Microscope) 4 or 8 h after the cells were seeded in the plate. 3.3. Identification of Physiological Function of Microvascular Endothelial Cells Uptake of acetylated low density of lipoprotein (Ac-LDL). Uptake of Ac-LDL is one of the physiolog- ical properties of EC. This property of EC was demonstrated in murine MEC in Figures 3A-C using DiI-labeled Ac-LDL. Again, uptake of DiI-Ac-LDL by rat MEC and HUVEC is displayed in Figure 3D and Figure 3E, respectively, as positive controls. g y Capillary-like tube formation. A capillary-like tube formation assay was performed to assess the property of angiogenesis for MEC. The cells isolated and purified by using GS-I and antibody against CD31/PECAM-1 were able to form capillary-like tubes, shown in Figure 3F. 3.1. Morphology of Primary Endothelial Cells The morphological properties of cultured primary mouse MEC (Figure 1A) were compared with both primary cultured rat MEC (Figure 1B) as well as human umbilical vein endothelial cells (HUVEC, purchased from Lonza, Figure 1C). 3.2. Identification of Expression of CD31/PECAM-1 of Endothelial Cell Marker CD31/PECAM-1 has been widely used as an endothelial cell marker. The immunofluorescence assay with monoclonal antibody specifically against CD31/PECAM-1 showed positive staining of CD31/PECAM-1 in cells isolated by the two-step method (n = 3). The representative images of the expression of CD31/PECAM-1 in isolated cells are shown in Figures 2A-C. The positive control that was used for im- munofluorescence in the isolated cells, was demonstrated by in vivo staining endothelial cells of an intact venule in cramaster skeletal muscle, shown in Figure 2D. Additionally, as a comparison, primary rat MEC stained by CD31/PECAM-1 were shown in Figure 2E. 2.5. Statistics Purification of MEC derived from C57BL/6J and P2Y2R-/- mice was analyzed by using student’s t-test (GraphPad Prism version 5, GraphPad Software, San Diego, CA). The level of significance was set at 0.05 Purification of MEC derived from C57BL/6J and P2Y2R-/- mice was analyzed by using student’s t-test (GraphPad Prism version 5, GraphPad Software, San Diego, CA). The level of significance was set at 0.05 J. Biomedical Science and Engineering https://doi.org/10.4236/jbise.2020.1312026 292 J. Bio J. Biomedical Science and Engineering 292 (p < 0.05). (p < 0.05). 3.4. Detection and Count of Endothelial Cells by Flow Cytomery The purified cells were labeled with CD31/PECAM-1 antibody and analyzed by flow cytometry. The cells labeled with only secondary antibody conjugated with fluorescence, but omission of primary antibody served as a negative control. The representative data from the flow cytometry in three cell samples derived from one culture dish (defined n = 1) are shown in Figures 4A-E. The count of CD31/PECAM-1 positive cells in primary cultured cell population was 88.02% ± 2.99% (n = 6) and 87.78% ± 6.78% (n = 6) for cells derived from wild type C57BL/6J mice and P2Y2 knockout mice, respectively, shown in Figure 4F. Figure 1. Representative morphology of primary and cultured mouse MEC derived from ab- dominal skeletal muscles (A). MEC derived from different species (rat), but the same tissue (abdominal skeletal muscle) (B) and human umbilical vein EC (HUVEC) (C) commercially purchased are presented for reference. Figure 1. Representative morphology of primary and cultured mouse MEC derived from ab- dominal skeletal muscles (A). MEC derived from different species (rat), but the same tissue (abdominal skeletal muscle) (B) and human umbilical vein EC (HUVEC) (C) commercially purchased are presented for reference. J. Biomedical Science and Engineering https://doi.org/10.4236/jbise.2020.1312026 https://doi.org/10.4236/jbise.2020.1312026 293 293 Figure 2. Representative images of immunofluorescence for identification of CD31/PECAM-1 expression in murine primary cultured EC (A-C) and in vivo murine venules (D). The green (A, B, and C; labeled with Alexa-488) and red color (D and E, labeled with Alexa-568) indicate expression of CD31/PECAM-1. The blue color (B, C, and F) denotes the cell nuclei stained with DAPI. The scale of image A-C is shown in image B. Rat primary cultured EC derived from the same tissue (abdominal skeletal muscle) served as a positive control (E). Figure 2. Representative images of immunofluorescence for identification of CD31/PECAM-1 expression in murine primary cultured EC (A-C) and in vivo murine venules (D). The green (A, B, and C; labeled with Alexa-488) and red color (D and E, labeled with Alexa-568) indicate expression of CD31/PECAM-1. The blue color (B, C, and F) denotes the cell nuclei stained with DAPI. The scale of image A-C is shown in image B. Rat primary cultured EC derived from the same tissue (abdominal skeletal muscle) served as a positive control (E). Figure 3. Physiological function assay of MEC. The representative images of uptake of DiI-Ac-LDL by primary cultured murine MEC is demonstrated in images of A-C. 3.4. Detection and Count of Endothelial Cells by Flow Cytomery The uptake of DiI-Ac-LDL by primary cultured rat EC (D) and HUVEC (E) served as positive controls. The blue color denotes nuclei stained with DAPI. The scale for the images from A to C is shown in B. The representative images of capillary-like tube formation, a unique intrinsic property of MEC, are shown in F (murine MEC) and G (rat MEC). Figure 3. Physiological function assay of MEC. The representative images of uptake of DiI-Ac-LDL by primary cultured murine MEC is demonstrated in images of A-C. The uptake of DiI-Ac-LDL by primary cultured rat EC (D) and HUVEC (E) served as positive controls. The blue color denotes nuclei stained with DAPI. The scale for the images from A to C is shown in B. The representative images of capillary-like tube formation, a unique intrinsic property of MEC, are shown in F (murine MEC) and G (rat MEC). https://doi.org/10.4236/jbise.2020.1312026 J. Biomedical Science and Engineering 294 Figure 4. Representative data of flow cytometry for detection and enumeration of CD31/PECAM-1 positive cells (A-E). The gate (A) of the cell population was applied for all of the samples. The cells incubated with secondary antibody alone served as a nega- tive control (B) for the three separate samples labeled with both primary and secondary antibodies (C-E). The count of CD31/PECAM-1 positive cells isolated and purified by the two-step scheme using DynabeadsTM-GS-I lectin followed by DynabeadsTM-CD31 monoclonal antibody is shown in F. The purity of isolated cell populations from skeletal muscle microvessels was 88.02% ± 2.99% (n = 6). WT indicates cells derived from wild type C57BL/6J mice and P2Y2 KO presents the cells from P2Y2 receptor knockout mice. Figure 4. Representative data of flow cytometry for detection and enumeration of CD31/PECAM-1 positive cells (A-E). The gate (A) of the cell population was applied for all of the samples. The cells incubated with secondary antibody alone served as a nega- tive control (B) for the three separate samples labeled with both primary and secondary antibodies (C-E). The count of CD31/PECAM-1 positive cells isolated and purified by the two-step scheme using DynabeadsTM-GS-I lectin followed by DynabeadsTM-CD31 monoclonal antibody is shown in F. The purity of isolated cell populations from skeletal muscle microvessels was 88.02% ± 2.99% (n = 6). WT indicates cells derived from wild type C57BL/6J mice and P2Y2 KO presents the cells from P2Y2 receptor knockout mice. 4. DISCUSSION We report a refined method to isolate MEC derived from murine skeletal muscle. This method em- ployed the use of a Dynabeads™-based two-step scheme using GS-I, to recognize the carbohydrate moiety We report a refined method to isolate MEC derived from murine skeletal muscle. This method em- ployed the use of a Dynabeads™-based two-step scheme using GS-I, to recognize the carbohydrate moiety J. Biomedical Science and Engineering https://doi.org/10.4236/jbise.2020.1312026 295 https://doi.org/10.4236/jbise.2020.1312026 https://doi.org/10.4236/jbise.2020.1312026 295 J. Biomedical Science and Engineering 295 and then monoclonal antibody against CD31/PECAM-1 following enzymatic dissociation of skeletal mus- cles. To the best of our knowledge, this is the first description to use combined methods to isolate MEC from murine skeletal muscles. Such a refined method met the requirements of simple and fast preparation, high yield and purity of MEC population, and consistency. 4.1. Enzymatic Dissociation of Skeletal Muscle Tissue The enzyme-based tissue digestion possibly affects the structure or negatively impacts function of the surface molecules expressed on the cells. The enzymatic digestion of tissue has been extensively used for the isolation of cells from intact tissue. It was found that dispase at 0.8 U/ml (equivalent to 1 mg/ml) for 45 seconds at 37˚C with 5% fetal calf serum affected the expression of numerous surface molecules and im- paired antigen-mediated detectability of the majority of surface markers in immune cells [13]. To over- come this problem, we first detected abundant surface carbohydrates which attach to the surface proteins and lipids on the membrane of MEC [14]. The expression level of carbohydrates is much greater than that of a surface antigen, such as CD31/PECAM-1, because CD31/PECAM-1 is only one of various surface proteins in MEC [15]. 4.2. Isolation of MEC Using Dynabead-Based Two-Step Scheme We reported the modified method with the two-step isolation scheme here. The first step was to use GS-I to bind a carbohydrate moiety specifically expressed on the surface of microvascular endothelial cells [16, 17]. The carbohydrate chains are covalently attached to the surface proteins or lipids, and these are abundantly present on the EC surface as components of the glycocalyx [14]. The glycocalyx is a thick coating on the surface of endothelial cells, demonstrated in vivo from microvascular endothelial surface in skeletal muscles [18]. It was demonstrated that GS-I specifically bound to microvascular endothelial cells, but not macrovascular endothelial cells [16]. Using GS-I to isolate MEC has been used in various physiol- ogy or pathophysiology studies focusing on MEC [10, 12, 19, 20]. The first step using GS-I to isolate MEC enabled us to obtain abundant cells, providing sufficient numbers of isolated cells. Unfortunately, GS-I was also found to bind to non-endothelial cells such as epithelia cells [16]. To further remove non-endothelial cells from the cell population we employed Dynabeads coated with monoclonal antibody specifically against CD31/PECAM-1. CD31/PECAM-1 has been recognized as an endothelial marker and is extensively used for the detection or isolation of vascular EC. As described above, cell surface markers such as CD31/PECAM-1 can be degraded by enzymes in the process of enzy- matic dissociation of tissue [13]. Thus, to avoid this issue, we targeted CD31/PECAM antigen following subculture of the cells after initial isolation via the GS-I agglutination method. The specificity of CD31 an- tibody used in this study was validated by in vivo immunofluorescence staining in a venule of murine cremaster muscle, shown in Figure 2D. It was found that CD31/PECAM-1 is also expressed at low level on platelets and leukocytes, in addition to EC [21]. Thus, this study combined two strategies, by using GS-I followed by CD31-based antibody isolation to improve the purity of primary cultured MEC. 4.3. Characterization of Primary Endothelial Cells The identification of EC, including cobblestone morphology, immunostaining of EC biomarkers, and physiological functions specific for MEC such as uptake of DiI-Ac-LDL and promotion of angiogenesis, has been performed in this study. Although the high level of DiI-Ac-LDL uptake has been extensively used for identification of EC, other cells, such as macrophages, can also take up DiI-Ac-LDL [22]. Thus, several lines of evidence to characterize the isolated cells in the study overcomes the problems that other cells share markers or functional properties with EC. Flow cytometry revealed the percentage of CD31 positive cells in the population of all cells to be about 90% as shown in Figure 4. The reduced florescence intensity of CD31 (90%, but not 100%) was, in part, likely caused by steric hindrance on the part of the antibody [23]. https://doi.org/10.4236/jbise.2020.1312026 296 J. Biomedical Science and Engineering https://doi.org/10.4236/jbise.2020.1312026 296 J. Biome J. Biomedical Science and Engineering 296 Figure 5. A schematic diagram of the two-step scheme method for efficient isolation of MEC from murine skeletal muscles. Figure 5. A schematic diagram of the two-step scheme method for efficient isolation of MEC from murine skeletal muscles. 4.4. Limitation of the Study The study used GS-I and CD 31 to isolate cells so that MEC are possibly the mixed endothelial cells derived from arterioles, capillaries, and venules. 5. CONCLUSION A summary of the isolation and characterization of primary microvascular endothelial cells illustrated in the manuscript is shown in Figure 5. This study demonstrates a simple, effective method to acquire primary cultured MEC derived from skeletal muscles in any murine model, such as those genetically engi- neered or various disease models. 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https://openalex.org/W2107804376	https://curis.ku.dk/ws/files/95720228/Spatial_Amphibian_Impact_Assessment_a_management.pdf	English	null	Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis)	Nature Conservation	2,013	cc-by	10,684	Citation for published version (APA): Pontoppidan, M-B., & Nachman, G. S. (2013). Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis). Nature Conservation, 5, 29-52. https://doi.org/10.3897/natureconservation.5.4612 university of copenhagen university of copenhagen Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis) Pontoppidan, Maj-Britt; Nachman, Gøsta Støger university of copenhagen Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis) Pontoppidan Maj-Britt; Nachman Gøsta Støger Download date: 24. Oct. 2024 Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis) Maj-Britt Pontoppidan1, Gösta Nachman1 1 Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark Corresponding author: Maj-Britt Pontoppidan (mbp@bio.ku.dk) Academic editor: D. Schmeller \| Received 30 December 2012 \| Accepted 25 April 2013 \| Published 13 November 2013 Citation: Pontoppidan M-B, Nachman G (2013) Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis). Nature Conservation 5: 29–52. doi: 10.3897/natureconservation.5.4612 Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis) P t id M j B itt N h G t St Pontoppidan, Maj-Britt; Nachman, Gøsta Støger Document version Publisher's PDF, also known as Version of record Citation for published version (APA): Pontoppidan, M-B., & Nachman, G. S. (2013). Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis). Nature Conservation, 5, 29-52. https://doi.org/10.3897/natureconservation.5.4612 Download date: 24. Oct. 2024 Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 29 Nature Conservation 5: 29–52 (2013) doi: 10.3897/natureconservation.5.4612 http://www.pensoft.net/natureconservation APPLIED ECOLOGY Launched to accelerate biodiversity conservation A peer-reviewed open-access journal Spatial Amphibian Impact As Nature Conservation 5: 29–52 (2013) doi: 10.3897/natureconservation.5.4612 http://www.pensoft.net/natureconservation Spatial Amphibian Impact As Nature Conservation 5: 29–52 (2013) doi: 10.3897/natureconservation.5.4612 http://www.pensoft.net/natureconservation Spatial Amphibian Impact As Nature Conservation 5: 29–52 (2013) doi: 10.3897/natureconservation.5.4612 http://www.pensoft.net/natureconservation ment – a management APPLIED ECOLOGY Introduction Over the last decade a growing amount of literature has documented the severe im- pacts of transport infrastructure on biodiversity, population persistence and gene flow. An expanding network of roads and railways divides natural habitat into smaller and smaller fragments, affecting the amount and quality of habitat and reducing connec- tivity between habitat patches (Coffin 2007; Fahrig and Rytwinski 2009; Forman and Alexander 1998; Holderegger and Di Giulio 2010; Spellerberg 1998; Trombulak and Frissell 2000). To ensure an ecologically sustainable transportation system, it is essen- tial to find agreement between nature conservation and land use. In Europe and the US, programs and policies have been developed to address this need in strategic and environmental impact assessments (Brown 2006; Iuell et al. 2003; Trocmé et al. 2003), However, sustainable road planning requires adequate tools for assessment, prevention and mitigation of the impacts of infrastructure (Beckmann 2010; Forman et al. 2003; Gontier et al. 2010). Movement is vital to the survival of animal populations. The persistence of a popu- lation depends on the amount and accessibility of its required resources and, within a metapopulation framework, also on sufficient dispersal between subpopulations (Dun- ning et al. 1992; Wiens 1997). Taylor et al. (1993) defined connectivity as the degree to which the landscape facilitates or impedes movement among resource patches’’ and measures of connectivity are often used as indicators of a landscape’s capability to sustain a population. However, habitat requirements and behaviours differ between animal species and, thus, the connectivity of a landscape must in essence be species specific. Geographical information systems (GIS) have proved to be an important tool when assessing the impact of roads on landscape fragmentation and/or connectivity (Beckmann 2010; Brown 2006; Calabrese and Fagan 2004). Methods using least cost modelling (Adriaensen et al. 2003; Epps et al. 2007) or graph theoretical approaches (Bunn et al. 2000; Minor and Urban 2008; Zetterberg et al. 2010) usually combine GIS data with some species specific data such as dispersal distances or habitat suit- ability. However, none of these methods considers the particular dispersal, survival and establishment of the animals, which depend not only on the quality of the habitat but also on the behaviour of the animals, their responses to habitat conditions, land- scape elements, interactions with other animals and many other factors. Individual based models (IBMs) have proved to be suitable for describing such processes (Grimm 1999; McLane et al. Keywords Rana arvalis, Individual-based modelling, Fragmentation, Connectivity, Pond-breeding amphibians, Landscape planning, mitigation, management tool, population persistence Abstract An expanding network of roads and railways fragments natural habitat affecting the amount and quality of habitat and reducing connectivity between habitat patches with severe consequences for biodiversity and population persistence. To ensure an ecologically sustainable transportation system it is essential to find agreement between nature conservation and land use. However, sustainable road planning requires adequate tools for assessment, prevention and mitigation of the impacts of infrastructure. In this study, we present a spatially explicit model, SAIA (Spatial Amphibian Impact Assessment), to be used as a stand- ardized and quantitative tool for assessing the impact of roads on pond-breeding amphibians. The model considers a landscape mosaic of breeding habitat, summer habitat and uninhabitable land. As input, we use a GIS-map of the landscape with information on land cover as well as data on observed frog popula- tions in the survey area. The dispersal of juvenile frogs is simulated by means of individual-based model- ling, while a population-based model is used for simulating population dynamics. In combination the two types of models generate output on landscape connectivity and population viability. Analyses of maps without the planned road constructions will constitute a “null-model” against which other scenarios can be compared, making it possible to assess the effect of road projects on landscape connectivity and popula- tion dynamics. Analyses and comparisons of several alternative road projects can identify the least harmful solution. The effect of mitigation measures, such as new breeding ponds and underpasses, can be evaluated by incorporating them in the maps, thereby enhancing the utility of the model as a management tool in Environmental Impact Assessments. We demonstrate how SAIA can be used to assess which management measures would be best to mitigate the effect of landscape fragmentation caused by road constructions by means of a case study dedicated to the Moor frog (Rana arvalis). Copyright M-B. Pontoppidan, G. Nachman. 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. 30 Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) Methods SAIA combines an individual based model with a population based model. The in- dividual based model provides estimates of landscape connectivity and immigration probabilities between all pair wise ponds. The population based model provides esti- mates of population size and persistence probability.i We use the terms dispersal and migration as defined by Semlitsch (2008), i.e. dis- persal is interpopulational, unidirectional movements from natal sites to other breeding sites and migration is intrapopulational, round-trip movements toward and away from aquatic breeding sites. The habitat of pond breeding amphibians, such as the Moor frog, includes terrestrial as well as aquatic habitat. Therefore, we define an adequate habitat patch of a subpopulation as containing not only the breeding pond but also all accessible summer habitat within migration distance from the pond (Dunning et al. 1992; Pope et al. 2000). Introduction 2011) and recently there has been an increase in IBM case stud- ies demonstrating the potential for analysing population dynamics emerging from the interactions between landscape settings and animal behaviour (e.g. Graf et al. 2007; Kramer-Schadt et al. 2004; Pe’er et al. 2011). Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 31 We have developed a strategic management tool to be used in assessment and mitigation of road effects on a regional population of pond-breeding amphibians. The model, called SAIA (Spatial Amphibian Impact Assessment), combines the use of GIS land cover maps with IBM and provides information on connectivity as well as esti- mates of population persistence. SAIA is to be used by the Danish road authorities when assessing how new road constructions may affect Moor frogs (Rana arvalis). In this paper we demonstrate how SAIA can be used for assessing which management measures would be best to mitigate the effect of landscape fragmentation caused by the construction of a road ca 90 km west of Copenhagen, Denmark. To achieve this goal the following specific research questions were addressed: 1. What is the structure of the regional habitat network before road construction?f . What is the structure of the regional habitat network before road construction 2. How is the habitat network affected by the new road? f 3. Which mitigation strategies are best suited to preserve the overall persistence of the regional population of Moor frogs? 3. Which mitigation strategies are best suited to preserve the overall persistence of the regional population of Moor frogs? Model species Moor frogs spend most of their life in terrestrial habitat; aquatic habitat is only used during the breeding season in early spring (Elmberg 2008; Glandt 2008; Hartung 1991). Soon after breeding, the frogs return to their summer habitat, which lies mostly within a 400 m radius from the breeding pond (Elmberg 2008; Hartung 1991; Ko- var et al. 2009). Adult frogs show strong site fidelity and often use the same breeding pond and summer habitat from year to year (Loman 1994). Long distance dispersal takes place predominantly during the juvenile life-stage (Semlitsch 2008; Sinsch 1990; 2006). Shortly after metamorphosis, the young frogs leave the natal pond and disperse into the surrounding landscape seeking suitable summer habitat. Dispersal distances Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 32 Table 1. List of variables characterizing the agents in SAIA. Variable Notation Value range Agent type Description Area Value W 0.5; 1 Cell Effective area of the cell Daily Survival Ds Cell Daily survival probability Frog Density D Cell Mean number of frogs in the cell Habitat Attraction Ha 1-5 Cell The cell’s relative attraction to frogs during movement Habitat Code Hc Cell The land cover category of the cell Habitat Survival Hs 1-5 Cell The cell’s relative survival index Summer Quality Hq 1-5 Cell The cell’s relative suitability as summer habitat Breeding Pond Frog Breeding pond of frog agents Natal Pond Frog Natal pond of frog agents Pond ID Pond ID number Pond Perimeter O Pond Perimeter of the pond Pond Quality Q 0.1-1 Pond Quality index of the pond Population Size N0 Pond Number of adult females (estimated as egg masses found in the pond during survey) Summer Habitat A Pond Summer habitat cells associated with the pond Summer Habitat Area A’ Pond Effective area of associated summer habitat Table 1. List of variables characterizing the agents in SAIA. are between a few hundred meters up to 1–2 kilometres (Baker and Halliday 1999; Hartung 1991; Sinsch 2006; Vos and Chardon 1998). The juveniles stay in terres- trial habitat 2–3 years until they reach maturity, although some observations indicate that juvenile frogs follow the adults during the spring migration, without entering the breeding ponds (Hartung 1991; Sjögren-Gulve 1998). Model overview Full model description following the protocol suggested by Grimm et al. (2006, 2010) and model parameterisation are provided in Appendix. Table 1 shows a full list of model variables while a list of model parameters can be found in Table A3 in the Ap- pendix. Netlogo v.4.1.3 (Wilensky 1999) was used as modelling environment (freely downloadable at http://ccl.northwestern.edu/netlogo). Map construction To construct a model landscape in which our virtual frogs can move we use a GIS raster map of the study area with land cover data. Each raster cell contains information about the cell’s land cover or habitat type (Hc). Moreover, three variables are associated with each category of land cover/habitat (Table 2): Hq, the category’s relative suitability as summer habitat; Ha, the category’s relative attraction to frogs during movement and Hs, the category’s relative survival index. For each cell, the assigned survival index is Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 33 Table 2. Land cover categories and the associated values of Habitat Survival, Habitat Attraction and Summer Quality. Habitat Code (Hc) Description Habitat Attraction (Ha) Habitat Survival (Hs) Summer Quality (Hq) 2 4-lane motorway 2 N/A 1 3 2-lane motorway 2 N/A 1 4 Road, width > 6m 3 N/A 1 5 Road, width 3-6 m 3 N/A 1 6 Other roads 3 2 2 8 Pathway 4 4 3 10 Multiple surface 3 3 3 11 Railway 4 2 3 12 Building 1 N/A N/A 15 Other made surface 2 3 2 18 Wetlands 5 5 5 20 Running water 4 4 3 22 Meadows 5 5 5 24 Grassland 4 4 4 25 Lakes 1 N/A N/A 28 Hedgerow 4 4 4 29 Heath land 5 5 4 32 Woodland 4 4 4 34 Stand of trees 4 4 3 36 Bare surface 2 2 1 40 Fallow land 4 4 4 42 Field crops 2 2 2 50 Drift fence 1 N/A N/A 60 Underpass 4 4 1 Table 2. Land cover categories and the associated values of Habitat Survival, Habitat Attraction and Summer Quality. Map construction Habitat Code (Hc) Description Habitat Attraction (Ha) Habitat Survival (Hs) Summer Quality (Hq) 2 4-lane motorway 2 N/A 1 3 2-lane motorway 2 N/A 1 4 Road, width > 6m 3 N/A 1 5 Road, width 3-6 m 3 N/A 1 6 Other roads 3 2 2 8 Pathway 4 4 3 10 Multiple surface 3 3 3 11 Railway 4 2 3 12 Building 1 N/A N/A 15 Other made surface 2 3 2 18 Wetlands 5 5 5 20 Running water 4 4 3 22 Meadows 5 5 5 24 Grassland 4 4 4 25 Lakes 1 N/A N/A 28 Hedgerow 4 4 4 29 Heath land 5 5 4 32 Woodland 4 4 4 34 Stand of trees 4 4 3 36 Bare surface 2 2 1 40 Fallow land 4 4 4 42 Field crops 2 2 2 50 Drift fence 1 N/A N/A 60 Underpass 4 4 1 Table 2. Land cover categories and the associated values of Habitat Survival, Habitat Attraction and Summer Quality. converted into a daily survival probability (Ds). When concerning paved roads, the daily survival probabilities ranges between 0.1 and 0.8 depending on road category; all other land cover/habitat categories are assigned values between 0.9820 and 0.9995 (see Appendix in the supplementary material for details). Cells with structures or habitats which are assumed inaccessible to the frogs (e.g. buildings, fences or large water bodies) are given a habitat attraction of 1. This will prevent the frogs from entering the cell. A point-data set containing information about potential breeding ponds found in the study area is used to create stationary pond agents. Each pond agent is character- ized by an ID number, the perimeter of the pond (O), initial population size (adult females) (N0) and a quality index (Q) indicating the suitability of the pond and the im- mediate surroundings (20 m) in regard to egg and larval survival. In addition the pond variable A is updated with a list of summer habitat cells within migration distance. Summer habitat cells are identified as cells with SummerQuality (Hq) > 3. Summer habitat cells can be completely surrounded by other summer habitat cells (core cells) Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 34 or have one or more neighbouring cells which are not summer habitat (edge cells). Individual based model The IBM is largely identical to the model described in Pontoppidan and Nachman (2013). The model simulates the dispersal of juvenile frogs and estimates immigra- tion probabilities between ponds. Immigration requires two events: 1) the successful dispersal of a juvenile frog to summer habitat outside its natal habitat patch and 2) subsequent successful migration from the new summer habitat to a nearby breeding pond. In real life dispersal starts just after metamorphosis in early summer and lasts until hibernation in autumn. The second part of the immigration event, migration, takes place in the spring 2.5 years later. For simplicity, we simulate the dispersal and breeding migration, as if they take place in the same year.h At the start of a simulation, 250 frog agents are created at each pond. The frogs then disperse through the landscape in random directions from the ponds until they find suit- able summer habitat; the movement of the frogs depends on the attractiveness of neigh- bouring cells and the cells’ suitabilities as summer habitat. Survival probabilities depend on the traversed habitat types. Unlike the former model, movement behaviour in SAIA also depends on weather conditions. A database containing data on daily precipitation (Cappelen 2009) is used to reflect natural weather patterns. At the start of a simulation, a random year is chosen from this database and at each time step, information on precipita- tion is drawn for the simulated day of the year. When daily precipitation exceeds a given threshold (α), the variables HabitatAttraction and DailySurvival of all accessible cells are given the highest value. An exception is paved roads where only HabitatAttraction, but not DailySurvival, is changed. The simulation runs for 240 time steps, each time step representing one day. After each simulation, immigration probabilities between all pairs of ponds are calculated and an immigration matrix is constructed. Map construction To account for edge effects, core cells are given an area value (W) of 1 while W is 0.5 for edge cells (Watts and Handley 2010) and the effective area of the summer habitat (A’) is found as the sum of W-values of the summer habitat cells belonging to the pond. The individual based model only uses information about pond ID, quality, and summer habitat while all of the variables enter into the population based model. Population based model After the individual based simulation, a population based model simulates the popula- tion dynamics in each pond through 40 iterations of a life cycle model. The elements of the life cycle model are 1) Reproduction, 2) Survival and 3) Immigration. For simplic- ity, we only model the female part of the population. We assume a sex ratio of 1:1 and that females always become mated once in a season. Pond populations are grouped by age from 0 through 6 years, and survival and reproductive rates are based on life-table Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 35 Table 3. Life table for Rana arvalis constructed by amphibian experts. Stage/Age Survival probability Fecundity ( eggs pr. female) Egg/larvae 0.005 - 0 0.55 0 1 0.55 70 2 0.55 945 3 0.55 1190 4 0.50 1250 5 0.40 1300 6 0.20 1300 Table 3. Life table for Rana arvalis constructed by amphibian experts. data constructed by amphibian experts (Fog and Hesselsøe 2009) (Table 3). The pond variable (N0) is set as the initial population size of the pond. Demographic but not environmental stochasticity is incorporated into the model. data constructed by amphibian experts (Fog and Hesselsøe 2009) (Table 3). The pond variable (N0) is set as the initial population size of the pond. Demographic but not environmental stochasticity is incorporated into the model. Individuals can start reproducing in their third year. As in Hels and Nachman (2002), the expected egg production of a female is assumed to follow a negative bi- nomial distribution with mean R¯ and clumping parameter k. R¯ is the mean number of eggs produced by a female of a given age. The number of two-weeks old frogs ready to disperse is considered as the reproductive output. This involves the survival of egg and larvae, as well as the survival of the young frogs the first two-weeks after meta- morphosis. The overall probability that an egg develops into a frog that survives until dispersal time is assumed to be negatively affected by the density of eggs in the pond and positively affected by the quality of the pond. The conditional probability that a frog survives from age a to age a+1 is assumed to depend on age. Furthermore, survival is assumed to depend on the frog density in the summer habitat. Population based model For simplicity, this is modelled as a “culling” process when frog density exceeds the carrying capacity of summer habitat. The carrying capacity is estimated as the mean initial frog density in summer habitat cells associated with the populated ponds. Immigration probabilities between all pairs of ponds are obtained from the immigration matrix constructed dur- ing the IBM. The actual number of immigrants a subpopulation receives depends on the reproductive output of each of the other ponds and the corresponding immigra- tion probability. Emigration rates are not modelled explicitly. Output At the end of the individual based simulation, the following output was recorded: the number of surviving frogs, the natal and breeding ponds of all frogs, and the immigration probabilities (pij) between all pair-wise ponds. Landscape connectivity (S) is found as (eq. 1) 36 Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) At the end of the population based simulation, the estimated population size of each pond is recorded as the resulting numbers of frogs of ages 2 through 6. The model was run 50 times and mean connectivity with 95% confidence intervals (CI) was computed. For each pond, we computed mean population size with 95% confidence intervals (CI). Pond persistence probability was computed as the proportion of replicates where the estimated population size was positive. The regional population size was computed as the sum of all ponds. Mean number of populated ponds with 95% CI was also calcu- lated. SAIA’s connectivity measure is an index of the potential connectivity between all ponds, whether they are populated or not. The population based model links the poten- tial connectivity with local population dynamics, and estimated abundances and persis- tence probabilities can be regarded as a result of the realised connectivity. The estimated landscape connectivity and population size are considered measures of the landscape's ecological performance in regard to the modelled species. g p g p Cluster analysis was used to identify highly connected groups of ponds. Ponds were grouped into clusters depending on their mutual connectivity, using unweighted, arithmetic, average clustering as described by Legendre and Legendre (1998). Since immigration probabilities between ponds are not necessarily symmetric, i.e. pij ≠ pji, we used summed immigrations probabilities as similarity measures (m), i.e. mij = pij + pji. The threshold at which a given pond or cluster no longer can be added to another cluster was set to mij ≤ 0.01. Connectivity between any pairs of clusters (Sk,l) is found as j (eq. 2) where nk and nl are the number of ponds in clusters k and l, respectively. Cluster abun- dance is computed as the sum of the member ponds’ estimated population size. where nk and nl are the number of ponds in clusters k and l, respectively. Cluster abun- dance is computed as the sum of the member ponds’ estimated population size. Case study We apply SAIA to a road project in Denmark and demonstrate the workflow of an impact assessment. The project concerns an area in the north-western part of Zea- land, 10 km east of the city of Kalundborg (55°40.14'N, 11°17.85'E) (Figure 1) and includes a broadening of an existing 2-lane motorway into a 4-lane motorway as well as an extension of the motorway. The assessment procedure can be divided into three parts – Initial analyses, Mitigation planning and Mitigation analysis. Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 37 Initial analyses This part involves the construction and analysis of two maps. The first map, Sce- nario 0, is a map of the landscape as it looks before the planned road project. This map serves as Null Scenario and the results of the analysis are considered the state of the landscape we wish to maintain. The second map, Scenario 1, is of the land- scape as expected after road construction. Analysis of this map gives indications of the effects the planned road construction can have on the ecological performance of the landscape. Model validation We have applied a bottom-up approach and used pattern oriented modelling (Grimm and Railsback 2005; Latombe et al. 2011) to parameterise and calibrate SAIA’s sub- models controlling movement, dispersal and survival. This is documented in detail in Appendix in the supplementary material. However, lack of field data and the specificity of the model’s emergent results to the modelled system complicate validation of the full model. Instead, we have used a heuristic approach, consulting amphibian experts during the whole modelling process to evaluate the plausibility of the emergent results. The population based model is based on expert data but is not validated against field data. Moreover, environmental stochasticity is not included in the population based model. Hence, the output should not be considered predictive, but rather as an index of the landscape’s potential carrying capacity. Scenario 0 Construction of the map is based on a GIS data set from the road project, supplied by the Danish Road Directorate and an environmental consultancy firm, Amphi Consult. The extent of the land cover map is 600 × 800 cells, and each cell is 10 × 10 m. All cells are assigned values of Ha, Hs and Hq depending on their land cover type, following a protocol designed by Amphi Consult (Hassingboe et al. 2012) (Table 2). A point data set contains information about potential breeding ponds found during a field survey of the area. The survey was conducted in spring 2012 by Amphi Consult in the initial phases of the road project following standard procedures (Fog and Hesselsøe 2009). The initial population (N0) was estimated as the number of egg masses found in the surveyed pond and is assumed to equal the number of breeding females in the pond. Pond quality (Q) was assessed visu- ally and includes factors such as the shape of the pond, degree of shading, vegeta- tion in as well as around the pond and presence of predators (fish). The data set contains 121 ponds, of which 23 ponds are of high quality (Q > 0.6). Six of the ponds are populated; population sizes ranging between 1 and 55 egg masses, in total 106 egg masses. The map with the location of the ponds is shown in Figure 2A, ponds containing egg masses are marked with a star shape and the number of egg masses found. Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 38 Figure 1. Location of two study areas in Denmark. KaB is an area near Kalundborg on Zealand and HoB is near Holstebro in Jutland. Only KaB is used in the present analysis, but both areas are used for the parameterisation of the model. Figure 1. Location of two study areas in Denmark. KaB is an area near Kalundborg on Zealand and HoB is near Holstebro in Jutland. Only KaB is used in the present analysis, but both areas are used for the parameterisation of the model. Scenario 1 The map used in Scenario 0 is modified by changing the land cover category of the existing road section from a 2-lane motorway to a 4-lane motorway. The new sec- tion of the road is added as well and categorised as a 4-lane motorway (Figure 3A). As a consequence of the change in land cover category the daily survival probability (Ds) of the road cells decreases from 0.20 to 0.10 (Table A5 in the Appendix). The road construction also involves removal of five unpopulated ponds along the road (Figure 3A). Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 39 Figure 2. Scenario 0. A Map of the landscape before road constructions. Black dots represent potential breeding ponds. Small dots are ponds with pond quality (Q) ≤ 6; large dots are ponds with Q ≥ 7. Populated ponds are indicated with a star shape. The number of egg masses found in the pond is shown in parenthesis B Result of cluster analysis showing clusters c1–c13. Ponds linked with black lines belong to the same cluster. Pond size and colour indicate the result of the population based model. Yellow circles represent ponds with an estimated population size ≥ 1. Ponds with larger yellow circles have a persistence probability > 0.75. Figure 2. Scenario 0. A Map of the landscape before road constructions. Black dots represent potential breeding ponds. Small dots are ponds with pond quality (Q) ≤ 6; large dots are ponds with Q ≥ 7. Populated ponds are indicated with a star shape. The number of egg masses found in the pond is shown in parenthesis B Result of cluster analysis showing clusters c1–c13. Ponds linked with black lines belong to the same cluster. Pond size and colour indicate the result of the population based model. Yellow circles represent ponds with an estimated population size ≥ 1. Ponds with larger yellow circles have a persistence probability > 0.75. Mitigation planning Once the analyses of Scenario 0 and Scenario 1 are done, planning of possible mitiga- tion measures can start. The results from the initial analyses can give insights in the structure of the pond network and can locate areas or subpopulation where the road construction will have the strongest impact. Likely source populations and their colo- nisation potential may be identified and possible sink population may be recognised. Interpretation of the results provides a basis for considerations about which mitigation measures are needed and where to place them. A series of scenarios with different sug- gestions for mitigation measures can then be constructed and analysed. Mitigation analysis In this case study we construct and analyse three alternative mitigation scenarios - Scenario 2, Scenario 3a and Scenario 3b. The choices of mitigation measures depend as just mentioned on the results of the initial analysis. Thus, the reasoning behind the different mitigation scenarios will be explained in the result section. However, the map construction of the three scenarios is briefly described below. Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 40 Figure 3. Scenario 1. A Map of the landscape after road constructions (red road). Black dots represent poten- tial breeding ponds. Small dots are ponds with pond quality (Q) ≤ 6; large dots are ponds with Q ≥ 7. Popu- lated ponds are indicated with a star shape. Pink ponds are ponds removed in connection with the construc- tions B Result of cluster analysis showing clusters c1–c13. Ponds linked with black lines belong to the same cluster. Pond size and colour indicate the result of the population based model. Yellow circles represent ponds with an estimated population size ≥ 1. Ponds with larger yellow circles have a persistence probability > 0.75. Figure 3. Scenario 1. A Map of the landscape after road constructions (red road). Black dots represent poten- tial breeding ponds. Small dots are ponds with pond quality (Q) ≤ 6; large dots are ponds with Q ≥ 7. Popu- lated ponds are indicated with a star shape. Pink ponds are ponds removed in connection with the construc- tions B Result of cluster analysis showing clusters c1–c13. Ponds linked with black lines belong to the same cluster. Pond size and colour indicate the result of the population based model. Yellow circles represent ponds with an estimated population size ≥ 1. Ponds with larger yellow circles have a persistence probability > 0.75. Figure 4. Analyses of mitigation measures. Result of cluster analyses showing clusters c3–c11. Ponds linked with black lines belong to the same cluster. Pond size and colour indicate the result of the popula- tion based model. Yellow circles represent populated ponds. Ponds with larger yellow circles have a persis- tence probability > 0.75. A Scenario 2 - Location of underpasses is shown with red arrows B Scenario 3a – Three new ponds in cluster c9 and five new ponds in c11 are shown with red dots C Scenario 3b – Eight new ponds connecting c9 and c11 are shown with red dots. Scenario 2: Construction of underpasses and drift fences In this scenario we add three underpasses to the map used in Scenario 1. Drift fences are established along the road for 100 m on each side of the underpass, except for underpass 2 which has a 300 m drift fence to the east. The location of the underpasses is shown in Figure 4A. Underpasses are constructed by changing the habitat code and the associated values of habitat attraction (Ha) and daily survival probability (Ds) of the affected road cells. Drift fences are created by changing the habitat attraction of the affected road section to 1, thus preventing access to the cells. Mitigation analysis Figure 4. Analyses of mitigation measures. Result of cluster analyses showing clusters c3–c11. Ponds linked with black lines belong to the same cluster. Pond size and colour indicate the result of the popula- tion based model. Yellow circles represent populated ponds. Ponds with larger yellow circles have a persis- tence probability > 0.75. A Scenario 2 - Location of underpasses is shown with red arrows B Scenario 3a – Three new ponds in cluster c9 and five new ponds in c11 are shown with red dots C Scenario 3b – Eight new ponds connecting c9 and c11 are shown with red dots. Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 41 Scenario 3a and 3b: Construction of artificial breeding ponds In these two scenarios eight artificial, high quality breeding ponds are added to the map used in Scenario 1. Scenario 3a and 3b represent two alternative locations of the eight ponds (Figure 4B and C). Each breeding pond is created as a pond agent and as- signed a pond quality (Q) of 0.7. The initial population size (N0) is set to 0. The pond perimeter (O) is set to 79 m, corresponding to the average size of a standard artificial breeding pond. Scenario 0 The average percentage of ponds populated during a simulation is 32%, although only 22% of the ponds have a more permanent status (pond persistence probability > 0.75). The regional abundance of adult female frogs is estimated to be 157; the percentage of frogs surviving during dispersal is 57 %, and landscape connectivity is 55 (Figure 5). The cluster analysis groups the 121 ponds into 13 clusters, cluster sizes ranging from 2-20 ponds (Figure 2B, Table 4). The six populated ponds found during field surveys are distributed on four different clusters. One pond with only one adult female is found in cluster c5. Another pond, with an initial population of five, belongs to cluster c4 and two other ponds, also with a N0 of five, are found in cluster c8. Cluster c11 contains the remaining two populated ponds with an initial total population of 90 adult females. Apart from cluster c5, all of these initially populated clusters ex- hibit high viability. Clusters c4, c8 and c11 have mean pond persistence probabilities between 77% - 93% and estimated cluster abundances from 23–51 adult females. Cluster c9 also shows high values of abundance and persistence. Although initially unpopulated, c9 contains several high quality ponds and is connected with c8 and c11 which may promote colonisation and establishment. In cluster c6 and c7, the mean Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 42 Table 4. Results from analysis of Scenario 0 (before road construction). Cluster ID c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 Number of ponds in cluster 9 5 2 8 20 13 19 7 7 11 12 5 3 Number of high quality ponds (Q> 0.6) 2 0 0 4 2 0 1 7 3 0 3 1 0 Connectivity to other clusters 0.45 0.54 0.30 0.57 2.19 1.13 2.94 0.36 1.20 0.07 0.10 0.05 0.06 Estimated cluster abundance 0 0 0 49 0 3 11 23 14 0 51 0 0 Mean pond persistence probability 0 0 0 0.82 0.03 0.29 0.35 0.93 0.73 0.01 0.77 0 0.13 Connectivity to c4 0 0.09 0.04 - 0.01 0.44 0 0 0 0 0 0 0 Connectivity to c8 0 0 0 0 0 0.23 0.04 - 0.10 0 0 0 0 Connectivity to c11 0 0 0 0 0 0.00 0 0 0.02 0.02 - 0 0.06 Table 4. Scenario 1 After construction of the road, the percentage of populated ponds is reduced to 26% and the number of ponds with a persistence probability > 0.75 is now down to 16%. Survival rate of dispersing frogs is 56% and estimated regional abundance is 136 adult females. Landscape connectivity decreases to 51 (Figure 5). The number of clusters is unchanged but connectivity between clusters is reduced (Table 5). Connectivity from c7 and c9 to their primary source (c8) decreases more than 80%. Moreover, three ponds are lost in c7 and c9 due to the road construction. Estimated abundance and mean pond persistence probability decreases in c7 and c9 and these clusters are no longer able to uphold viable populations (Figure 3B). However, the initially populated clusters c4, c8 and c11 are not affected by the road construction. Scenario 0 Results from analysis of Scenario 0 (before road construction). in cluster 9 5 2 8 20 13 19 7 7 11 12 5 3 Number of high quality ponds (Q> 0.6) 2 0 0 4 2 0 1 7 3 0 3 1 0 Connectivity to other clusters 0.45 0.54 0.30 0.57 2.19 1.13 2.94 0.36 1.20 0.07 0.10 0.05 0.06 Estimated cluster abundance 0 0 0 49 0 3 11 23 14 0 51 0 0 Mean pond persistence probability 0 0 0 0.82 0.03 0.29 0.35 0.93 0.73 0.01 0.77 0 0.13 Connectivity to c4 0 0.09 0.04 - 0.01 0.44 0 0 0 0 0 0 0 Connectivity to c8 0 0 0 0 0 0.23 0.04 - 0.10 0 0 0 0 Connectivity to c11 0 0 0 0 0 0.00 0 0 0.02 0.02 - 0 0.06 Figure 5. Key results from analysis of the five scenarios. Upper and lower 95% confidence limits are indicated with black triangles. Figure 5. Key results from analysis of the five scenarios. Upper and lower 95% confidence limits are indicated with black triangles. Figure 5. Key results from analysis of the five scenarios. Upper and lower 95% confidence limits are indicated with black triangles. Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 43 pond persistence probability is considerably lower (29–35%) as is the estimated cluster abundance. While the two clusters, especially c6, are connected with other populated clusters, they lack high-quality ponds and the clusters may function as sinks. In the remaining clusters the estimated abundance is less than one individual. Mitigation planning The initial analyses reveal that the landscape contains three viable populations (c4, c8 & c11) centred on the initially populated ponds. These populations appear not to be affected by the road construction and in the simulations the clusters seem to func- tion as sources enabling colonisation and establishment of populations in c9 and c7. Cluster c4 has a large and viable population, but even though it is well connected with the neighbouring clusters their qualities are not high enough to enable establishment of new populations. Since c4 is not connected with c7 and c9, its potential as source cluster is low. Cluster c8 seems to be the primary source cluster to c9 and c7; however, expansion of the road heavily reduces its value as a source. Furthermore, the removal of three ponds between c9 and c7 may diminish the connectivity between these clusters. Cluster c11 has a viable population and although situated somewhat remotely there is still some connectivity to c7 and c9. The results indicate that, in order to compensate or mitigate the effect of the road project, the best strategies will be either to re-establish connectivity across the road between cluster c8 and clusters c7/c9 and between cluster c7 and c9 or to take advan- tage of the viability of cluster c11 and its source potential. Based on this, we create and analyse the following scenarios: Scenario 2: Connectivity across the road is re-established by constructing three un- derpasses and drift fences along the middle section of the motorway (Figure 4A). Two of the underpasses (including drift fences) were placed between cluster c6 and c7; the Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 44 Table 5. Results from analysis of Scenario 1 (after road construction). Mitigation planning Cluster ID c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 Number of ponds in cluster 9 5 2 8 19 13 17 7 6 9 12 5 3 Number of high quality ponds (Q> 0.6) 2 0 0 4 2 0 1 7 2 0 3 1 0 Connectivity to other clusters 0.02 0.10 0.27 0.52 1.69 0.67 1.92 0.23 0.55 0.06 0.09 0.05 0.06 Estimated cluster abundance 0 0 0 50 2 2 1 24 5 0 49 0 0 Mean pond persistence probability 0 0 0.03 0.84 0.05 0.24 0.08 0.93 0.26 0 0.78 0 0.15 Connectivity to c4 0 0.08 0 - 0 0.44 0 0 0 0 0 0 0 Connectivity to c8 0 0 0 0 0 0.20 0.01 - 0.02 0 0 0 0 Connectivity to c11 0 0 0 0 0 0 0 0 0.03 0 - 0 0.06 Table 5. Results from analysis of Scenario 1 (after road construction). third between cluster c8 and c9. The expectation is that connectivity between cluster c8 and c9/c7 will improve and enable establishment of populations in cluster c9.h third between cluster c8 and c9. The expectation is that connectivity between cluster c8 and c9/c7 will improve and enable establishment of populations in cluster c9.h Scenario 3a: The quality of cluster c9 and c11 is improved by establishing three, and then five, new high quality ponds within the range of the clusters (Figure 4B). The three new ponds in cluster c9 are expected to improve the probability of successful establish- ment of immigrants as well as reconnect c9 with c7.We expect an increase in abun- dance in cluster c11, and hence increased immigration to and colonisation of cluster c9.i g Scenario 3b: In this modification of scenario 3a the quality of cluster c9 and c11 is still improved but with only one and two ponds, respectively. The remaining five ponds are used to create a dispersal corridor between cluster c11 and c9 (Figure 4C). This strategy is expected to enhance the abundance in cluster c11 and to improve con- nectivity to c9, thereby increasing the probability of colonisation. Scenario 2 Quite unexpectedly, the creation of drift fences and underpasses do not improve the condition of the landscape. The mean percentage of populated ponds is 26% and the percentage of ponds with persistence probability > 0.75 is 16% as in Scenario 1. How- ever, the estimated regional abundance of female adults decreases to 115, landscape connectivity is 48 and dispersal survival rate 53% (Figure 5). As expected, connectivity between c8 and c9 is greatly improved. Cluster c9 now spans the road and has annexed one of the ponds in the periphery of c8 (Figure 4A). Abundance and mean pond per- sistence probability of cluster c9 increase; this, however, is due to the inclusion of a pond from cluster c8. Persistence and abundance do not improve on the original con- Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 45 Table 6. Results from analysis of mitigation measures. Scenario 2: Drift fences and underpasses; Scenario 3a: Implementing new ponds in clusters c9 and c11; Scenario 3b: Implementing new ponds as corridor between c9 and c11. Cluster ID Estimated cluster abundance Mean pond persistence probability Connectivity to c8 Connectivity to c11 S2* S3a S3b S2* S3a S3b S2* S3a S3b S2 S3a S3b c4 40 48 49 0.82 0.82 0.82 0 0 0 0 0 0 c7 1 3 4 0.08 0.11 0.11 0.002 (0.004) 0 0 0 0 0 c8 17 (21) 23 23 0.90 (0.95) 0.90 0.92 - - - 0 0 0 c9 6 (2) 11 10 0.38 (0.27) 0.44 0.58 0.19 (0.29) 0.02 0.02 0.025 0.03 0.22 c11 40 50 61 0.74 0.80 0.85 0 0 0 - - - * In scenario 2 one pond originally belonging to c8 is annexed by c9. Entries in parentheses are values based on the original cluster configurations. * In scenario 2 one pond originally belonging to c8 is annexed by c9. Entries in parentheses are values based on the original cluster configurations. figuration of cluster c9 (Table 6). Apart from cluster c9, connectivity between initially populated clusters and other clusters does not improve. Connectivity to cluster c4 and c11 is unchanged, while connectivity to cluster c8 actually decreases. Finally, the abun- dance of frogs in cluster c4 and c11 decreases to 20% even though connectivity both within the cluster and to other clusters is unchanged. figuration of cluster c9 (Table 6). Scenario 2 Apart from cluster c9, connectivity between initially populated clusters and other clusters does not improve. Connectivity to cluster c4 and c11 is unchanged, while connectivity to cluster c8 actually decreases. Finally, the abun- dance of frogs in cluster c4 and c11 decreases to 20% even though connectivity both within the cluster and to other clusters is unchanged. Scenario 3a Establishment of eight new ponds has a positive effect on landscape condition. The estimated number of adult females in the region increases to 143, percentage of popu- lated ponds is 31% and 19% of the ponds are permanently populated. Landscape con- nectivity is 56 and dispersal survival rate 56% (Figure 5). The five new ponds in cluster c11 perform well and contain permanent populations. However, the performance of the cluster does not improve, apart from a slightly higher persistence probability (Table 6). Cluster c9 seems to benefit from the additional ponds, although none of the new ponds contain permanent populations. Cluster abundance and connectivity are nearly restored to their original conditions although mean pond persistence probability is still below 50%. Connectivity from cluster c7 to other ponds improves somewhat, but not enough to restore the cluster to its former performance (Figure 4B). Discussion This study demonstrates how initial analyses of the landscape before and after the planned road constructions can help to identify which areas will be most affected by the construction. The analyses enable the user to recognise the colonisation potential of the clusters and to identify source or sink clusters and to use this knowledge for plan- ning mitigation measures. In the present case study, the simulations indicated that the six populations recorded during the field survey will be largely unaffected by the road construction. Nevertheless, the road construction will severely impair the colonisation potential of cluster c8, thereby reducing the ecological performance of the landscape. Of the three mitigation strategies tested, the analysis showed that Scenario 3b is the best solution. This strategy of connecting clusters c9 and c11 restores the landscape to its former ecological performance. Even though not all individual ponds or clusters will be in the same condition as before, the strategy promotes viable populations on both sides of the road. The strategy is not strictly aimed at mitigating the impaired connectivity across the road, but rather tries to compensate for the effects of road construction by improving other areas. Still, the populations on either side of the road are not totally iso- lated from each other; some dispersal does take place making genetic exchange possible. p p g g g p Comparing the results from the analyses of Scenarios 3a and 3b suggests that the location of compensating new ponds is not trivial. In both scenarios cluster c11 gets five new ponds, all of high quality. Nevertheless, the results differ quite a lot. Scenario 3a places the new ponds within the cluster sharing the summer habitat of other ponds. Even though the new ponds are colonized and support viable populations, the abundance of frogs within the cluster does not improve. In Scenario 3b, where the abundance of frogs within cluster c11 increases, the new ponds were placed between c9 and c11 and only partly share summer habitat with other ponds. This result emphasizes that for the Moor frog the carrying capacity of an area is not improved by adding new ponds, only new or better summer habitat can achieve this. Hence, we may improve cluster performance by creating new ponds in unutilized summer habitat within dispersal distance. In Scenarios 3a and 3b, cluster c9 is also enlarged with three new ponds. Scenarios 3b With this strategy we succeed in restoring the landscape to its original ecological per- formance. The number of adult female frogs in the region is 159. Mean percentage of Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 46 populated ponds is 32% and 22% are populated permanently. Dispersal survival rate is 56% and landscape connectivity is 55 (Figure 5). Three of the new ponds are now part of cluster c9 while the remaining five new ponds belong to cluster c11. Connectiv- ity between cluster c9 and c11 is strong and six of the new ponds contain permanent populations (Figure 4C). The abundance and mean persistence probability of cluster c11 increase and are now better than before the road construction. Conditions in clus- ter c9 also improve, compared to Scenario 1, but its original performance is not quite restored. The performance of cluster c7 does not change and is still at the same level as found in Scenario 1 (Table 6). Discussion In these cas- es there was no difference in frog abundance in the cluster whether the new ponds were placed in unused summer habitat or not. In both scenarios, though, mean pond persis- Spatial Amphibian Impact Assessment – a management tool for assessment of road effects... 47 tence probability greatly improved compared to Scenario 1. So, while adding ponds to a cluster did not improve carrying capacity, it ensured a more viable cluster population.h a cluster did not improve carrying capacity, it ensured a more viable cluster population. The analysis of Scenario 2 showed that drift fences and underpasses have nega- tive effects on the ecological performance of this landscape. This result is highly surprising as well as controversial since fences and underpasses are standard mitiga- tion measures used in many road projects (Iuell et al. 2003). Even though fences and underpasses are supposed to prevent road mortality and promote connectivity, the overall annual survival rate, as well as connectivity, decreased. These effects are prob- ably mostly due to the fences. Underpasses per se do not change movement patterns, but fences do. Moreover, we did see increased connectivity locally across the road between c8 and c9. Fences may force individuals to move along the road, exposing them to low quality habitat for a longer time. Furthermore, the mitigation meas- ures may be counterproductive if the combination of fences and underpasses lead individuals into low quality habitat or areas without ponds to colonize. The popula- tion dynamics in the ponds is an emergent property, dependent on local conditions as well as regional dynamics. The change in connectivity and movement patterns caused by the mitigation measures, therefore, seems to be able to affect abundances even in clusters farther away. Very little is known about the effects of mitigation measures on connectivity and local and regional population persistence. Once mitigation measures are implemented, efforts are seldom put into discovering how well they work. Recordings of animals using wild life passages reveal nothing about effects on local and regional persistence (Lesbarreres and Fahrig 2012; van der Grift et al. 2013). In a simulation study on a generic species, Jaeger and Fahrig (2004) found that fencing, while preventing road mortality, did not necessarily improve population persistence and they recommended fencing only when road mortality is 100 %. Discussion In a study on moose (Alces alces), Olsson and Widen (2008) found that fences resulted in decreased use of wildlife passages. Our simulation results underscore the need for a better understanding of how mitigation measures affect animal behaviour and population dynamics. Conclusion When planning road constructions, it is important to integrate mitigation measures right from the start. Often there are economic constraints on which measures are pos- sible, certain structures as viaducts or bridges may already be in place or land available for compensation measures is restricted. SAIA offers a tool to evaluate different scenar- ios to find the best combination of mitigation measures for a given set of conditions. The model is meant to be used by non-specialists – all that is needed are GIS maps of the different scenarios. We attempted to find a balance between detailed and yet intui- tive and easy interpretable output. Even though SAIA was developed for the Danish Road Directorate, its use is not restricted to road constructions but can be applied to other structures affecting the landscape and their potential impacts on wildlife. Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 48 Acknowledgments The work was funded by the Danish Road Directorate. We thank Amphi Consult for providing us with amphibian expertise and field data. We are grateful for continuous and enthusiastic feed-back from M. Ujvári, M. Hesselsøe, A. Jørgensen and M. Sch- neekloth during model development. Special thanks are due to Uta Berger for encour- aging and inspiring discussions. We thank Michal J. Reed for linguistic assistance. 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Trombulak SC, Frissell CA (2000) Review of ecological effects of roads on terrestrial and aquatic communities. Conservation Biology 14: 18–30. doi: 10.1046/j.1523-1739.2000.99084.x van der Grift EA, van der Ree R, Fahrig L, Findlay S, Houlahan J, Jaeger JAG, Klar N, Madri- nan LF, Olson L (2013) Evaluating the effectiveness of road mitigation measures. Biodi- versity and Conservation 22: 425–448. doi: 10.1007/s10531-012-0421-0 Vos CC, Chardon JP (1998) Effects of habitat fragmentation and road density on the distribu- tion pattern of the moor frog Rana arvalis. Journal of Applied Ecology 35: 44–56. doi: 10.1046/j.1365-2664.1998.00284.x j Watts K, Handley P (2010) Developing a functional connectivity indicator to detect change in fragmented landscapes. Ecological Indicators 10: 552–557. doi: 10.1016/j.ec- olind.2009.07.009 Wiens JA (1997) Metapopulation Dynamics and Landscape Ecology. In: Hanski I, Gilpin ME (Eds) Metapopulation Biology: ecology, genetics, and evolution. Academic press, Inc., Wilensky U (1999) NetLogo. Center for Connected Learning and Computer-Based Modeling, Northwestern University, Evanston, IL. http://ccl.northwestern.edu/netlogo Zetterberg A, Mortberg UM, Balfors B (2010) Making graph theory operational for landscape ecological assessments, planning, and design. Landscape and urban planning 95: 181–191. doi: 10.1016/j.landurbplan.2010.01.002 Maj-Britt Pontoppidan & Gösta Nachman / Nature Conservation 5: 29–52 (2013) 52 Citation: Pontoppidan M-B, Nachman G (2013) Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis). Nature Conservation 5: 29–52. doi: 10.3897/natureconservation.5.4612.app 10.3897/natureconservation.5.4612.app Appendix Full model description following the protocol suggested by Grimm et al. (2006, 2010) and model parameterisation. (doi: 10.3897/natureconservation.5.4612.app). File for- mat: Adobe PDF document (pdf). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Citation: Pontoppidan M-B, Nachman G (2013) Spatial Amphibian Impact Assessment – a management tool for assessment of road effects on regional populations of Moor frogs (Rana arvalis). Nature Conservation 5: 29–52. doi: 10.3897/natureconservation.5.4612.app
https://openalex.org/W4213440259	https://www.research.ed.ac.uk/files/200551280/OA_jad_prepress_jad_1_1_jad201256_jad_1_jad201256.pdf	English	null	Variation in VKORC1 Is Associated with Vascular Dementia	Journal of Alzheimer's disease	2,021	cc-by	7,809	Variation in VKORC1 is associated with vascular dement Citation for published version: Mur, J, McCartney, DL, Chasman, DI, Visscher, PM, Terrera, GM, Cox, S, Russ, T & Marioni, RE 2021, 'Variation in VKORC1 is associated with vascular dementia', Journal of Alzheimer's Disease. https://doi.org/10.3233/JAD-201256 General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Digital Object Identifier (DOI): 10.3233/JAD-201256 Document Version: Publisher's PDF, also known as Version of record Published In: Journal of Alzheimer's Disease Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Journal of Alzheimer’s Disease xx (20xx) x–xx DOI 10.3233/JAD-201256 IOS Press Abstract. 17 ted Background: The genetic variant rs9923231 (VKORC1) is associated with differences in the coagulation of blood and consequentially with sensitivity to the drug warfarin. Variation in VKORC1 has been linked in a gene-based test to dementia/Alzheimer’s disease in the parents of participants, with suggestive evidence for an association for rs9923231 (p = 1.8 × 10–7), which was included in the genome-wide signiﬁcant KAT8 locus. 18 19 20 21 cte Objective: Our study aimed to investigate whether the relationship between rs9923231 and dementia persists only for certain dementia sub-types, and if those taking warfarin are at greater risk. 22 23 ect Methods: We used logistic regression and data from 238,195 participants from UK Biobank to examine the relationship between VKORC1, risk of dementia, and the interplay with warfarin use. 4 5 orrec Results: Parental history of dementia, APOE variant, atrial ﬁbrillation, diabetes, hypertension, and hypercholesterolemia all had strong associations with vascular dementia (p < 4.6 × 10–6). The T-allele in rs9923231 was linked to a lower warfarin dose (perT-allele = –0.29, p < 2 × 10–16) and risk of vascular dementia (OR = 1.17, p = 0.010), but not other dementia sub-types. However, the risk of vascular dementia was not affected by warfarin use in carriers of the T-allele. 26 27 28 29 cor Conclusion: Our study reports for the ﬁrst time an association between rs9923231 and vascular dementia, but further research is warranted to explore potential mechanisms and specify the relationship between rs9923231 and features of vascular dementia. 30 31 32 nc Keywords: Alzheimer disease, genetics, vascular dementia, warfarin 33 Un Variation in VKORC1 is Associated with Vascular Dementia Proof Jure Mura,b,c, Daniel L. McCartneyb, Daniel I. Chasmand, Peter M. Visschere, Graciela Muniz-Terreraf,g, Simon R. Coxa, Tom C. Russc,f,g and Riccardo E. Marionib,∗ 3 4 aLothian Birth Cohorts group, Department of Psychology, University of Edinburgh, Edinburgh, UK 5 bCentre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 6 7 Author Proo Jure Mura,b,c, Daniel L. McCartneyb, Daniel I. Chasmand, Peter M. Visschere, Graciela Muniz-Terreraf,g, Simon R. Coxa, Tom C. Russc,f,g and Riccardo E. Marionib,∗ 3 4 aLothian Birth Cohorts group, Department of Psychology, University of Edinburgh, Edinburgh, UK 5 bCentre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 6 7 cAlzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, UK 8 dDivision of Preventive Medicine, Brigham and Women’s Hospital & Harvard Medical School, Boston, MA, USA 9 10 eInstitute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia 11 fEdinburgh Dementia Prevention, University of Edinburgh, Edinburgh, UK 12 gDivision of Psychiatry, Centre for Clinical Brain Science, University of Edinburgh, Edinburgh, UK 13 Handling Associate Editor: M. Arfan Ikram 14 Accepted 27 January 2021 15 Pre-press 27 February 2021 16 r P cAlzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, UK 8 dDivision of Preventive Medicine, Brigham and Women’s Hospital & Harvard Medical School, Boston, MA, USA 9 0 hor eInstitute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia fEdinburgh Dementia Prevention, University of Edinburgh, Edinburgh, UK ho gDivision of Psychiatry, Centre for Clinical Brain Science, University of Edinburgh, Edinburgh, UK 13 d Auth Accepted 27 January 2021 Pre-press 27 February 2021 ISSN 1387-2877 © 2021 – The authors. Published by IOS Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). Sample r Proo We used data from UK Biobank, a large and detailed prospective study of over 500,000 partici- pants aged 37–73 that were recruited between the years 2006 and 2010. UK Biobank has been described in detail before [13]. The Research Ethics Committee (REC) granted ethical approval for the study (ref- erence 11/NW/0382) and the current analysis was conducted under data application 10279. rrec In a recent genome-wide association study (GW AS) meta-analysis of parental dementia and case- control Alzheimer’s dementia (ADem) [9], VKORC1 was associated (after Bonferroni correction) with ADem in a gene-based test (p = 5.1 × 10–8); the T- allele in rs9923231, which is related to the need for a lower dose of warfarin, was not a genome-wide signiﬁcant ﬁnding, but was both located within a genome-wide signiﬁcant locus and nominally associ- ated with an increased risk of ADem (p = 1.8 × 10–7). Pure Alzheimer’s disease pathology, characterized by amyloid plaques and neuroﬁbrillary tangles in the grey matter, is uncommon, and most patients exhibit a mixed pathology in which vascular factors often play a prominent role [10]. In fact, there is exten- sive evidence directly linking vascular dysfunction to ADem [11]. Thus, a possible explanation for the ﬁndings [9] is that vascular factors played a crucial role in a proportion of the ADem cases/family history cases observed. If that is the case, then there should be an even stronger relationship between VKORC1 and vascular dementia (VaD) that is mostly due to cardiovascular factors. Genotyping cted Autho Details on genotyping in the UK Biobank have been reported before [14, 15]. Brieﬂy, for 49,950 par- ticipants, genotyping was performed using the UK BiLEVE Axiom Array, and for 438,427 participants, genotyping was performed using the UK Biobank Axiom Array. The released data contained 805,426 markersfor488,377participants.Furtherqualitycon- trol steps were performed as previously reported [9]. They included the removal of outliers, of incongru- ent data points, and of related participants using a relationship cut-off of 0.025 (GCTA GREML) [16]. This left an unrelated cohort of 314,278 individuals of white British ancestries (Fig. 1). INTRODUCTION Warfarin is the most prescribed anticoagulant worldwide [1] and is commonly used as a treatment for atrial ﬁbrillation (AF) [2]. The drug functions by inhibiting the enzyme vitamin K epoxide reductase ∗Correspondence to: Riccardo E. Marioni, Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK. Tel.: +1 44 0 1316518528; E-mail: riccardo.marioni@ed.ac.uk. ISSN 1387-2877 © 2021 – The authors. Published by IOS Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). J. Mur et al. / Variation in VKORC1 is Associated with Vascular Dementia 2 (VKOR), effectively interfering with the vitamin K cycle requiredfor coagulationofblood[3]. Asaresult of variations in age, height, weight, genotype, and other factors [4–6], patients vary up to 20-fold in their sensitivity to warfarin [7]. Clinically, the optimum dose is estimated using tests of blood coagulation, commonly the International Normalized Ratio (INR). The strongest genetic predictor of warfarin sensitivity is the gene VKORC1, which encodes for the vitamin K epoxide reductase subunit 1 (VKORC1) and acco- unts for approximately a third of the variance in warfarin sensitivity [3]. Three VKORC1 SNPs, rs99 23231, rs9934438, and rs2359612—which are in very high linkage disequilibrium—are the best gen- etic predictors of warfarin sensitivity [3, 7, 8]. (VKOR), effectively interfering with the vitamin K cycle requiredfor coagulationofblood[3]. Asaresult of variations in age, height, weight, genotype, and other factors [4–6], patients vary up to 20-fold in their sensitivity to warfarin [7]. Clinically, the optimum dose is estimated using tests of blood coagulation, commonly the International Normalized Ratio (INR). The strongest genetic predictor of warfarin sensitivity is the gene VKORC1, which encodes for the vitamin K epoxide reductase subunit 1 (VKORC1) and acco- unts for approximately a third of the variance in warfarin sensitivity [3]. Three VKORC1 SNPs, rs99 23231, rs9934438, and rs2359612—which are in very high linkage disequilibrium—are the best gen- etic predictors of warfarin sensitivity [3, 7, 8]. T-allele status is associated with an increased risk of VaD and explore whether carriers of the T-allele are at a greater risk of VaD than non-carriers when prescribed warfarin. Models cted Au All analyses where the outcome variable was con- tinuous were performed using linear regression; all models where the outcome variable was binary were performed using logistic regression. All models were controlled for the assessment center in which the par- ticipant was tested, the genotyping- batch and array, 40 genetic principal components, the age, sex, edu- cation,socioeconomicdeprivation,alcoholconsump- tion, smoking, physical activity, and body mass index (BMI) of the participants. The models predicting VaD were subsequently additionally controlled for APOE variant, concentration of triglycerides (mmol/L), and the diagnoses of hypertension, hypercholesterolemia, and diabetes. All covariates were ascertained imme- diately prior to or during the participants’ recruitment to the UK Biobank. For education, a binary classiﬁ- cation was used that indicated whether a graduate degree had been attained. For socioeconomic depri- vation, the Townsend index [17] was used, where higher values indicate greater socioeconomic depri- vation (range in the sample: –6.3–10.8). For alcohol consumption, a 6-level scale of frequency of alco- hol consumption was used, where 1: “daily or almost daily”, 2: “three or four times a week”, 3:“one or two times a week”, 4: “one to three times a month”, 5: “special occasions only”, 6: “never”. For smok- ing, the participants were classiﬁed as non-smokers, past smokers, or current smokers. For physical activ- ity, the scale provided by the UK Biobank was ncorrec without a date, and duplicate prescriptions (deﬁned as identical prescriptions issued to the same person on the same day) were removed from the sample. This resulted in the removal of 1,467,547 prescrip- tions. Three participants were completely removed from the dataset (Fig. 1). Warfarin prescriptions were extracted by searching for the word “warfarin” under the name/content of each prescription. For each par- ticipant, we calculated warfarin use by summing the number of days on which warfarin was prescribed, and warfarin dose by averaging the prescribed dose over all prescriptions of warfarin. c Warfarin prescription data hor Proof These data included Hospital Episode Statistics for England, Scottish Morbidity Records for Scotland, and the Patient Episode Database for Wales. Peo- ple with record of any dementia were included in a broad dementia category of “general dementia” that included ADem and VaD, as well as other types of dementia. Furthermore, narrower, more speciﬁc categories (ADem, VaD) were also identiﬁed. Infor- mation on the codes used in the extraction of each diagnosis is provided in Supplementary Table 1. We excluded from our analyses all participants that were 60 years old or younger on the last date of sampling (June 30, 2020) since dementia risk increases steeply with age. Parental diagnoses were ascertained during the initial assessment by asking participants about the presence of “Alzheimer’s disease/dementia” for both mother and father. In our analyses, the parental diag- nosis of dementia was considered positive if at least one parent was reported to have suffered from the disorder. Fig. 1. The data cleaning procedure. The left path (orange boxes) represents the genotyping and associated quality control, the mid- dle path (blue box) represents the ascertainment of primary care- and inpatient diagnoses, and the right path (yellow boxes) repre- sents the linkage to primary care prescriptions and the cleaning of the latter. The last two steps (grey boxes) involve the inclusion of only those participants that were older than 60 at the end of sampling and who passed through the left and middle paths (ﬁrst grey box, 238,195 participants), or through all three data-cleaning paths (second grey box, 115,206 participants). All analyses that did not include prescribing data in the models were performed using the 238,195 participants, while the analyses that utilized warfarin prescription history used the 115,206 participants. c Warfarin prescription data The UK Biobank obtained data on prescriptions for 222,111 participants via primary care computer system suppliers (EMIS Health and Vision for Scot- land, and Wales, Vision and The Phoenix Partnership for England) and has engaged other intermediaries (Albasoft, a third-party data processor, for Scotland and the SAIL databank for Wales). All participants provided written consent for linkage to their health records upon recruitment to UK Biobank. The data were extracted in May 2017 for Scotland, in Septem- ber 2017 for Wales, and in June, in July, and in August 2017 for England. The data include the exact dates of prescriptions, drug codes (BNF, Read v2, CTV3, and dm + d), names of drugs as written on the prescription, and, where available, the dosages of prescribed drugs. Empty prescriptions, prescriptions Uncor Most strokes in western countries are due to occlu- sions in blood vessels (ischemic), and some are due to ruptures in blood vessels (hemorrhagic) [12]. If carriers of the T-allele in rs9923231 experience a reduction of blood coagulation and subsequent seq- uential minor hemorrhagic strokes, the resulting pa- thology could manifest in dementia and explain the observedlink.Furthermore,comparedtonon-carriers of the T-allele, patients with AF that carry the T-allele could be at an increased risk of intracerebral hem- orrhage and consequentially VaD when prescribed warfarin. Here, we study the same UK Biobank cohort as previously [9], but consider both individ- ual and parental dementia status. We test whether J. Mur et al. / Variation in VKORC1 is Associated with Vascular Dementia 3 3 Fig. 1. The data cleaning procedure. The left path (orange boxes) represents the genotyping and associated quality control, the mid- dle path (blue box) represents the ascertainment of primary care- and inpatient diagnoses, and the right path (yellow boxes) repre- sents the linkage to primary care prescriptions and the cleaning of the latter. The last two steps (grey boxes) involve the inclusion of only those participants that were older than 60 at the end of sampling and who passed through the left and middle paths (ﬁrst grey box, 238,195 participants), or through all three data-cleaning paths (second grey box, 115,206 participants). All analyses that did not include prescribing data in the models were performed using the 238,195 participants, while the analyses that utilized warfarin prescription history used the 115,206 participants. RESULTS reduced to a 3-level scale, indicating light, moderate, 205 or strenuous physical activity, as has been used before 206 [18]. For APOE genotype based on the nucleotides 207 at SNP positions rs429358 and rs7412, participants 208 with the 3/3 haplotype were denoted as carrying 209 variant 3, participants with the 2/2 or 2/3 hap- 210 lotypes were denoted as carrying variant 2, and 211 participants with the 3/4 or 4/4 haplotypes were 212 denoted as carrying variant 4. Brain imaging data, 213 including the volume of white matter hyperintensi- 214 ties (WMH), were available for 18,251 participants 215 in the sample. For analysis where WMH was mod- 216 elled as an outcome, WMH was log-transformed 217 and corrected for intracranial volume. For analyses 218 where parental diagnoses were modelled as out- 219 comes, the ages of each parent (current age or age 220 at death) were included in the models. In all cases 221 where we tested for associations between rs9923231 222 (VKORC1) and any form of dementia, we assumed 223 an additive genetic effect for rs99232331. All covari- 224 ates were simultaneously added to the model and 225 the models were not corrected for multiple com- 226 parisons. The effects are reported in odds ratios 227 (OR’s) or unstandardized beta-coefﬁcients. All anal- 228 yses were performed in R version 3.6.3. The code 229 for preparing and analyzing the data is available at 230 https://github.com/Logos24/VKORC1-and-VaD. 231 232 Disease status U Data on diagnoses for 465,510 participants were obtained by the UK Biobank from two sources: 1) from primary care similarly to the prescriptions described above, and 2) from hospital inpatient admissions data. Inpatients are deﬁned as people who are admitted to hospital and occupy a hospital bed. J. Mur et al. / Variation in VKORC1 is Associated with Vascular Dementia 4 reduced to a 3-level scale, indicating light, moderate, 205 or strenuous physical activity, as has been used before 206 [18]. For APOE genotype based on the nucleotides 207 at SNP positions rs429358 and rs7412, participants 208 with the 3/3 haplotype were denoted as carrying 209 variant 3, participants with the 2/2 or 2/3 hap- 210 lotypes were denoted as carrying variant 2, and 211 participants with the 3/4 or 4/4 haplotypes were 212 denoted as carrying variant 4. Brain imaging data, 213 including the volume of white matter hyperintensi- 214 ties (WMH), were available for 18,251 participants 215 in the sample. For analysis where WMH was mod- 216 elled as an outcome, WMH was log-transformed 217 and corrected for intracranial volume. For analyses 218 where parental diagnoses were modelled as out- 219 comes, the ages of each parent (current age or age 220 at death) were included in the models. In all cases 221 where we tested for associations between rs9923231 222 (VKORC1) and any form of dementia, we assumed 223 an additive genetic effect for rs99232331. All covari- 224 ates were simultaneously added to the model and 225 the models were not corrected for multiple com- 226 parisons. The effects are reported in odds ratios 227 (OR’s) or unstandardized beta-coefﬁcients. All anal- 228 yses were performed in R version 3.6.3. The code 229 for preparing and analyzing the data is available at 230 https://github.com/Logos24/VKORC1-and-VaD. 231 Table 2 Table 2 Table 2 Results of the additive models with T as the effect allele, using rs9923231 to predict parental dementia, general dementia, ADem, and VaD sub-sample used for analyses utilizing prescription history (Fig. 1) were very similar to the entire sample (Supplementary Table 2). A total of 13,361 (5.6%) participants had been diagnosed with AF and among the 115,206 participants with data on prescriptions, 5,513 (4.8%) had a history of being prescribed war- farin (Supplementary Table 3). oof Effect per T allele rs9923231 OR 95% CI p n cases Parental dementia 1.04 1.02–1.06 3.7 × 10–5 34,737 General dementia 1.02 0.98–1.07 0.33 4,259 ADem 1.02 0.94–1.10 0.60 1,531 VaD 1.17 1.04–1.32 0.010 669 pp y There were 145,186 (61.0%) carriers of the T-allele 246 in the sample: 111,756 (46.9%) were heterozygous 247 for the T-allele, and 33,430 (14.0%) were homozy- 248 gous for the T-allele; the allele frequencies were in 249 Hardy-Weinberg equilibrium (χ2 = 0.23, df = 1, p = 250 0.63). Among the participants, 4,259 (1.8%) had 251 suspected general dementia, 1,531 (0.64%) had 252 suspected ADem (Supplementary Table 4, Supple- 253 mentary Figure 1), and 669 0.28%) had suspected 254 VaD (Supplementary Table 4, Supplementary Fig- 255 ure 1); 152 participants (0.03%) had been diagnosed 256 with both ADem and VaD. People with at least 257 one parent with dementia were more likely develop 258 ADem (OR = 3.0, 95% CI = 2.6–3.4, p < 2.0 × 10–16) 259 and more likely to develop VaD (OR = 2.1, 95% 260 CI = 1.7–2.7, p < 1.9 × 10–9). 261 cted Author Pro When limited to the speciﬁc outcome of VaD, the additive effect of the T-allele was much larger (OR = 1.17, 95% CI = 1.04–1.32, p = 0.010, Table 2, Fig. 2). The full breakdown of all allele groups is shown in Supplementary Table 5. We repeated the models for VaD, with rs9923231 as a predictor and with the simultaneous addition of concentration of triglycerides, APOE variant, diagnoses of hyperten- sion (n = 84,694), hypercholesterolemia (n = 40,363), and diabetes (n = 20,990) as additional covariates. While triglycerides, APOE variant, hypertension, hypercholesterolemia, and diabetes were signiﬁcant predictors, this did not affect the relationship between rs9923231 and VaD (Supplementary Table 6). rs9923231 polymorphism and warfarin dose 262 correc Carrying the T-allele was negatively associated 263 with the average dose of warfarin (perT-allele = –0.29, 264 SE = 0.015, p < 2.0 × 10–16). Individuals heterozy- 265 gous for the T-allele were prescribed a dose of war- 266 farin that was on average 0.23 mg smaller than the 267 dose prescribed to non-carriers (SE = 0.022, p < 268 2.0 × 10–16), while individuals homozygous for the 269 T-allele were prescribed a dose of warfarin that was 270 on average 0.62 mg smaller than the dose presc- 271 ribed to non-carriers (SE = 0.032, p < 2.0 × 10–16). 272 The average dose of warfarin was also negatively 273 associated with age ( = –0.010, SE = 2.0 × 10–3, 274 p = 4.1 × 10–7), and was higher in males ( = 0.062, 275 SE = 0.022, p = 5.7 × 10–3). 276 Warfarin use and VaD in carriers of the T-allele In our sample, participants diagnosed with AF 314 were at greater risk for ADem (OR = 1.55, 95% 315 CI = 1.31–1.81, p = 1.7 × 10–7) and for VaD (OR = 316 2.92, 95% CI = 2.38–3.57, p < 2.0 × 10–16). The 317 effect remained signiﬁcant for both ADem (OR = 318 1.29, 95% CI = 1.08–1.53, p = 4.5 × 10–3) and VaD 319 (OR = 2.17, 95% CI = 1.74–2.69, p = 1.9 × 10–12) 320 when APOE status, triglycerides, and diagnoses of 321 hypercholesterolemia, hypertension, and diabetes 322 were included in the model as covariates. To test 323 whether warfarin use in T-allele carriers diagnosed 324 Table 2 Beca- use of the importance of cardiovascular events in the etiology of VaD, the T-allele was also used to predict stroke, with the full set of covariates as above. The models were not signiﬁcant for ischemic (n = 8,087, OR = 0.98, 95% CI = 0.94–1.01, p = 0.21), nor for hemorrhagic (n = 2,146, OR = 0.94, 95% CI = 0.88–1.01, p = 0.073) stroke. Due to the likely causal link between WMH and dementia [19], rs9923231 was related to WMH in the sample. When all the above covariates were included in the model, the association was signiﬁcant, with the T-allele neg- atively associated with WMH (beta = –2.3 × 10–8, SE = 7.5 × 10–9, p = 2.8 × 10–3). Sample characteristics 233 of Among the 238,195 participants, 129,034 (54.2%) were female and 109,161 (45.8%) were male (Table 1). The age range at recruitment was 46–74 years (Fig. 2) and the median age was 60.9 years (IQR = 9.1). The demographic characteristics of the Author Proo d Author Proo Fig. 2. Odds ratios for parental dementia, ADem, and VaD per rs9923231 genotype status. Depicted are the additive effect and the effects of each allele group. The tails represent 95% conﬁdence intervals for the ORs. d A Fig. 2. Odds ratios for parental dementia, ADem, and VaD per rs9923231 genotype status. Depicted are the additive effect and the effects of each allele group. The tails represent 95% conﬁdence intervals for the ORs. Uncorrected Table 1 Demographic characteristics of the sample Variable Level Median (IQR) or n (%) All General dementia ADem VaD (n = 238,195) (n = 4259) (1531) (669) Age 60.9 (9.1) 65.5 (5.8) 65.9 (5.3) 66.3 (4.7) Sex Female 129,034 (54.2) 1,939 (45.5) 795 (51.9) 267 (39.9) Male 109,161 (45.8) 2,320 (54.5) 736 (48.1) 402 (60.1) Education Graduate degree 72,385 (30.7) 947 (22.6) 313 (20.9) 115 (17.6) No graduate degree 163,563 (69.3) 3,235 (77.4) 1,199 (79.1) 539 (82.4) Deprivation –2.5 (3.6) –2.2 (4.2) –2.3 (4.1) –2.2 (3.8) Alcohol consumption Daily or almost daily 54,261 (22.8) 990 (23.3) 311 (20.3) 152 (22.8) 3 or 4 times a week 57,255 (24.1) 814 (19.1) 313 (20.5) 117 (17.5) 1 or 2 times a week 60,106 (25.2) 952 (22.4) 357 (23.3) 147 (22.0) 1–3 times a month 24,778 (10.4) 396 (9.3) 156 (10.2) 59 (8.8) Special occasions only 25,409 (10.7) 576 (13.5) 215 (14.1) 91 (13.6) Never 16,239 (6.8) 524 (12.3) 177 (11.6) 101 (15.1) Smoking Current smoker 21,470 (9.0) 413 (9.8) 115 (7.6) 74 (11.2) Previous smoker 89,608 (37.8) 1,839 (43.4) 647 (42.6) 310 (46.8) Non-smoker 126,288 (53.2) 1,983 (46.8) 757 (49.8) 278 (42.0) Physical activity Strenuous 19,441 (8.7) 186 (4.9) 74 (5.2) 27 (4.7) Moderate 148,812 (66.6) 2,350 (62.1) 908 (64.2) 359 (62.2) Light 55,137 (24.7) 1,247 (33.0) 432 (30.6) 191 (28.6) BMI 26.8 (5.7) 27.1 (6.0) 26.8 (5.6) 27.8 (7.1) APOE variant 2 30,818 (13.3) 306 (8.2) 79 (5.3) 51 (7.8) 3 138,634 (59.7) 1,559 (42.0) 505 (33.6) 275 (42.1) 4 62,665 (27.0) 1,846 (49.7) 917 (61.1) 327 (50.1) te Demographic characteristics of the sample J. Mur et al. / Variation in VKORC1 is Associated with Vascular Dementia 5 5 DISCUSSION ec In this study, we explored the relationship between suspected dementia, atrial ﬁbrillation, warfarin use, and rs9923231, whose T-allele is associated with a reduction in the dose of warfarin [3, 7, 8]. We found a signiﬁcant association between rs9923231 and sus- pected VaD, but not between rs9923231 and either suspected general dementia or suspected ADem. While AF was linked to VaD, the use of warfarin in patients that have AF and carry the T-allele did not increase the risk for VaD. cted A Based on our results and considering the impo- rtance of cardiovascular abnormalities in the pathol- ogy of dementia [10, 11], any future studies exploring the association between rs9923231 and dementia must strongly consider the role of cardiovascular factors: The relationship between genotype and dem- entia might hold only for cases of pure VaD or for thoseinwhichvascularpathologyrepresentsthemain cause of the disorder. Uncorrec While there have been reports of variants for mono- 356 genic forms of VaD [20], data on the genetics of 357 sporadic VaD are sparse. To our knowledge, only two 358 GWAS have been conducted to investigate this: One 359 (n = 5,700) [21] found only rs12007229 on the X- 360 chromosome to be linked to incident VaD, while the 361 other (n = 284) [22] did not ﬁnd any signiﬁcant asso- 362 ciations for VaD. A systematic review of all genetic 363 association studies for the broader term of vascular 364 cognitiveimpairmentfoundanassociationfor6SNPs 365 in 6 genes: APOE, ACT, ACE, MTHFR, PON1, and 366 PSEN-1 [23]. 367 There is an established association between dem- entia and both stroke [27] and WMH [28]; thus, stroke or WMH could act as mediators between rs9923231 genotype and VaD. However, we found no evidence for a positive association between either rs9923231 and stroke or rs9923231and WMH. Moreover, the latter association was statistically signiﬁcant and neg- ative in direction, suggesting participants carrying the T-allele were less likely to exhibit WMH. While we did not directly test for the effects of other rel- evant processes, including microbleeds and covert stroke, in the relationship between rs9923231 and VaD, given the lack of evidence for an association between rs9923231 and stroke, they are unlikely to act as prominent mediators. Unc rs9923231 polymorphism and dementia risk 277 Un Parents of carriers of the T-allele were more li- 278 kely to have developed dementia (additive effect 279 per T-allele: OR = 1.04, 95% CI = 1.02–1.06, p = 3.7 280 × 10–5). When the presence of the T-allele was used 281 to predict general dementia in participants, the effect 282 was not signiﬁcant, nor was the effect signiﬁcant 283 when the presence of the T-allele was used to predict 284 ADem in participants (Table 2, Fig. 2). 285 J. Mur et al. / Variation in VKORC1 is Associated with Vascular Dementia 6 Author Proof rs9923231 and VaD, although it is important to note that this is not at a genome-wide signiﬁcant thresh- old. The lack of a relationship between rs9923231 and either ADem or general dementia in the present study suggests that the association between the T- allele and ADem, as reported previously [9], might have been partly due to the classiﬁcation of parental dementia. The UK Biobank questionnaire admin- istered to participants did not distinguish between different types of dementia and it is not known how many of the 42,034 parents that were reportedly diag- nosed with “Alzheimer’s/dementia” [9] may have suffered from VaD. This hypothesis is further sup- ported by the estimated effect sizes for the association between rs9923231 and ADem, which were not num- erically larger than those for the association between rs9923231 genotype and parental dementia. Since parental dementia was used as a proxy for ADem in participants, the effect for ADem in participants sho- uld have been substantially greater than for parental dementia (even in the absence of statistical signif- icance) if there truly was an association between rs9923231 and ADem (as opposed to an association between rs9923231 and VaD). Furthermore, in a rec- ent GWAS of clinically diagnosed ADem (n = 94,437) [26] there was no association between rs 9923231 and ADem. with AF increases the risk of VaD, we performed a 5 logistic model with AF, warfarin use, and rs9923231 6 predicting VaD, with the inclusion of a 3-way interac- 7 tion term between AF, warfarin, and rs9923231. The 8 interaction between AF, warfarin use, and rs9923231 9 was not signiﬁcant (OR = 0.99, 95 % CI = 0.98–1.00, 0 p = 0.063). Unc rs9923231 polymorphism and dementia risk 277 The two-way interactions between the 1 above variables were also not signiﬁcant and effect 2 sizes (main effects) were not substantially attenuated 3 by the addition of the other variables into the mod- 4 els (Supplementary Tables 7 and 8). Due to the small 5 number of people with VaD and very limited statis- 6 tical power for these analyses (Supplementary Text 7 1), we repeated the analysis by modelling parental 8 dementia as an outcome and including the 3-way 9 interaction term as above; parental dementia was 0 thus treated as a proxy for VaD in the participa- 1 nts. The interaction between AF, warfarin use, and 2 carrier-status was not signiﬁcant (OR = 0.999, 95% 3 CI = 0.996–1.00, p = 0.77). 4 Interplay between AF, VaD and warfarin use 429 AF has been previously associated with cogni- tive decline and dementia. In our study, AF was associated with VaD and with ADem, even after con- trolling for hypertension and hypercholesterolemia. The association between AF and VaD is unsurprising, considering the inclusion of either vascular disease or history of stroke in almost all deﬁnitions of VaD [29]. Despite a substantial overlap of risk factors for AF and ADem, there is some evidence for an inde- pendent relationship between the two disorders [30, 31] ed Author The knowledge of genetic risk factors for diseases enables the generation of more accurate hypothe- ses about underlying biological mechanisms and illuminates potential targets for pharmacological intervention. Moreover, it allows for more informed stratiﬁcation of participants in clinical trials. Stud- ies that build on our research should aim to replicate the ﬁndings in a bigger sample and with greater pre- cision determine the effect size for the association between rs9923231 and VaD. Additionally, further work is required to identify possible associations between rs9923231 and features of VaD, such as lacu- nar infarction, intracerebral hemorrhage, and white matter hyperintensities. ec Due to the positive association between AF and VaD, the relationship between rs9923231 and VaD, and between rs9923231 and required warfarin dose, T-allele carriers that take warfarin to treat their AF might be at an increased risk of VaD than non-carriers due to warfarin-related brain hemorrhages. To test this, we studied an interaction between warfarin use, AF, and VKORC1 genotype with VaD. We observed no variation in dementia risk by different combina- tions of these predictors. Due to reduced coagulation of blood in carriers of the T-allele, these individu- als could be at greater risk of internal bleeding when taking warfarin. However, the required dose of war- farin is regularly estimated and adjusted using tests of blood coagulation and based on the results of the present paper, this approach is just as efﬁcient in patients carrying the T-allele. cte ACKNOWLEDGMENTS c DLM and REM are supported by Alzheimer’s Research UK major project grant ARUK-PG2017B- 10. JM is supported by funding from the Wellcome Trust 4-year PhD in Translational Neuroscience— training the next generation of basic neuroscientists to embrace clinical research [108890/Z/15/Z]. PMV acknowledges funding from the Australian National Health and Medical Research Council (1113400) and the Australian Research Council (FL180100072). JM and TCR are members of the Alzheimer Scot- land Dementia Research Centre funded by Alzheimer Scotland. TCR is employed by NHS Lothian and the Scottish Government. SRC is supported by Age UK (Disconnected Mind project), the UK Medical Research Council [MR/R024065/1] and a National Institutes of Health (NIH) research grant R01AG054628. The authors thank all participants of the UK Biobank for providing data for the study DISCUSSION These results fur- ther complicate the potential relationship between U Previous research has associated variation in rs9923231 with warfarin dose [3, 7, 8, 24], and with various adiposity-related traits, such as hip cir- cumference, arm- and leg fat mass, and BMI (Gene Atlas [25]). To our knowledge the present study for the ﬁrst time describes an association between J. Mur et al. / Variation in VKORC1 is Associated with Vascular Dementia 7 rs9923231 and VaD and reinforce the need for addi- tional studies to conﬁrm this association and to test alternative mechanism distinct from stroke or WMH. r Proof average of their prescribed dose, despite some indi- viduals possibly taking more or less of the medicine depending on their individual drug regimes. Fourth, clinical diagnoses of dementia subtypes are difﬁ- cult and are prone to errors due to the presence of comorbidities and cardiovascular factors [32]. In the present paper, imaging data to conﬁrm the diagnoses was unavailable and all diagnoses were based solely on records from primary care and hospitals. Finally, while most deﬁnitions of VaD include both dementia and a history of stroke or cardiovascular disease, VaD is very heterogeneous [33]; in the present study, we did not explore potential mechanisms and mediators of the association between rs9923231 and VaD, nor did we test the relationship for different subtypes of VaD. 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W4353062105.txt	https://www.researchsquare.com/article/rs-2447422/latest.pdf	en		Effect of Water Absorption on Graphene Nanoplatelet and Multiwalled Carbon Nanotubes-impregnated Glass Fibre-Reinforced Epoxy Composites	Journal of inorganic and organometallic polymers and materials	2,023	cc-by	182	WITHDRAWN: Effect of water absorption on graphene nanoplatelet and multiwalled carbon nanotubes- impregnated glass-reinforced epoxy composites Research Article Keywords: Posted Date: February 6th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2447422/v2 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 Journal of Inorganic and Organometallic Polymers and Materials on March 22nd, 2023. See the published version at https://doi.org/10.1007/s10904-023-02610-2. 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 Abstract The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Full Text The authors have withdrawn this preprint from Research Square. Page 2/2
https://openalex.org/W1785097165	https://www.hal.inserm.fr/inserm-00092484/file/1471-213X-5-6.pdf	English	null	Delta activity independent of its activity as a ligand of Notch	BMC developmental biology	2,005	cc-by	10,093	To cite this version: Lee-Peng Mok, Tielin Qin, Boris Bardot, Matthew Lecomte, Asal Homayouni, et al.. Delta activ- ity independent of its activity as a ligand of Notch.. BMC Developmental Biology, 2005, 5, pp.6. 10.1186/1471-213X-5-6. inserm-00092484 Delta activity independent of its activity as a ligand of Notch. Lee-Peng Mok, Tielin Qin, Boris Bardot, Matthew Lecomte, Asal Homayouni, Francois Ahimou, Cedric Wesley Lee-Peng Mok, Tielin Qin, Boris Bardot, Matthew Lecomte, Asal Homayouni, Francois Ahimou, Cedric Wesley HAL Id: inserm-00092484 https://inserm.hal.science/inserm-00092484v1 Submitted on 12 Sep 2006 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. BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-213X/5/6 This article is available from: http://www.biomedcentral.com/1471-213X/5/6 © 2005 Mok 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: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner. Results: Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta. Conclusion: Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo. Op Research article Delta activity independent of its activity as a ligand of Notch Lee-Peng Mok1, Tielin Qin2, Boris Bardot3, Matthew LeComte1, Asal Homayouni4, Francois Ahimou5 and Cedric Wesley1 Address: 1Department of Microbiology and Molecular Genetics, 322 Stafford Hall, 95 Carrigan Drive, The University of Vermont, Burlington, VT 05405, USA, 2Rollins School of Public Health, Emory University, Atlanta, GA 30322, USA, 3INSERM E365, Faculte de Medecine Lariboisiere, 10 avenue de Verdun, Paris 75010, France, 4College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA and 5Department of Civil Engineering, The University of Minnesota, Minneapolis, MN 55455, USA Email: Lee-Peng Mok - lmok@uvm.edu; Tielin Qin - tqin@sph.emory.edu; Boris Bardot - bbardot@uvm.edu; Matthew LeComte - Matthew.LeComte@uvm.edu; Asal Homayouni - ahomayou@vet.k-state.edu; Francois Ahimou - ahimo001@tc.umn.edu; Cedric Wesley - cwesley@uvm.edu * Corresponding author Received: 15 August 2004 Accepted: 10 March 2005 Published: 10 March 2005 BMC Developmental Biology Open Access BMC Developmental Biology 2005, 5:6 doi:10.1186/1471-213X-5-6 BMC Developmental Biology 2005, 5:6 doi:10.1186/1471-213X-5-6 Page 1 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 BMC Developmental Biology 2005, 5:6 all Drosophila cells [8] and up regulated in proneural cells specified to differentiate the neurons [5]. Whether or not the up regulation of Da expression is part of lateral inhibi- tion is not clear in Drosophila. In Caenorhabditis elegans, however, the differential accumulation of the Da homolog HLH-2 is the earliest detectable difference between the cells taking up alternate fates during lateral inhibition [9]. As N and Dl are known to regulate Da expression [10], it is very possible that Da expression is regulated during lateral inhibition in flies as well. by having one effect through N and a different one through Dl. Therefore, we addressed the following ques- tions in this study. Does Sca bind Dl? If yes, does it affect any Dl activity? Are there Dl activities independent of its activity as a ligand of N? Is Sca capable of activating N in the absence of Dl? N and Dl are expressed in almost all cells in vivo and N receptor activities in response to Dl binding are widely used during development. In developmental instances where Sca is present, the expression data suggest that both N and Dl will have access to Sca. Thus, it is very difficult to separate in vivo the activities of N alone, Dl alone, N on Dl, Dl on N, Sca on N, Sca on Dl, and Sca on N and Dl together. Therefore, we addressed the questions posed above in an in vitro model system based on Drosophila Schneider (S2) cells. S2 cells do not express the endog- enous N, Dl, or Sca [14,16]. S2 cells expressing N (S2-N cells) mixed with S2 cells expressing Dl (S2-Dl cells) reproduce all aspects of lateral inhibition [16-22]. Using these cells and the medium prepared from S2 cells expressing Sca into the medium [14], we show that Sca binds Dl, Dl has activity independent of its activity as a lig- and of N, Sca can affect this activity of Dl, and Sca can acti- vate N in the absence of Dl. These observations would be useful for undertaking the challenging task of determining how the various activities of N, Dl, and Sca are integrated during tissue differentiation. When N expressed on one proneural cell binds Dl expressed on the neighboring proneural cell, N is proteo- lytically cleaved to release the Notch intracellular domain (Nintra) from the plasma membrane. http://www.biomedcentral.com/1471-213X/5/6 Nintra translocates to the nucleus and, in association with the transcription fac- tor Suppressor of Hairless (SuH), activates transcription of the Enhancer of split Complex (E(spl)C) genes. Cells that express a high level of E(spl)C RNA suppress their neuro- nal predisposition, become the epidermal precursor cells (EPCs), and differentiate the epidermis. Cells that express a low level of E(spl)C RNA and a high level of Da protein become the Neuronal Precursor Cells (NPCs) and differ- entiate the nervous system [1,2,5,11]. From here onwards, we refer to this SuH dependent N activity that promotes expression of E(spl)C RNA as SuH/Nintra signaling. A 1.5 to 2-fold difference in the level of SuH/Nintra signaling is suf- ficient to initiate specification of the EPCs and the NPCs [11]. This difference is amplified by subsequent activities of N and Dl, or activities of other genes responding to the initial difference in the level of SuH/Nintra signaling. The lateral inhibition process described above is repeatedly used during development for differentiation of various tis- sues with minor variations or changes in target genes. Sca associates with Dl Although N and Sca complexes could be immuno-precip- itated [14], we, and others [23], had failed to detect Sca on S2-N cells. We suspected that some factor present in the tissue culture medium was washed away when the cells were processed for immuno-fluorescent detection of Sca. To overcome such problems, we made Sca-GFP and estab- lished stable S2 cells expressing it (S2-Sca-GFP cells). S2- Sca-GFP cells produced the Sca-GFP protein of the expected size (as determined by western blotting) and both Sca and GFP antibodies recognized this protein (data not shown). We concluded that S2-Sca-GFP cells expressed the expected Sca-GFP protein and used the con- ditioned medium from these cells to treat live S2-N, S2- Dl, and S2 cells. Scabrous (Sca) is a secreted factor that is produced at high levels in the NPCs and functions non-autonomously to promote specification of the EPCs during differentiation of the compound eye and the bristle organ [12,13]. In its absence, lateral inhibition is not abolished but is reduced in strength or becomes imprecise indicating that Sca only refines the process. Sca binds N and stabilizes it. These actions promote formation of sharp boundaries between neuronal and non-neuronal cells during development of the compound eye [14]. The possibility that Sca might bind Dl as well is suggested by the observation that simul- taneous over expression of Sca almost completely blocks the effect of Dl over-expression on wing margin develop- ment but hardly modifies the effect of N over-expression [15]. Dl and Sca have also been observed to co-localize in intracellular vesicles in vivo [13]. The observations that Sca can promote N activity [14] but block Dl activity are paradoxical as SuH/Nintra signaling is very much depend- ent on the activities of both N and Dl. One explanation for this paradox could be, that Sca promotes lateral inhibition Live S2-Dl cells showed the strongest GFP signals, fol- lowed by live S2-N cells, and then live S2 cells (Fig. 1A– C). The signals were so strong on the S2-Dl cells that the signals on S2-N cells were not obvious at the same bright- ness/contrast settings. When cells were simultaneously fixed and rinsed with 1X PBS, the signals were comparable at the same settings (insets in Fig. 1A–C). Page 2 of 12 (page number not for citation purposes) Background ation of the neuronal and epidermal tissues from prone- ural cells that are predisposed to making the neuronal tissue. Proneural cells express high levels of the neuronal transcription co-factors from the Achaete Scute Complex (ASC) or related genes [3,4]. These factors require their partner Daughterless (Da) to activate transcription of the neurogenesis genes [5-7]. Da is expressed at low levels in g Notch (N) and Delta (Dl) are cell surface proteins that are required for differentiation of almost all tissues in the fruit fly Drosophila melanogaster. They are evolutionarily con- served, functioning similarly in animals from worms to humans [1,2]. The best-known instance of their function is the process of lateral inhibition that initiates differenti- Page 1 of 12 (page number not for citation purposes) Page 1 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 http://www.biomedcentral.com/1471-213X/5/6 Sca associates with Dl Signals could not be detected on S2-N or S2-Dl cells after three 5-minute washes with 1X PBS, confirming our suspicion that the Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 BMC Developmental Biology 2005, 5:6 ca associates with Dl Figure 1 ca associates with Dl. A-C. Fluorescent photomicrographs of different cell lines treated with Sca-GFP medium for 30 minut Cells simultaneously fixed and rinsed in 4% paraformaldehyde/1X PBS are shown in the insets. Experiments were repeated hree times. D. Western blots showing recovery of Dl in Sca immuno-precipitates from total protein extracts prepared fro 2-Dl cells treated with S2-Sca cells. S2-Sca cells were used instead of Sca conditioned medium to maximize the ratio of bou o unbound Sca. Cross-linker = membrane insoluble and cleavable 3,3'- Dithiobis (sulfosuccinimidylpropionate) (DTSSP), wh ross-links proteins interacting at the cell surface. IP Ab = antibody used for immunoprecipitation; W Ab = antibody used o he western blot; ppt = immunoprecipitate; super = supernatant. Experiments were repeated two times. a associates with Dl gure 1 Sca associa Figure 1 This is not consistent with the in vivo find- ings that the Dl intracellular domain (lacking in Dl∆I) is required for SuH/Nintra signaling, possibly for promoting Dl internalization that results in exerting a 'pull' on N and increased production of Nintra [21,25-29]. However, our results are consistent with other S2 cell studies showing that even fixed S2-Dl cells can promote production of SuH/Nintra signaling in S2-N cells [19]. Thus, it is possible that that Dl internalization and pulling is not required for SuH/Nintra signaling in S2 cells. In any case, in our S2 cell system, the S2-N and S2-Dl cells require shaking for for- mation of cell aggregates. As a consequence, we shake all cell mixtures, including those containing the secreted lig- and Sca. This shaking might have simulated the pulling effect and overcome any deficiency Dl∆I might have in this regard thereby resulting in a level of SuH/Nintra signal- ing that is comparable to that produced by the full length Dl. standard immuno-fluorescence procedure is inappropri- ate for detecting Sca binding on S2-N or S2-Dl cells. Secreted GFP did not bind the surfaces of any of these cells (data not shown). This indicated that the Sca part of Sca- GFP fusion protein bound the S2-N and S2-Dl cell sur- faces. In all experiments conducted to determine the activ- ity of Sca, N, or Dl, that are described below, we used only S2 cells expressing the wild type Sca because (1) we do not perform washes to remove non-specifically bound pro- teins and (2) we wanted to avoid possible GFP associated effects (stability, etc.). We have previously shown that Sca and N form complexes [14]. To determine whether Sca forms complexes with Dl, we performed immuno-precipitation experiments with S2-Dl cells that were co-cultured with S2-Sca cells. Pro- teins interacting at the cell surfaces were either cross- linked or un-linked prior to cell lysis for protein extrac- tion. Membrane insoluble cross-linkers improve recovery of cell surface complexes [18,24]. Sca immuno-precipita- tion recovered Dl strongly in the presence of cross-linkers and relatively weakly in the absence of cross-linkers (Fig. 1D). No bands were observed when S2 cells were used instead of S2-Dl cells (data not shown). In the reverse experiments, Dl immuno-precipitations failed to recover Sca, possibly because there was too much unbound Dl in the extracts. Dl and Sca were not detected in the absence of immuno-precipitation antibodies (Fig. Sca associa Figure 1 Sca associates with Dl Figure 1 Sca associates with Dl. A-C. Fluorescent photomicrographs of different cell lines treated with Sca-GFP medium for 30 minutes. Cells simultaneously fixed and rinsed in 4% paraformaldehyde/1X PBS are shown in the insets. Experiments were repeated three times. D. Western blots showing recovery of Dl in Sca immuno-precipitates from total protein extracts prepared from S2-Dl cells treated with S2-Sca cells. S2-Sca cells were used instead of Sca conditioned medium to maximize the ratio of bound to unbound Sca. Cross-linker = membrane insoluble and cleavable 3,3'- Dithiobis (sulfosuccinimidylpropionate) (DTSSP), which cross-links proteins interacting at the cell surface. IP Ab = antibody used for immunoprecipitation; W Ab = antibody used on the western blot; ppt = immunoprecipitate; super = supernatant. Experiments were repeated two times. Sca associates with Dl Figure 1 Sca associates with Dl. A-C. Fluorescent photomicrographs of different cell lines treated with Sca-GFP medium for 30 minutes. Cells simultaneously fixed and rinsed in 4% paraformaldehyde/1X PBS are shown in the insets. Experiments were repeated three times. D. Western blots showing recovery of Dl in Sca immuno-precipitates from total protein extracts prepared from S2-Dl cells treated with S2-Sca cells. S2-Sca cells were used instead of Sca conditioned medium to maximize the ratio of bound to unbound Sca. Cross-linker = membrane insoluble and cleavable 3,3'- Dithiobis (sulfosuccinimidylpropionate) (DTSSP), which cross-links proteins interacting at the cell surface. IP Ab = antibody used for immunoprecipitation; W Ab = antibody used on the western blot; ppt = immunoprecipitate; super = supernatant. Experiments were repeated two times. Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 BMC Developmental Biology 2005, 5:6 http://www.biomedcentral.com/1471-213X/5/6 E(spl)C m3 expression appeared to be solely dependent on N activation and the Notch intracellular domain as it was promoted in S2-N cells treated with either S2-Dl cells or S2-Dl∆I cells (Fig. 2A, lanes 7–12). As Dl∆I lacks the intra- cellular domain, it is expected to behave only as a ligand of N and not generate any intracellular signal of its own in response to N binding. We observe comparable levels of SuH/Nintra signaling with S2-Dl and S2-Dl∆I cells (Fig. 2A, lanes 10, 12). Sca associa Figure 1 1D, lanes 1 and 4) or in the absence of Scabrous (data not shown). We also recovered Sca in Dl immuno-precipitations and Dl in Sca immuno-precipitations from protein extracts of wildtype embryos (data not shown). These observations indicated that Sca associates with Dl. We explored the consequence of this association. We examined the expression of various proteins known to be involved in lateral inhibition to find out if Dl expres- sion affected them. They were Numb, Dishevelled, Sup- pressor of Hairless, Wingless, Hairless, Hairy, Achaete, Da, and Armadillo. We found a relatively high level of Da pro- tein in S2-Dl cells compared with the level in S2-Dl∆I cells (Fig. 2B, lanes 1–2). Similar levels of Da were expressed in S2-Dl∆I and S2 cells (data not shown). Two independ- ently transfected S2-Dl cell lines also showed high levels of Da, and un-induced S2-Dl cells showed background levels of Da, indicating that Dl expression promotes Da expression (Fig. 2B, lanes 3–6). Increase in Da levels appeared to be specifically linked to Dl expression, as S2- N cells did not show an increase (Fig. 2C, compare lanes 1 & 3). Overall, Da expression in S2-Dl cells was 2.18X higher (+/- 0.37, p < 0.05) than the level in S2 cells, some- times more than 5X higher. Here and in all cases to follow, the blots shown in the figures are the most representative blots among replications. Graphs show quantification, relative to standards or other proteins (as indicated), of signals on the blots composing the figures as the response can be assessed only in comparison to the control lanes in the same experiment. Pooling data from all replications of an experiment obscured the response, or misrepresented the data, due to variation between different batches of cells. Therefore, we computed error variance for the degree of response over all replications of an experiment. These values for important responses are mentioned in the text. Da expression in Dl cells is reduced in response to Sca N promotes expression of E(spl)C m3 gene in response to Dl [19,20]. We examined whether Sca promoted expres- sion of E(spl)C m3 in S-N cells or S2-Dl cells and found that it was indeed the case with S2-N cells, but not with S2-Dl cells (Fig. 2A, lanes 1–6). Page 4 of 12 (page number not for citation purposes) Sca associa Figure 1 S2-N cells showed a low level of E(spl)C m3 expression when S2-Dl or S2-Dl∆I cells were replaced with S2 cells, in the absence of Sca (Fig. 2A, lanes 1, 7–8); S2-Dl or S2-Dl∆I cells mixed with S2 cells did not show any accumulation (Fig. 2A, lanes 13–16). The low level of E(spl)C m3 RNA expression in S2-N cells in the absence of ligands is due to the low level of Nintra produced upon induction of N expression in S2 cells [18]. This expression increases upon ligand treatment [18], resulting in increased expression of E(spl)C m3 RNA expression (Fig. 2A, lanes 2, 10, 12). Numerous repeti- tions of the experiments indicated that Dl is a more potent ligand of N than Sca with respect to induction of E(spl)C m3 expression (data not shown). Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) BMC Developmental Biology 2005, 5:6 http://www.biomedcentral.com/1471-213X/5/6 -regulates Daughterless protein expression, and N up-regulates E(spl)C m3 gene expression, in response to Sca 2 -regulates Daughterless protein expression, and N up-regulates E(spl)C m3 gene expression, in response to Sca hern blots of total RNA from the indicated cell mixtures extracted at 0 or 45 minutes after treatment with me ng Sca (+) or not (-). Gene probes used are shown on the right. m3 = E(spl)C m3 and rp 49 = a ribosomal protei show the levels of total RNA in the lanes in all northern blots. Sca = conditioned medium prepared from the S2 ll line in all experiments here onwards. The control medium used along side Sca medium (-) was prepared from S2 cells. Experiments were repeated two times. For unknown reasons, the medium collected from heat shocke ed in lanes 1, 3, and 5) produced higher background levels of E(spl)C m3 RNA in S2-N cells (lane 1). B. Western the levels of Da and Dl in different Dl cell lines. S2-Dl, S2-Dl(1) and S2-Dl(2) are independently established hsD = un-induced (i.e., not heat shocked). Hsp70 = heat shock 70 protein used to show the levels of proteins in the stern blots. Dl and Dl∆I were detected with αDlEC. Da signals here (and the indicated signals elsewhere) were Dl regulates expression of fringe and pangolin g p f g p g To gather additional evidence for Dl activity independent of its activity as a ligand of Notch, we performed microar- ray experiments using the Affymetrix Drosophila Gene- Chip Arrays to compare gene expression in S2 cells and S2-Dl cells. Many genes relevant to known Dl functions responded in S2-Dl cells (at p < 0.05, n = 3 × 2 pooled samples): axonal path finding genes (e.g., Gef64C, 39.38X,Up; Tenascin major, 6.77XUp), actin-based cell motility and kinases (Rho-Kinase, 15.08XUp; Rhophilin 3.4XUp; nemo 1.72XUp, basket 1.69XUp; pointed 2.2XUp), N signaling pathway genes (e.g., reaper, 2.26XUp; sanpodo, 1.91XUp), and oogenesis genes (e.g., swallow, 8.12XUp; sprouty, 3.58Xup). Expression of transformer, was also up (1.76X) and it is significant in the light of our observation that Dl promotes expression of Da: both Da and transformer are involved in sex determi- nation. Expression of da RNA was not significantly increased in S2-Dl cells, possibly due to the negative part of the da gene autoregulation system [34]. The detailed analyses with validations will be published elsewhere. The experiment also identified fringe (fng) and pangolin (pan) as responding to Dl expression. fng is a glycosyl trans- ferase that regulates the affinity of N for Dl [35-37], and possibly also the affinity of Dl for N [38]. pan is a tran- scription factor functioning in the Wingless (Wg) pathway [39,40]. Notch and Wg pathways interact closely at many differentiation events during development [24,41-44]. Therefore, we chose fng and pan for further investigation. When S2-N and S2-Dl cells were together in the presence of Sca, the levels of Da protein and E(spl)C m3 RNA were very variable (data not shown). This was possibly due to the varying combinations of Sca effect on S2-Dl cells, Sca effect on S2-N cells, Dl effect on S2-N cells, and N effect on S2-Dl cells. Dl is processed to produce Dl intracellular domain (DlIC), constitutively, and the levels of DlIC increase upon N treatment [30-33]. Therefore, we examined the levels of DlIC following treatment of S2-Dl cells with S2- N cells or Sca medium. We found that the DlIC levels increased by 25 to 50% (relative to Dl levels) with both treatments (Fig. 3A and 3B). We examined the levels of Da in S2 cells expressing DlIC and DlTMIC (lacking the extra- cellular domain only and including the transmembrane domain). Dl down-r Figure 2 Dl down-regulates Daughterless protein expression, and N up-regulates E(spl)C m3 gene expression, in response to Scabrous Figure 2 Dl down-regulates Daughterless protein expression, and N up-regulates E(spl)C m3 gene expression, in response to Scabrous. A. Northern blots of total RNA from the indicated cell mixtures extracted at 0 or 45 minutes after treatment with medium containing Sca (+) or not (-). Gene probes used are shown on the right. m3 = E(spl)C m3 and rp 49 = a ribosomal protein gene used to show the levels of total RNA in the lanes in all northern blots. Sca = conditioned medium prepared from the S2-Sca stable cell line in all experiments here onwards. The control medium used along side Sca medium (-) was prepared from heat shocked S2 cells. Experiments were repeated two times. For unknown reasons, the medium collected from heat shocked S2 cells (used in lanes 1, 3, and 5) produced higher background levels of E(spl)C m3 RNA in S2-N cells (lane 1). B. Western blots showing the levels of Da and Dl in different Dl cell lines. S2-Dl, S2-Dl(1) and S2-Dl(2) are independently established hsDl cell lines. Ui = un-induced (i.e., not heat shocked). Hsp70 = heat shock 70 protein used to show the levels of proteins in the lanes of all western blots. Dl and Dl∆I were detected with αDlEC. Da signals here (and the indicated signals elsewhere) were quan- tified relative to Hsp70 (western blots), rp49 (northern blots), or other indicated molecules, using the NIH Image 1.63 pro- gram. These experiments were repeated more than ten times. C. Western blots showing Da levels in the indicated cell mixtures, with (+) or without (-) Sca. These experiments were repeated five times. Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 http://www.biomedcentral.com/1471-213X/5/6 BMC Developmental Biology 2005, 5:6 The number of repetitions of an experiment is indicated in the figure legends. shown, as we cannot clearly separate the effects of N, Dl, and Sca, the way we can do in S2 cells. The experiments described in this section indicate that Da accumulation is promoted by the full-length Dl, not by the Dl intracellular domains (DlIC or DlTMIC), and Sca suppresses the activ- ity of the full length Dl. Dl down-r Figure 2 The experiments also indicate that Sca promotes E(spl)C m3 RNA expression in S2-N cells even in the absence of Dl. We examined Da levels in S2-Dl and S2-N cells that were treated or not treated with Sca conditioned medium. We found that Sca treatment decreased the levels of Da in S2- Dl cells (Fig. 2C, lanes 3–4). The levels in S2-N cells were low and unaffected by Sca treatment (Fig. 2C, lanes 1–2). These experiments suggested that Sca blocks accumula- tion of Da in S2-Dl cells (2.81X, +/- 0.59, p < 0.05). We also determined the levels of Da when S2-N and S2-Dl cells were together in the absence of Sca. The level of Da never increased (Fig. 2C, lanes 6–7). As N activation sup- presses daughterless RNA expression [18], it was possible that N activation suppressed Da expression in S2-N cells and masked an increase in S2-Dl cells. To determine if this was the case, we compared the level of Da in mixtures of S2-Dl∆I cells and S2 cells with mixtures of S2-Dl∆I cells and S2-N cells. As S2-Dl and S2-Dl∆I cells activate N equally well (see Fig. 2A, lanes 9–12), any change in Da level would be due to N activation. We found comparable levels of Da in the two samples (Fig 2C, lanes 8–9). Thus, S2-Dl cells do not appear to increase Da expression in response to S2-N cells. Page 6 of 12 (page number not for citation purposes) Dl regulates expression of fringe and pangolin Western blots (from a 12% gel wing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C. Western blots showing levels of Da in the indicated cell mixtures. D. Western blots showing the levels of Da, Dl, and DlIC at different times fol- wing heat shock induction of Dl in S2-Dl cells. All experiments were repeated at least three times. The levels of cleaved Dl intracellular domain is not associated with high levels of Da Figure 3 The levels of cleaved Dl intracellular domain is not associated with high levels of Da. A. Western blots (from a 8% SDS-PAGE) showing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B. Western blots (from a 12% gel) showing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C. Western blots showing the levels of Da in the indicated cell mixtures. D. Western blots showing the levels of Da, Dl, and DlIC at different times fol- lowing heat shock induction of Dl in S2-Dl cells. All experiments were repeated at least three times. The levels of cleaved Dl intracellular domain is not associated with high levels of Da igure 3 The levels of cleaved Dl intracellular domain is not associated with high levels of Da. A. Western blots (from a 8% SDS-PAGE) howing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B. Western blots (from a 12% gel) howing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C. Western blots showing The levels Figure 3 The levels of cleaved Dl intracellular domain is not associated with high levels of Da Figure 3 The levels of cleaved Dl intracellular domain is not associated with high levels of Da. A. Western blots (from a 8% SDS-PAGE) showing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B. Western blots (from a 12% gel) showing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C. Western blots showing the levels of Da in the indicated cell mixtures. D. Western blots showing the levels of Da, Dl, and DlIC at different times fol- lowing heat shock induction of Dl in S2-Dl cells. Dl regulates expression of fringe and pangolin The levels of Da in S2-DlIC and S2-DlTMIC were always comparable to or lower than the level in S2 or S2-Dl∆I cells (Fig. 3C). Also, we found Da levels to be neg- atively correlated with the accumulation of DlIC in time- course experiments (Fig. 3D). This negative correlation could be a direct consequence of the accumulation of DlIC or due to autoregulation of the da gene [34]. We examined the levels of Da in flies expressing heat shock induced Dl, N, or Sca, in flies heterozygous for the null alleles of N or Dl, and in flies homozygous for a null allele of Sca. We found that Da expression was strongly associ- ated with Dl expression rather than with N expression, and inconsistently associated with Sca expression. These data are consistent with our findings in S2 cells but are not Northern blot analyses showed that the expression of fng and pan was higher in S2-Dl cells compared with S2-N or S2-Dl∆I cells (Fig. 4A). DlIC and DlTMIC promoted expression of pan and fng weakly, if at all (Fig. 4B). Two independently established S2-Dl cell lines also showed higher levels of fng and pan RNAs (Fig. 4C). Sca treatment S2-Dl cells reduced the levels of fng and pan RNA (Fig. 4D). This reduction was expected as Sca reduces the levels of the full length Dl (see Fig. 3). Thus, just as it was the case with Da expression, the full length Dl, not any of its parts, strongly promoted pan and fng expression. We examined the levels of fng and pan RNA in flies expressing heat shock induced Dl, N, or Sca, in flies heterozygous for the null alleles of N or Dl, and in flies homozygous for a null allele of Sca. We found that fng and pan RNA expression was strongly associated with Dl expression Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) BMC Developmental Biology 2005, 5:6 http://www.biomedcentral.com/1471-213X/5/6 e levels of cleaved Dl intracellular domain is not associated with high levels of Da gure 3 e levels of cleaved Dl intracellular domain is not associated with high levels of Da. A. Western blots (from a 8% SDS-PAGE wing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B. Dl regulates expression of fringe and pangolin All experiments were repeated at least three times. Page 7 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 BMC Developmental Biology 2005, 5:6 promotes expression of fng and pan gure 4 promotes expression of fng and pan. A. Northern blots showing fng and pan expression in the indicated cell mixtures at d 45 minutes after cell mixing. B. Northern blots showing fng and pan expression in the indicated cell lines two hours afte uction of expression. C. Northern blots showing fng and pan expression in two other independently established S2-Dl ce es. Cells used for lanes 1–2 were uninduced (ui); cells used for lanes 3–4 were heat shock induced. D. Northern blots sho fng and pan expression in S2-Dl cells that were either untreated or treated with Sca medium. All experiments were peated at least three times. The fng band marked with an asterisk corresponds to the published mRNA [35]. Only this ban s used for fng quantification. The pan band shown is consistent with the information described in van de Wetering et al. [4 d Brunner et al [39] Dl promot Figure 4 Discussion O i Our experiment addressed four questions. Does Sca bind Dl? If yes, does it affect any Dl activity? Are there Dl activ- ities independent of its activity as a ligand of N? Is Sca capable of activating N in the absence of Dl? Results described in Figure 1 show that Sca binds Dl. This binding is not dependent on N as S2-Dl cells do not express N. We have previously shown that Sca binds N [14]. It is possible that Sca binds N or Dl stronger when they are present together on the same cell or on neighboring cells. It would be possible to test this in the future using Atomic Force Microscopy that is best suited for determining binding strengths of cell surface proteins like N or Dl [21]. Results in Figure 2A show that Sca can promote SuH/Nintra signal- ing through N in the absence of Dl, as S2-N cells do not express Dl. However, numerous repetitions of the experi- ment indicate that Sca is not as potent as Dl in this regard. This is consistent with the fact that lateral inhibition is blocked in the absence of zygotic Dl, which does not affect proneural cluster formation and thereby Sca expression. It would have been relatively easy to determine if over- expression of Sca in the absence of Dl rescues SuH/Nintra signaling phenotypes, and the extent of this rescue, if Dl did not have any activity independent of N. Hopefully, it would be possible in the future, when we better under- stand this Dl activity and are able to circumvent it. Results in Figure 2A also show that the expression of E(spl)C m3 gene, a target of SuH/Nintra signaling pathway, is respon- sive only to N indicating that this pathway is unlikely to be involved in mediating Dl activities. Dl activity that is independent of its N ligand activity has been speculated for some time. Efforts to identify it have intensified since the discovery that Dl gets proteolytically processed in the same manner as N [30-33]. However, it is extremely difficult to separate these two activities of Dl. The proof that the Dl activity we have identified actually functions during development in the expected manner, the details of the mechanisms underlying this function, and a better integration of the known functions of N, Dl, and Sca, will have to await more work which is neither quick nor simple. Discussion O i We hope that this work stimulates more efforts towards this task and makes this task a bit easier by identifying the potential of Sca as a regulator of Dl activity and the possibility that the full length Dl might be important for Dl activity independent of N, or Sca. Sca could also serve as a great tool for in vivo dissection of Dl response to N, as Sca and N appear to have a similar effect on Dl. It is quite likely that our experiments did not pick up Dl receptor activity in response to N or Sca. In any case, the potential developmental significance of our findings is briefly discussed below. Results described in Figures 2, 3, 4 and our microarray analysis show that Dl has activity independent of its activ- ity as a ligand of N and Dl could be a receptor of Sca. This is clearly shown in experiments with S2-Dl cells that do not express N and we do not provide either N or Sca (Fig. 2B, C; 4A). The Dl activities we have described- promo- tion of expression of Da protein, fng RNA, and pan RNA- can be detected in vivo as well although the interpretation here is not simple due to the many possible interactions among N, Dl, and Sca that cannot be easily sorted out. However, these data (which we do not show) strongly suggest that the Dl activities we have described in S2 cells represent the in vivo Dl activities during development. Da is a widely expressed protein and cells requiring its function show only a modest increase in its levels [5,6,8] indicating that, just like Nintra/SuH signaling, small changes in Da levels might be sufficient for initiating or augmenting NPC specification and promoting neuronal differentiation. Small changes in Da levels might also be imposed by the built-in autoregulation of the da gene [34]. According to the well-accepted lateral inhibition model in the field, Dl activity as a ligand of N is postulated to increase in the NPCs and N receptor activity in response to Dl is postulated to increase in the EPCs [11]. Accord- ingly, Dl expression has been observed to increase in the NPCs or their equivalent cell types in certain instances involving N and Dl functions [50,51]. Dl promot Figure 4 Dl promotes expression of fng and pan Figure 4 Dl promotes expression of fng and pan. A. Northern blots showing fng and pan expression in the indicated cell mixtures at 0 and 45 minutes after cell mixing. B. Northern blots showing fng and pan expression in the indicated cell lines two hours after induction of expression. C. Northern blots showing fng and pan expression in two other independently established S2-Dl cell lines. Cells used for lanes 1–2 were uninduced (ui); cells used for lanes 3–4 were heat shock induced. D. Northern blots show- ing fng and pan expression in S2-Dl cells that were either untreated or treated with Sca medium. All experiments were repeated at least three times. The fng band marked with an asterisk corresponds to the published mRNA [35]. Only this band was used for fng quantification. The pan band shown is consistent with the information described in van de Wetering et al. [40] and Brunner et al. [39]. Page 8 of 12 (page number not for citation purposes) BMC Developmental Biology 2005, 5:6 http://www.biomedcentral.com/1471-213X/5/6 http://www.biomedcentral.com/1471-213X/5/6 is based on the activity of its intracellular domain [45-47]. Accordingly, treatment with Sca, which promotes produc- tion of the Dl intracellular domain, suppresses Dl activity related to Da rather than promote it (Figs. 2C; 3). This observation also indicates that Sca is able to affect Dl activities. A clean dissection of Sca effects through N from its effects through Dl would require identification of Sca binding sites on Dl, and N and Dl binding sites on Sca. We know that Dl and Sca bind different regions of N [14,48,49]. It would not be too surprising if Dl bound N and Sca in different regions, and if Sca bound N and D in different regions. With that knowledge and suitable mutants, we might be able to determine whether N, Dl, and Sca activities function in a mutually exclusive or com- binatorial manner in vivo. rather than with N expression, and inconsistently associ- ated with Sca expression. These data are consistent with our S2-Dl cells data but are not shown, as we cannot clearly separate the effects of N, Dl, and Sca, the way we can do in S2 cells. Methods DNA constructs Sca-Gfp: The stop codon of sca was replaced with a glycine codon and fused in-frame with GFP to obtain Sca-GFP. A Bam HI-KpnI fragment containing this sca sequence was cloned into pEGFP vector (Clontech). The XbaI fragment containing Sca-GFP coding fragment was cloned into the pCaSpeR-hs vector. Dl∆I: A stop codon and a XbaI restric- tion site was introduced after the transmembrane domain using PCR. The PCR product was checked for mutations and used to replace the BstEII-BcgI fragment in the Dl cDNA. An Eco RI-XbaI fragment from this construct (Dl amino acid 1 to 620) was cloned into the pCaSpeR-hs vec- tor. DlIC: The Dl intracellular region (codon 619 to the stop codon 881) was PCR amplified, checked for errors, and cloned into the BglII-XbaI sites in the pCaSpeR-hs vector. N/Dl binding and SuH/Nintra signaling are strongly affected by the functions of glycosyl transferases such as fng. The possibility that Dl, and not N, regulates fng RNA expression might explain some of the very complex func- tions of these glycosyl transferases and the complex inter- actions between N and Dl during lateral inhibition. As N and Dl activities are known to strongly interact with the Wg signaling pathway, it is interesting that Dl promotes pan expression. It is possible that Dl activity independent of N accounts for some of the interactions between the N and the Wg pathways. So far, these interactions have been considered only from the perspective of N receptor activity. Lastly, our data suggest interesting interactions among Dl, N, and Sca in instances of lateral inhibition and tissue dif- ferentiation when their functions overlap. The full length Dl promotes Da accumulation, not any of its parts that might result from processing in response to N or Sca bind- ing. Thus, both the processed N and Dl might promote EPC specification- processed N through E(spl)C RNA and processed Dl through suppression of Da expression. Con- sequently, lateral inhibition might initiate with symmetri- cal actions of N and Dl promoting EPC specification in all proneural cells. Sca might boost N and Dl processing in the incipient EPCs while suppressing them or not affect- ing them in the incipient NPCs. Thus, it is possible that Sca or Sca-like molecule have a role in breaking the sym- metrical actions of N and Dl during certain lateral inhibi- tion instances. Discussion O i Our data suggest that an increase in Da levels in these instances could be due to the accumulation of the full length Dl, not any its parts such as DlIC, Dl∆I, or DlTMIC. The requirement for The N independent Dl activity we have described is dependent on the full length Dl, not just on its intracellu- lar domain or the extracellular domain (Figs. 2C; 3; 5A-B). This is different from the situation with N whose activity Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) BMC Developmental Biology 2005, 5:6 http://www.biomedcentral.com/1471-213X/5/6 http://www.biomedcentral.com/1471-213X/5/6 the intracellular and the extracellular domains to be linked might mean that we have detected Dl activity requiring Dl's presence at the membrane or in the cyto- plasm. This is consistent with the report that the cellular transformation ability of Jagged 1, a mammalian Dl homolog, requires an intact protein containing both the extracellular and the intracellular domains [52]. It is pos- sible that DlIC, in the nucleus [31], promotes other activ- ity that is different from the one described here. It is also possible that Da, fng, or pan might not be the direct target of the full length Dl activity. Our microarray data indicate that many other genes (including some in the RAS or EGFR signaling pathways) are strongly up regulated in Dl expressing cells. It is possible that one of these genes is the primary target. It is also possible that Da, fng, or pan accu- mulation is significant only in the context of these other genes. We will have to await validation of other putative targets of Dl activity, and evaluation of their role in lateral inhibition or other activities involving N and/or Dl, to determine if Da, fng, or pan are typical or atypical targets of Dl activity. the intracellular and the extracellular domains to be linked might mean that we have detected Dl activity requiring Dl's presence at the membrane or in the cyto- plasm. This is consistent with the report that the cellular transformation ability of Jagged 1, a mammalian Dl homolog, requires an intact protein containing both the extracellular and the intracellular domains [52]. It is pos- sible that DlIC, in the nucleus [31], promotes other activ- ity that is different from the one described here. Discussion O i It is also possible that Da, fng, or pan might not be the direct target of the full length Dl activity. Our microarray data indicate that many other genes (including some in the RAS or EGFR signaling pathways) are strongly up regulated in Dl expressing cells. It is possible that one of these genes is the primary target. It is also possible that Da, fng, or pan accu- mulation is significant only in the context of these other genes. We will have to await validation of other putative targets of Dl activity, and evaluation of their role in lateral inhibition or other activities involving N and/or Dl, to determine if Da, fng, or pan are typical or atypical targets of Dl activity. might be possible to develop strong hypotheses for testing in vivo, cleanly sort the different activities of N, Dl, and Sca, and understand the fascinating in vivo developmental mechanisms involving N, Dl, and Sca. Cell lines and conditioned medium S2-N, S2-Dl, and S2-Sca cells have been previously described [14,20,49]. Other cell lines were established using the standard calcium phosphate transfection proce- dure and hygromycin selection. Conditioned medium was produced as described in Powell et al. [14], using serum-free or serum-containing Shields and Sang's M3 medium. For experiments, cells were heat shocked at 37°C for 30 minutes in a water bath, allowed to synthe- size proteins for 2 hours, washed in culture medium with- out serum, mixed with the appropriate cell lines, and shaken gently in 14 ml falcon tubes for two hours or the indicated time. See Wesley and Mok [20] for more details. Methods DNA constructs It is also possible that Sca mediates long range N signaling during differentiation of some other tis- sues, either alone or in association with Dl, as proposed by Renaud and Simpson [13]. By extending our results, it Conclusion S bi d Dl Sca binds Dl and suppresses a Dl activity that is independ- ent of Dl's activity as a ligand of N. This Dl activity requires the full length Dl and is not enhanced by expres- sion of just the Dl intracellular domain, which is different from the mechanism underlying Notch activity. Da pro- tein, fng RNA, and pan RNA responds positively to the N independent Dl activity we have discovered. These could be direct or indirect targets. Our microarray analysis has identified many more putative targets of N independent Dl activity that can be explored for a better understanding of the complex interactions among Dl, Sca, and N during Drosophila development. Page 10 of 12 (page number not for citation purposes) Immunoprecipitations, western blotting, northern blotting, RNA in situ, and protein staining Procedures described in Lieber et al. [43], Wesley [24], Wesley and Saez [18], and Wesley and Mok [20] were fol- Page 10 of 12 (page number not for citation purposes) Page 10 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/5/6 BMC Developmental Biology 2005, 5:6 lowed. Eight per cent SDS-PAGE systems were used for western blotting, unless otherwise indicated; 1% formal- dehyde-MOPS agarose system for northern blottings. fringe cDNA (from Dr. Ken Irvine), rp49 cDNA, and rt- PCR amplified pangolin cDNA were used to prepare probes for northern blots. Incubation times with ligands were two hours for western blots and 45 minutes for all northern blots; it was three hours for fng and pan northern blot showing the effect of Sca (Fig. 4D). 2. Mumm JS, Kopan R: Notch Signaling: From the Outside. Dev Biol 2000, 228:151-165. lowed. Eight per cent SDS-PAGE systems were used for western blotting, unless otherwise indicated; 1% formal- dehyde-MOPS agarose system for northern blottings. fringe cDNA (from Dr. Ken Irvine), rp49 cDNA, and rt- PCR amplified pangolin cDNA were used to prepare probes for northern blots. Incubation times with ligands were two hours for western blots and 45 minutes for all northern blots; it was three hours for fng and pan northern blot showing the effect of Sca (Fig. 4D). 3. Cabrera CV: Lateral inhibition and cell fate during neurogen- esis in Drosophila : the interactions between scute, Notch and Delta. Development 1990, 109:733-742. p 4. Jarman AP, Grell EH, Ackerman L, Jan LY, Jan YN: Atonal is the proneural gene for Drosophila photoreceptors. Nature 1994, 369:398-400. 5. Vaessin H, Brand M, Jan LY, Jan YN: daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo. Develop- ment 1994, 120:935-945. 6. Brown NL, Paddock SW, Sattler CA, Cronmiller C, Thomas BJ, Car- roll SB: daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression. Dev Biol 1996, 179:65-78. Antibodies: αSca (mAb sca1) and αDlEC (C594.9B) were obtained from the Developmental Studies Hybridoma Bank; αGFP (G-6539) and αHsp70 (H-5147) from Sigma; αDlIC (GPC2) from Dr. Marc Muskavitch, αDlIC (dC-19) from Santa Cruz Biotechnology, αDa (DAM 109-10) from Dr. Claire Cronmiller; and αNI from Dr. Toby Lieber. p g , 7. Authors' contributions 15. Lee E-C, Yu S-Y, Baker NE: The Scabrous protein can act as an extracellular antagonist of Notch signaling in the Drosophila wing. Curr Biol 2000, 10:931-934. LPM and TQ designed and carried out many of the exper- iments in cultured cells and flies; BB carried out the immuno-precipitation experiments with cultured cells and helped in interpretation of data; MLC made the Sca GFP construct and assisted in many experiments; AH per- formed some experiments in cultured cells and prepared and maintained cell lines; FA assisted in statistical analy- ses and interpretation of data; and CSW conceived the study, designed experiments, and performed or partici- pated in many of experiments in cultured cells and flies. LPM, BB, and MLC helped CSW in drafting the manu- script. All authors read and approved the final manuscript. 16. Fehon RG, Kooh PJ, Rebay I, Regan CL, Xu T, Muskavitch M, Arta- vanis-Tsakonas S: Molecular interaction between the protein products of the neurogenic loci Notch and Delta, two EGF- homologous genes in Drosophila. Cell 1990, 61:523-534. g g p 17. Klueg KM, Muskavitch MAT: Ligand-receptor interactions and trans-endocytosis of Delta, Serrate and Notch: members of the Notch signaling pathway in Drosophila. Development 1999, 112:3289-3297. 18. Wesley CS, Saez L: Analysis of Notch lacking the carboxyl ter- minus identified in Drosophila embryos. J Cell Biol 2000, 149:683-696. 19. Mishra-Gorur K, Rand MD, Perez-Villamil B, Artavanis-Tsakonas S: Down-regulation of Delta by proteolytic processing. J Cell Biol 2002, 159:313-324. 20. Wesley CS, Mok L-P: Regulation of Notch signaling by a novel mechanism involving Suppressor of Hairless stability and carboxyl terminus-truncated Notch. Mol Cell Biol 2003, 23:5581-5593. Acknowledgements 21. Ahimou F, Mok L-P, Bardot B, Wesley CS: The adhesion force of Notch with Delta and the rate of Notch signaling. J Cell Bio 2004, 167:1217-1229. g We thank Drs. Claire Cronmiller, Nick Baker, Toby Lieber, and Matt Rand for materials; Dr. Matt Rand and Uma Wesley for critical comments on the manuscript. We also thank the two reviewers and a member of the Edito- rial Board for helpful comments and advice. The work was supported by the grant R 01 NS43122-03 to C. S. Wesley. 22. Bardot B, Mok L-P, Thayer T, Ahimou F, Wesley CS: The Notch amino terminus regulates protein levels and Delta-induced clustering of Drosophila receptors. Exp Cell Res 2005, 304:202-223. 23. Lee E-C, Baker NE: GP300Sca is not a high affinity Notch lig- and. Biochem. Biophysical Res Comm 1996, 225:720-725. 23. Lee E-C, Baker NE: GP300Sca is not a high affinity Notch lig- and. Biochem. Biophysical Res Comm 1996, 225:720-725. Immunoprecipitations, western blotting, northern blotting, RNA in situ, and protein staining Giebel B, Stuttem I, Hinz U, Campos-Ortega JA: Lethal of Scute requires overexpression of daughterless to elicit ectopic neuronal development during embryogenesis in Drosophila. Mech Dev 1997, 63:75-87. 8. 8. 8. 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Renaud O, Simpson P: scabrous modifies epithelial cell adhesion and extends the range of lateral inhibition signaling during development of the spaced bristle pattern in Drosophila. Dev Biol 2001, 240:361-376. 14. Powell PA, Wesley C, Spencer S, Cagan RL: Scabrous complexes with Notch to mediate boundary formation. Nature 2001, 409:626-630. 1. Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: Cell fate control and signal integration in development. Science 1999, 284:770-776. BMC Developmental Biology 2005, 5:6 http://www.biomedcentral.com/1471-213X/5/6 25. Parks AL, Kleug KM, Stout JR, Muskavitch MA: Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 2000, 127:1373-1385. 48. Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsa- konas S: Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multi- functional receptor. Cell 1991, 67:687-699. p y p 26. Struhl G, Adachi A: Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins. Mol Cell 2000, 6:625-636. 49. Lieber T, Wesley CS, Alcamo E, Hassel B, Krane JF, Campos-Ortega JA, Young MW: Single amino acid substitutions in EGF-like ele- ments of Notch and Delta modify Drosophila development and affect cell adhesion in vitro. Neuron 1992, 9:847-859. 27. Pavlopoulos E, Pitsouli C, Kleug KM, Muskavitch MA, Moschonas NK, Delidakis C: neuralized encodes a peripheral membrane pro- tein involved in delta signaling and endocytosis. Dev Cell 2001, 1:807-816. 50. Huppert SS, Jacobsen TL, Muskavitch MAT: Feedback regulation is central to Delta-Notch signaling required for Drosophila wing vein morphogenesis. Development 1997, 124:3283-3291. 28. Overstreet E, Chen X, Wendland B, Fischer JA: Either part of a Drosophila epsin protein, divided after the ENTH domain, functions in endocytosis of delta in the developing eye. Curr Biol 2003, 13:854-860. 51. de Celis JF, Bray S, Garcia-Bellido A: Notch signaling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophhila wing. Development 1997, 124:1919-1928. 29. Wang W, Struhl G: Drosophila Epsin mediates a select endo- cytic pathway that DSL ligands must enter to activate Notch. Development 2004, 131:5367-5380. 52. Ascano JM, Beverly LJ, Capobianco AJ: The C-terminal PDZ-lig- and of JAGGED 1 is essential for cellular transformation. J Biol Chem 2003, 278:8771-8779. p 30. Ikeuchi T, Sisodia S: The Notch ligands, Delta 1 and Jagged 2, are substrates for Presenilin-dependent "γ-Secretase" cleavage. J Biol Chem 2003, 278:7751-7754. g J 31. Bland CE, Kimberly P, Rand MD: Notch-induced proteolysis and Nuclear localization of the Delta ligand. J Biol Chem 2003, 278:13607-13610. 32. La Voie MJ, Selkoe DJ: The Notch ligands, Jagged and Delta, are sequentially processed by alpha-Secretase and Presenilin/ gamma-Secretase and release signaling fragments. J Biol Chem 2003, 278:34427-34437. 33. Six E, Ndiaye D, Laabi Y, Brou C, Gupta-Rossi N, Israel A, Logeat F: The Notch ligand Delta 1 is sequentially cleaved by an ADAM protease and γ-secretase. Proc Nat Acad Sci USA 2003, 100:7638-7643. 34. References 24. Wesley CS: Notch and Wingless regulate expression of cuticle patterning genes. Mol Cell Biol 1999, 19:5743-5758. 24. Wesley CS: Notch and Wingless regulate expression of cuticle patterning genes. Mol Cell Biol 1999, 19:5743-5758. Page 11 of 12 (page number not for citation purposes) Page 11 of 12 (page number not for citation purposes) BMC Developmental Biology 2005, 5:6 BMC Developmental Biology 2005, 5:6 Smith JEIII, Cronmiller C: The Drosophila daughterless gene autoregulates and is controlled by both positive and negative cis regulation. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 43. Cadigan KM, Nusse R: wingless signaling in the Drosophila eye and embryonic epidermis. Development 1996, 122:2801-2812. p p 44. Axelrod JD, Matsuno K, Artavanis-Tsakonas S, Perrimon N: Interac- tion between Wingless and Notch signaling pathways medi- ated by Dishevelled. Science 1996, 271:1826-1832. y 45. Fortini ME, Rebay I, Caron LA, Artavanis-Tsakonas S: An activated Notch receptor blocks cell-fate commitment in the develop- ing Drosophila eye. Nature 1993, 365:555-557. g p y , 46. Struhl G, Fitzgerald K, Greenwald I: Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 1993, 74:331-345. , 47. Lieber T, Kidd S, Alcamo E, Corbin V, Young MW: Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev 1993, 7:1949-1965. Page 12 of 12 (page number not for citation purposes)
https://openalex.org/W1986980314	https://europepmc.org/articles/pmc2010888?pdf=render	English	null	Circulating immune complexes as markers of response to chemotherapy in malignant teratomas and gestational trophoblastic tumours	British journal of cancer	1,982	cc-by	3,958	Received 27 July 1981 Accepted 26 October 1981 Summary.-Concentrations of circulating immune complexes (CIC) were measured serially during chemotherapy of 22 patients with gestational trophoblastic tumours (GTT) and 11 patients with malignant teratoma (MT) by the polyethylene glycol precipitation and CIq solid-phase assays. Results were correlated with tumour res- ponse as measured by serum concentrations of human chorionic gonadotrophin (hCG) and a-foetoprotein (AFP). CIC concentrations correlated with disease status in the early stages of treatment in 4/22 patients with GTT and 5/11 with MT. CIC assays were less sensitive than hCG and AFP as a monitor of disease, and also less specific, in that 8 patients with GTT and 5 with MT developed raised CIC concentra- tions during chemotherapy in spite of sustained complete remission. Measurements of CIC concentrations by present methods are neither sufficiently sensitive nor speci- fic to be of clinical value as a tumour marker in GTT and MT, and this casts doubt on their potential value in other malignancies. Attention should be directed to identifi- cation of the components of CIC, some of which may be more cancer-specific. that measuremnnt of CIC concentrations may provide a tumour-marker system which will be of value in the management of patients with cancer (Baldwin & Robins, 1980). In order to be useful, such a tumour marker must either be more sensitive or more specific than current methods of tumour assessment. In malignancies such as gestational trophoblastic tumours (GTT) or malignant teratoma (MT), for which satisfactory biochemical markers are already available (Javadpour, 1979; Bagshawe & Searle, 1977), it is clear that measurement oftumour volume by clinical examination or conventional radiology can give a misleading assessment of the number of viable malignant cells (Bag- shawe, 1973; Newlands et al., 1980). In order to assess the value of CICs as a tumour marker we have therefore com- pared them with established biochemical markers the concentrations of which are related to viable tumour mass, namely human chorionic gonadotrophin (hCG) in RAISED CONCENTRATIONS of circulating immune complexes (CIC) are found in several types of cancer of man and experi- mental animals (for review see Baldwin & Robins, 1980). There is evidence that in some cases the antigen component of the complexes may be a tumour product (for review see Theofilopoulos & Dixon, 1980; Nydegger, 1980). Br. J. Cancer (1982) 45, 217 Br. J. Cancer (1982) 45, 217 CIRCULATING IMMUNE COMPLEXES AS MARKERS OF RESPONSE TO CHEMOTHERAPY IN MALIGNANT TERATOMAS AND GESTATIONAL TROPHOBLASTIC TUMOURS R. H. J. BEGENT, K. A. CHESTER, L. C. WALKERt AND D. F. TUCKERt From the Department of Medical Oncology, Charing Cross Hospital, and the tImperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London WC2 Received 27 July 1981 Accepted 26 October 1981 Received 27 July 1981 Accepted 26 October 1981 PEG precipitation assay Results for the normal group are shown in Fig. 1. Values above 20-6 jig IgG/ml of serum (mean+ 2 s.d.) were considered abnormal. 29 patients with rheumatoid arthritis served as a positive control group (Fig. 1). y Circulating immune complexes-.Two tests were used in parallel: the CIq solid-phase assay (CIqSP) and polyethylene glycol (PEG) precipitation. The CIqSP was performed as described by Hardin et al. (1979). The PEG precipitation assay was modified from the method of Kazatchkine et al. (1980). One hundred and fifty pl of4% PEG in barbitone- buffered saline containing 60 mm EDTA (pH 7 6) was added to duplicate 150u1l samples of serum. After 16 h at 4°C, samples were centrifuged at 1500 g for 20 min at 4°C, washed in 2% PEG and redissolved at room temperature in 150 pl of barbitone buffer. The amount of IgG in the redissolved precipitate was determined by radial immune diffusion in 0.8%/ agarose (Mercia Brocades MX agarose for gel electrophoresis) contain- ing suitable amounts of antisera (Dako anti- human IgG heavy-chain specific). RESULTS Patients and samples. Serum samples for measurement of CIC concentrations were taken serially from 22 patients with GTT requiring chemotherapy for invasive mole or choriocarcinoma (age 18-52, median 25 years) and 11 patients with advanced MT (age 17-41, median 25 years) before and during chemotherapy. The MT originated in the testis in 9 patients, the mediastinum in 1 and the ovary in 1. Control sera were ob- tained from 43 healthy volunteers of both sexes (age 19-68, median 31 years) and 29 patients with rheumatoid arthritis (whose sera were kindly donated by Professor R. N. Maini, the Kennedy Institute, London, W6). Immediately after separation, serum was aliquoted and frozen at -20°C, or at -70°C for storage in excess of 4 weeks. Patients were all treated with cytotoxic chemotherapy as described for GTT by Bag- shawe & Begent (1981) and for MT as des- cribed by Newlands et al. (1980). Concentrations of CIC were measured serially by the PEG precipitation and CIqSP assays in patients receiving chemo- therapy for GTT and MT. Results were correlated with values of the conventional serum tumour markers, hCG and AFP, and complete remission (CR) was defined by normal hCG and AFP concentrations. The validity of responses and remissions defined by hCG or AFP values was con- firmed by clinical examination and con- ventional radiology, including computeri- zed tomography where appropriate. Received 27 July 1981 Accepted 26 October 1981 Furthermore it has been suggested that concentrations of CIC correlate with tumour volume, and this has been demonstrated in some instances of viral and chemically induced tumours of experimental animals (Jennette & Feldman, 1977; Jennette, 1980). Less well defined correlations with tumour volume have been found in patients with several types of cancer. High values are common in relapse or before treatment, particularly with advanced stages of disease, and there is a tendency for the results to be normal in remission (for review see Baldwin & Robins, 1980). that measuremnnt of CIC concentrations may provide a tumour-marker system which will be of value in the management of patients with cancer (Baldwin & Robins, 1980). In order to be useful, such a tumour marker must either be more sensitive or more specific than current methods of tumour assessment. In malignancies such as gestational trophoblastic tumours (GTT) or malignant teratoma (MT), for which satisfactory biochemical markers are already available (Javadpour, 1979; Bagshawe & Searle, 1977), it is clear that measurement oftumour volume by clinical examination or conventional radiology can give a misleading assessment of the number of viable malignant cells (Bag- shawe, 1973; Newlands et al., 1980). In order to assess the value of CICs as a tumour marker we have therefore com- pared them with established biochemical markers the concentrations of which are related to viable tumour mass, namely human chorionic gonadotrophin (hCG) in , These studies have led to the suggestion R. H. J. BEGENT ET AL: 218 were performed twice wveekly during treat- ment. Total serum IgG.-This was quantitated by solid-phase radioimmunoassay (Walker et al., 1978). were performed twice wveekly during treat- ment. i GTT and a-foetoprotein (AFP) and hCG in MT. GTT and a-foetoprotein (AFP) and hCG in MT. Total serum IgG.-This was quantitated by solid-phase radioimmunoassay (Walker et al., 1978). Gestational trophoblastic tumours CIC concentrations were raised before treatment in 6/22 patients, this being significantly more than in the normal group (P= 0.025 by x2). There was no correlation with tumour load as measured 80 70 60 30 20 10 0 a 0 l~~~ O I Nrll I ep rdws BemC End of wthrltls bh mn rer obmnist O-MfG.T.T, FIG. 1. CIC concentrations measured by PEG precipitation assay in patients with GTT and controls. I I 80 70 60 30 20 10 0 a 0 l~~~ O I Nrll I ep rdws BemC End of wthrltls bh mn rer obmnist O-MfG.T.T, I I Tumour markers.-Human chorionic gona- dotrophin (normal range < 10 iu/l) was measured by automated radioimmunoassay using an antiserum directed against the f subunit (Kardana & Bagshawe, 1976; Bag- shawe, 1975). x-Foetoprotein (normal range <10 jtg/l) was measured using a double- antibody radioimmunoassay. These assays O-MfG FIG. 1. CIC concentrations measured by PEG precipitation assay in patients with GTT and controls. IMMUNE COMPLEXES AS TUMOUR MARKERS 219 0 0 0 * 6' I. ., IO uv .. ', X.4 0 ~~~~~..-O 1 .o\|-:f FIG. 2. Relationship of pre-treatment CIC concentrations to AFP in MIT (A) and to hCG in MT (0) and in GTT (0). When both markers were present in one patient with MT (6 cases) only the marker giving the higher value is shown. I I I 11 Cytotoxic chemothrp le .~~~~~~~40 1 ~301 g102 lop 020' I loS 10 3 4 5 Monft 0 0 0 * 6' I. ., IO uv .. ', X.4 0 ~~~~~..-O 1 .o\|-:f I I I 11 Cytotoxic chemothrp le .~~~~~~~40 1 ~301 g102 lop 020' I loS 10 3 4 5 Monft Monft Monft FIG. 3. Chart showing conicentrations of htCG and CIC in a patient during chemo- therapy foi GTT. CIC concentrations were raised before treatment, falling to normal in association with a response to treatment, shown by a fall in hCG. CIC values later rose falling to normal after chemotherapy was stopped. hCG remained undetectable or at very low levels during this time, indicating CR. values before treatment is included in Group 2. p 2. Raised CIC concentrations (in 2 or more consecutive specimens) in spite of sustained CR.-7/8 patients in whom this occurred had normal values before treatment. Gestational trophoblastic tumours The remaining patient had high values before treatment which be- came normal when CR was achieved and later rose once more. The increases occur- red after 4-19 (median 10) weeks' chemo- therapy, and did not appear to predict relapse, since 7/8 patients remain in CR 18-22 months after entry to the study. The 8th patient did relapse after stopping chemotherapy, but CIC concentrations had become consistently normal by this time. FIG. 2. Relationship of pre-treatment CIC concentrations to AFP in MIT (A) and to hCG in MT (0) and in GTT (0). When both markers were present in one patient with MT (6 cases) only the marker giving the higher value is shown. by concentrations of hCG (Fig. 2). Se- quential studies of the 22 patients (3-35, median 12, assays per patient) during treatment (lasting 2-36, median 19, weeks) identified 3 patterns of behaviour: cor- relation with hCG, raised CIC concentra- tions in spite of sustained CR and normal values throughout treatment. A simplified representation of the results in the 20 patients who achieved CR is given in Fig. 1. Analysis of individual patients included some with raifibd values between CR and the end of treatment, which cannot be shown in Fig. 1. This more detailed analysis gave the following results for the 3 groups: '301 20 110 - FIG. 4. Chart showing concentrations of AFP and CIC in a patient with MT. CIC concentrations were normal before treat- ment, rising during chemotherapy and returning to normal after finishing treat- ment. The patient then relapsed with rising AFP values but not CIC concentrations. I 'k i '301 20 110 - I 'k i '301 20 110 - I 'k i Normal values of CIC throughout treatment One of the 2 patients in this group attained sustained CR and the other a partial remission. A detailed example of a further patient is given in Fig. 6. In order to investi- gate whether raised values in the PEG precipitation assay could be explained by raised total serum IgG concentra- tions, the results of these two measure- ments were compared in all patients before treatment. No statistically signifi- cant correlation was found by Spearman's rank-correlation test for the patients with GTT (rs = 0.24) nor for those with MT (r. = 0.3). Total serum IgG was also measured serially in 4 patients in whom raised values were found in the PEG precipitation assay during the course of the disease. No correlation was seen be- tween changes in PEG assay results and total serum IgG. 1. Correlation with HCG.-In 4/6 patients in whom CIC concentrations were raised before treatment, values returned to normal either by CR or the end of treatment (Fig. 1). In a further patient there was no response to chemo- therapy and she is therefore excluded from Fig. 1. CIC concentrations remained high. The 6th patient with raised CIC FIG. 4. Chart showing concentrations of AFP and CIC in a patient with MT. CIC concentrations were normal before treat- ment, rising during chemotherapy and returning to normal after finishing treat- ment. The patient then relapsed with rising AFP values but not CIC concentrations. FIG. 4. Chart showing concentrations of AFP and CIC in a patient with MT. CIC concentrations were normal before treat- ment, rising during chemotherapy and returning to normal after finishing treat- ment. The patient then relapsed with rising AFP values but not CIC concentrations. R. H. J. BEGENT ET AL. 220 cheCktdwnmoterpy I1 1II1 1 .1 10 i 10 10 FIG. 6.-Chart showing concentrations of hCG and CIC in a patient with MT. CIC values returned to normal in associa- tion with a response to chemotherapy, but gave a less sensitive indication of disease status than hCG. 40 \| d FIG. 5. CIC concentrations measured by PEG precipitation in patients with MT. 80 70 60 50, `40 20 cheCkt*dwnmoterpy I1 1II1 1 .1 10 i 10 10 40 \| d FIG. 6.-Chart showing concentrations of hCG and CIC in a patient with MT. CIC values returned to normal in associa- tion with a response to chemotherapy, but gave a less sensitive indication of disease status than hCG. FIG. 5. CIC concentrations measured by PEG precipitation in patients with MT. 3. Normal values of CIC throughout treatment.-8 patients in this group achieved sustained CR and the 9th died after 2 weeks without responding to treatment. 3. Normal values of CIC throughout treatment.-8 patients in this group achieved sustained CR and the 9th died after 2 weeks without responding to treatment. later rose again before treatment was completed. The rises occurred 1-16 weeks (median 8) after starting chemotherapy and did not appear to predict relapse, since 4 of the patients remain in CR 15-21 months (median 18) after entry to the study. The 5th relapsed after finishing chemotherapy, but CIC concentrations had become consistently normal by then (Fig. 4). A detailed profile of one representative patient is shown in Fig. 3. Malignant teratoma CIC concentrations were raised before treatment in 5 of 11 patients, but there was no correlation with concentrations of hCG or AFP (Fig. 2). Sequential studies of all patients (6-31 assays per patient, median 21) during treatment (lasting 12-48 weeks, median 26) showed similar patterns of behaviour to GTT and a simplified representation of the results in the 10 patients who achieved CR is given in Fig. 5. Normal values of CIC throughout treatment DISCUSSION The results show that measurement of CIC concentrations in GTT and MT does not provide a satisfactory tumour-marker system for clinical use. Even in those patients in whom falls in CIC concentra- tion correlated with response to treatment, the sensitivity was inferior to that of hCG or AFP, and probably no better than clini- cal examination or radiological assessment. The most serious deficiency, however, is in specificity; there were rises in CIC concentration during cytotoxic chemo- therapy in patients with sustained and well documented CR. In all 4 of the patients in whom serum samples were available after treatment, CIC concen- trations returned to normal, suggesting that the chemotherapy itself might be responsible. , There are unfortunately few malignan- cies other than GTT and MT which are chemosensitive and also have a satis- factory biochemical tumour-marker system. Analysis of the value of CIC assays in the more common cancers cannot, therefore, be readily assessed by such stringent criteria. Given the small amount of data published in conditions in which relapses during chemotherapy are common (such as carcinoma of the ovary), it may be difficult to be certain whether rises in CIC concentrations which appear to predict relapse (Poulton et al., 1978) are caused by recurrent tumour or by the chemotherapy itself. p The effects of chemotherapy on CIC formation are likely to be complex. For example, eradication of tumour leads to removal of tumour products which might be antigen components of CIC, thus accounting for a fall in CIC concentrations during the early stages of chemotherapy. Cytotoxic drugs also affect antibody production, and it has been suggested that the effect is particularly marked on T-suppressor-cell function (Diamantstein et al., 1979; Athanassiades et al., 1978). Rheumatoid-factor levels have been shown to rise in patients receiving treatment for breast and bronchial cancer (Twomey et al., 1976) and this may reflect a more generalized disturbance of antibody pro- duction leading to increased autoantibody formation and perturbation of idiotype- anti-idiotype interactions. A recent review by Roitt et al. (1981) illustrates the y py The various currently available assays for CIC are based on recognition of the alterations which occur to antibody when it comes complexed, but may nevertheless give different results in the same disease state (Lambert et al., 1978). IMMUNE COMPLEXES AS TUMOUR MARKERS 221 Correlation with disease status In the 5 patients with raised concen- trations of CIC before treatment, values fell to normal at CR, as defined by normal hCG and AFP levels. One patient later relapsed with rising hCG and CIC con- centrations rose at the same time. Raised CIC concentrations (in 2 or more consecutive specimens) in spite of sustained remission This occurred in 4 patients with normal values before treatment. In a further patient they had been raised before treat- ment then become normal with CR and IMMUNE COMPLEXES AS TUMOUR MARKERS CIqSP assay complexity of idiotypic networks in con- trol of antibody production and hence the difficulty of predicting the effects of cytotoxic chemotherapy in human disease. The picture is further complicated by specific and nonspecific immunosuppres- sive effects of the tumour which are re- moved during successful chemotherapy. Results in patients with GTT and MT did not differ significantly from those of the normal group, and there was a good correlation with results of the PEG precipitation assay in patients with rheumatoid arthritis (rs= 0 72, P < 0 001, by Spearman's rank-correlation test). Impairment of reticuloendothelial func- tion by cytotoxic chemotherapy, as pre- viously demonstrated in rats (Sharbaugh & Grogan, 1969) and man (Magarey & Baum, 1970; Lokich et al., 1974), may also impair clearance of normally occurring or tumour-related CIC from the circula- tion. Cytotoxic drugs could achieve this by depletion of monocytes of marrow origin which probably develop into tissue- fixed macrophages, such as Kupffer cells, responsible for clearing CIC for the cir- culation (for review see Lancet, 1980). REFERENCES p LOKICH, J. J., DRUM, D. E. & KAPLAN, NV. (1]974) Hepatic toxicity of nitrosourea analogues. Clin. Pharm. Therap., 16, 363. ATHANASSIADES, P. H., PLATTs-MILLs, T. A. E., ASHERSON, G. L. & OLIVER, R. T. D. (1978) Effect of antileukaemic chemotherapy on helper and suppressor activity of T cells on immuno- globulin production by B cells. Eur. J. Cancer, 14,971. p , , MAGAREY, C. J. & BAUM, M. (1970) Reticuloendo- thelial activity in humans with cancer. Br. J. 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K., GUARNOTTA, G. & 6 others (1981) Idiotypic networks and their possible exploitation for manipulation of the immune response. Lancet, i, 1041. g p BALDWIN, R. W. & ROBINS, R. A. (1980) Circulating immune complexes in cancer. In Cancer Markers, (Ed. Sell). Clifton, New Jersey: Humana Press, p. 507. p SHARBAUGH, R. J. & GROGAN, J. B. (1969) Suppres- sion of reticuloendothelial function in the rat with cyclophosphamide. J. Bacteriol., 100, 117. (1980) p DIAMANTSTEIN, T., WILLINGER, E. & REIMAN, J. (1979) T-suppressor cells sensitive to cyclo- phosphamide and to its in vitro active derivative 4-hydroperoxycyclo-phosphamide control the mi- togenic response of murine splenic B cells to dextran sulfate. A direct proof for different sensitivities of lymphocyte subsets to cyclo- phosphamide J. Exp. Med., 150, 1571. y p p THEOFILOPOULOS, A. N. & DIXON, F. J. (1980) The biology and detection of immune complexes. Adv. Immunol., 28, 89. , , TWOMEY, J. J., ROSSEN, R. D., LEWIS, V. M., LAUGHTER, A. H. & DOUGLAS, C. C. DISCUSSION These methods do not distinguish between CIC with different antigen components such as tumour products, normal-tissue antigens, non-antigen-containing y-globulin aggre- gates and specific antiglobulin complexes. It therefore seems unlikely that any of these assays will be much more specific than those used in this study. This should R. H. J. BEGENT EY AL. 222 Clq solid plhase anld Raji cell assays. J. Clin. Invest., 63, 468. Clq solid plhase anld Raji cell assays. J. Clin. Invest., 63, 468. not be allowed to obscure the possibility that some CIC may contain as yet un- recognized tumour products which are relatively tumour-specific. 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J., & MOWBRAY, J. F. (1980) Circulating immune complexes containing anti-VIII antibodies in multi-transfixed patients with haemophilia A. Clin. Exp. Immunol., 39, 315. WVe wishl to thiank Professor K. 1). Bagshtawe for hiis support and advice, Dr E. S. Newlands for permission to study his patients, Yong Lan Pookim for technical assistance and our colleagues in the Department of Medical Oncology, Charing Cross Hospital, who performed the assays for hCG and AFP. We are grateful to the Cancer Research Campaign for its support. p LAMBERT, P. H., DIXON, F. J., ZUBLER, R. H., & 16 others. (1978) A W.H.O. collaborative study for the evaluation of eighteen methodls for detecting immune complexes in serum. J. Clin. Lab. Immunol., 1, 1. , , LANCET EDITORIAL (1980) Bone-marrow origin of Kupffer cells. Lancet, i, 130. REFERENCES (1976) Rheumatoid factor and tumour-lhost interaction. Proc. Natl Acad. Sci., 73, 2106. WALKER, L. A., AHLIN, T. D., TUNG, K. S. & WILLIAMS, R. C. 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https://openalex.org/W4312200336	https://www.biorxiv.org/content/biorxiv/early/2022/12/25/2022.12.25.521899.full.pdf	English	null	Formal cooperation in macaque monkeys obtained via single-payoff change from formal coordination games	bioRxiv (Cold Spring Harbor Laboratory)	2,022	cc-by	24,057	. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint 1 Formal cooperation in macaque monkeys obtained via single-payoff change from formal 2 coordination games 3 4 Charlotte van Coeverden and Wolfram Schultz 5 6 Department of Physiology, Development and Neuroscience 7 University of Cambridge 8 Cambridge CB2 3DY 9 United Kingdom 10 11 Corresponding Author: Wolfram Schultz 12 Email: Wolfram.Schultz@protonmail.com 13 14 Author Contributions: CvC and WS designed the experiment, CvC conducted experiments and 15 analyzed data, CvC and WS wrote the paper. 16 17 Competing Interest Statement: The authors declare no competing interests. 18 19 Keywords: social, choice, temptation, defection, Prisoner’s Dilemma, Stag Hunt 20 21 Acknowledgements: We thank Aled David and Christina Thompson for animal and technical 22 support and Colin Camerer, Raymundo Báez-Mendoza, Alexandre Pastor-Bernier and Fabian 23 Grabenhorst for discussions on game theory and the design of this experiment. This study was 24 supported by Wellcome Trust (WT 095495, WT 204811, WT 206207), European Research Counci 25 (ERC; 293549) and US National Institutes of Mental Health (NIMH) Caltech Conte Center 26 (P50MH094258). For the purpose of Open Access, the authors have applied a CC BY public 27 copyright licence to any Author Accepted Manuscript version arising from this submission. 28 29 Formal cooperation in macaque monkeys obtained via single-payoff change from formal coordination games 29 D c b r 25 2022 2 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint D b 25 2022 2 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 Introduction Cooperation characterizes social behavior that results in the common good and thus is of societal importance. Cooperation has two key characteristics: players make common choices that allow them to obtain the ultimate highest collective gain, but they need to forego an immediate higher selfish gain. Thus, cooperation results in an ultimate optimal collective benefit by outweighing an initial suboptimal individual gain (Nowak 2006). By contrast, coordination characterizes common choices for maximal gain without foregoing an immediate selfish gain. Thus, coordination results in immediate optimal collective outcome without foregoing an initial selfish gain. Overall, by acting beyond the short term, cooperation is more demanding than coordination. y p g The quintessential abstraction and operational definition of cooperation is provided by the Prisoner’s Dilemma (PD) that had originally been designed for performance in single trials. The most beneficial choice for an individual player in such single-shot PD is selfish defection in which the other player receives only a small payoff, thus resulting in limited common benefit. However, social behavior usually depends on experience and thus involves repeated choices during which long-term gains can be evaluated. Correspondingly, monkeys in the laboratory make repeated choices, in particular in tasks suitable for neurophysiology with appropriate statistics. The repeated experience is captured in the iterated PD in which players perform an unknown number of PD repetitions. The players come to cooperate over repeated trials, as the long-term common outcome exceeds the short-term individual gain, and the short term losses from getting defected by a selfish player have a chance to give way to an overall mutual gain for both players. All players in iterated and unlimited PD gain over the long run by cooperating. Thus, iterated PD captures well the notion of cooperation: two players obtain the highest common outcome by foregoing higher short-term individual gains (Rapoport 1974). PD-style cooperation games contrast with coordination games in which each of two players chooses the best option without having to forego any initial higher gain. p y p g g y g g Cooperation and coordination are not unique to humans and have been studied in animals (Stephens et al. 2002). In iterated versions of these games, macaque and cebus monkeys perform well in coordination games (de Waal & Davis 2003; Brosnan et al. 2012; Smith et al. Abstract ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 December 25, 2022 Abstract Coordination and cooperation are hallmarks of the behavior of social animals. Coordination 33 requires common choices to obtain maximum benefit, whereas cooperation requires to forgo 34 immediate selfish outcome for later common maximum benefit. A well validated economic game 35 for investigating cooperation is Prisoner Dilemma (PD). Recent studies show that monkeys 36 cooperate to a limited extent when playing an iterated PD. In our experiment, macaque monkeys 37 made choices on a touchscreen to obtain juice reward whose amount depended on the choices of 38 both animals. We designed four coordination games and two cooperation games (iterated PD) that 39 differed only in a single payoff (the so-called temptation) while all other payoffs remained constant. 40 The increasing temptation payoff resulted in performance that varied somewhat in the coordination 41 game (probability of common choice between p = 0.55 and p = 0.70) but dropped in both 42 cooperation games while nevertheless remaining significant (p = 0.28 to p = 0.68). The response 43 time of the second player increased significantly when the first player chose the cooperative option 44 across all games, suggesting reciprocation; further, the animals seemed to benefit from seeing the 45 action of the other player, indicating that the choices incorporated a social component. Taken 46 together, our results demonstrate good cooperation in the iterated PD by macaque monkeys after 47 being primed with coordination games. 48 December 25 2022 3 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25 2022 3 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint D b 25 2022 3 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 4 y g , , This research has been ethically reviewed, approved, regulated and supervised by the following individuals and institutions in the UK and at the University of Cambridge (UCam): the Minister of State at the UK Home Office, the Animals in Science Regulation Unit (ASRU) of the UK Home Office implementing the Animals (Scientific Procedures) Act 1986 with Amendment Regulations 2012, the UK Animals in Science Committee (ASC), the UK Home Office Inspector for our UCam laboratory, the UK National Centre for Replacement, Refinement and Reduction of Animal Experiments (NC3Rs), the UCam Animal Welfare and Ethical Review Body (AWERB), the UCam Governance and Strategy Committee, the Home Office Establishment License Holder of the UCam Biomedical Service (UBS), the UBS Director for Governance and Welfare, the UBS Named Information and Compliance Support Officer, the UBS Named Veterinary Surgeon (NVS), and the UBS Named Animal Care and Welfare Officer (NACWO). The animals were housed together with other adult male rhesus monkeys in home cages with enrichment, as follows: Monkeys T and V were cage mates together with two other monkeys; Monkey L was housed in a different holding room in a cage of his own together with two to three other monkeys in the same room. Before behavioral testing, Monkeys T and L had a headpost that had been surgically implanted for later neuronal recordings using general anesthesia and aseptic procedures (the early implantation served for allowing gradual osteointegration); however, the currently described behavioral tests were performed without head fixation. When seated in their respective primate chairs, Monkey L dominated Monkey T who dominated Monkey V, as tested by their tendency to grab food morsels offered by an experimenter; the dominance hierarchy appeared to correlate with their respective body weights. In the common home cage of Monkeys T and V, another monkey not used for the current experiments dominated Monkey T (who dominated Monkey V). For each daily session, the animal entered from their home cage into an individually adjusted and comfortably fitting purpose-made primate chair (Crist Instruments); each animal had its own primate chair for the duration of the experiment. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 The chair with the animal inside was 140 wheeled into the laboratory, where the animal was trained in three pair-wise combinations (dyads) 141 with one of the other experimental monkeys to perform the behavioral task in which it received 142 juice reward by contacting a touch key and subsequently touching a horizontally mounted 143 touchscreen. After an initial habituation of several months to the primate chair, laboratory, touch 144 key, touchscreen and juice spout, the animals were trained in the specific social task. 145 146 Behavioral task. Two monkeys in any of the three possible dyads faced each other across the 147 common horizontal touchscreen. Both animals performed in a binary choice task for juice reward 148 (Fig. 1A). A trial started when both animals contacted a touch-sensitive key, upon which the same 149 two fractal stimuli appeared on the touchscreen for each animal. The two fractals indicated the two 150 choice options; they had grey color, equal size, normalized luminosity and were visible to both 151 animals (unless otherwise stated) At 500 ms after fractal presentation a small grey rectangle 152 actions and choices (di Pellegrino et al. 1992; Hosokawa & Watanabe 2012; Báez-Mendoza et al. 02 2013; Haroush & Williams 2015; Chang et al. 2015; Grabenhorst et al. 2019; Báez-Mendoza et al. 03 2021). 04 0 actions and choices (di Pellegrino et al. 1992; Hosokawa & Watanabe 2012; Báez-Mendoza et al. 2 2013; Haroush & Williams 2015; Chang et al. 2015; Grabenhorst et al. 2019; Báez-Mendoza et al. 3 2021). 4 Animals and ethics. Three adult male rhesus monkeys (Macaca mulatta) served in this study. 8 Monkey L40 (‘Monkey L’), weighing 12.0 – 13.6 kg, was trained beginning May 25, 2014 and 9 tested in the full task from Sep 8, 2014 to May 22, 2017. Trident (‘Monkey T’), weighing 8.9 – 14.2 0 kg, was trained beginning May 25, 2014 and tested in the full task from Sep 8, 2014 to Feb 8, 2017. 1 Virtue (‘Monkey V’), weighing 5.1 kg – 9.0 kg, was trained beginning August 14, 2015 and tested 2 in the full task from Oct 28, 2015 to May 22, 2017. Thus, all three animals were tested in the full 3 task simultaneously from August 14, 2015 to Feb 8, 2017. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 actions and choices (di Pellegrino et al. 1992; Hosokawa & Watanabe 2012; Báez-Mendoza et al. 102 2013; Haroush & Williams 2015; Chang et al. 2015; Grabenhorst et al. 2019; Báez-Mendoza et al. 103 2021). 104 105 Methods 106 107 Animals and ethics. Three adult male rhesus monkeys (Macaca mulatta) served in this study. 108 Monkey L40 (‘Monkey L’), weighing 12.0 – 13.6 kg, was trained beginning May 25, 2014 and 109 tested in the full task from Sep 8, 2014 to May 22, 2017. Trident (‘Monkey T’), weighing 8.9 – 14.2 110 kg, was trained beginning May 25, 2014 and tested in the full task from Sep 8, 2014 to Feb 8, 2017. 111 Virtue (‘Monkey V’), weighing 5.1 kg – 9.0 kg, was trained beginning August 14, 2015 and tested 112 in the full task from Oct 28, 2015 to May 22, 2017. Thus, all three animals were tested in the full 113 task simultaneously from August 14, 2015 to Feb 8, 2017. 114 This research has been ethically reviewed, approved, regulated and supervised by the 115 following individuals and institutions in the UK and at the University of Cambridge (UCam): the 116 Minister of State at the UK Home Office, the Animals in Science Regulation Unit (ASRU) of the 117 UK Home Office implementing the Animals (Scientific Procedures) Act 1986 with Amendment 118 Regulations 2012, the UK Animals in Science Committee (ASC), the UK Home Office Inspector 119 for our UCam laboratory, the UK National Centre for Replacement, Refinement and Reduction of 120 Animal Experiments (NC3Rs), the UCam Animal Welfare and Ethical Review Body (AWERB), 121 the UCam Governance and Strategy Committee, the Home Office Establishment License Holder of 122 the UCam Biomedical Service (UBS), the UBS Director for Governance and Welfare, the UBS 123 Named Information and Compliance Support Officer, the UBS Named Veterinary Surgeon (NVS), 124 and the UBS Named Animal Care and Welfare Officer (NACWO). 125 The animals were housed together with other adult male rhesus monkeys in home cages with 126 enrichment, as follows: Monkeys T and V were cage mates together with two other monkeys; 127 Monkey L was housed in a different holding room in a cage of his own together with two to three 128 other monkeys in the same room. Introduction It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 4 actions and choices (di Pellegrino et al. 1992; Hosokawa & Watanabe 2012; Báez-Mendoza et al. 2 2013; Haroush & Williams 2015; Chang et al. 2015; Grabenhorst et al. 2019; Báez-Mendoza et al. 3 2021). 4 5 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 December 25, 2022 4 Introduction 2019) but are just above chance level or instable in cooperation games (iterated PD; Haroush & Williams 2015; Smith et al. 2019). The results indicate that macaque monkeys can coordinate their choices for a common good but have difficulties cooperating in formal iterated PD. As birds and rats do not usually cooperate in straightforward iterated PD (Gardner et al. 1984; Clements & Stephens 1995; Stevens & Stephens 2004), cooperation may evolve across species. Thus, the evolutionary aspect and the societal importance of cooperation warrants further exploration of the capacity of cooperation in monkeys. Our study asked how macaque monkeys can come to cooperate more efficiently, using the 86 formal iterated PD in which reward depends on the combined choices of two animals. We benefitted 87 from a previous approach that achieved cooperation in birds by priming them with maximum 88 reward for common choice (Stephens et al. 2002). Our monkeys played coordination games in 89 which the benefit from choosing the ‘Cooperate’ option and the disadvantage from choosing the 90 ‘Defect’ option were particularly well visible. We tested a series of coordination games that differed 91 only in a single payoff (the so-called ‘temptation’ payoff for choosing ‘Defect’ instead of 92 ‘Cooperate’). We stepwise increased the temptation payoff across games until the coordination 93 games became cooperation games (iterated PD). The temptation payoff encourages immediate 94 defection and thus discourages cooperation, which challenges an agent’s appreciation of long-term 95 gain from cooperation. Thus, we translated the easier ability of immediate coordination into the 96 more demanding ability of cooperation for common long-term gain. We tested these games in 97 monkeys, as they are the species of choice for ultimate investigations of neuronal mechanisms of 98 sophisticated social behavior. The animals made cooperative choices with probabilities between p = 99 0.28 and p = 0.68 that significantly exceeded chance (p = 0.25). These results should be useful for 100 investigating neuronal mechanisms of social behavior beyond the already known coding of other’s 101 December 25 2022 4 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 4 . CC-BY 4.0 International license perpetuity. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 Before behavioral testing, Monkeys T and L had a headpost that 129 had been surgically implanted for later neuronal recordings using general anesthesia and aseptic 130 procedures (the early implantation served for allowing gradual osteointegration); however, the 131 currently described behavioral tests were performed without head fixation. When seated in their 132 respective primate chairs, Monkey L dominated Monkey T who dominated Monkey V, as tested by 133 their tendency to grab food morsels offered by an experimenter; the dominance hierarchy appeared 134 to correlate with their respective body weights. In the common home cage of Monkeys T and V, 135 another monkey not used for the current experiments dominated Monkey T (who dominated 136 Monkey V). 137 For each daily session, the animal entered from their home cage into an individually 138 adjusted and comfortably fitting purpose-made primate chair (Crist Instruments); each animal had 139 its own primate chair for the duration of the experiment. The chair with the animal inside was 140 wheeled into the laboratory, where the animal was trained in three pair-wise combinations (dyads) 141 with one of the other e perimental monkeys to perform the behavioral task in which it received 142 actions and choices (di Pellegrino et al. 1992; Hosokawa & Watanabe 2012; Báez-Mendoza et al. 102 2013; Haroush & Williams 2015; Chang et al. 2015; Grabenhorst et al. 2019; Báez-Mendoza et al. 103 2021). 104 105 Methods 106 107 Animals and ethics. Three adult male rhesus monkeys (Macaca mulatta) served in this study. 108 Monkey L40 (‘Monkey L’), weighing 12.0 – 13.6 kg, was trained beginning May 25, 2014 and 109 tested in the full task from Sep 8, 2014 to May 22, 2017. Trident (‘Monkey T’), weighing 8.9 – 14.2 110 kg, was trained beginning May 25, 2014 and tested in the full task from Sep 8, 2014 to Feb 8, 2017. 111 Virtue (‘Monkey V’), weighing 5.1 kg – 9.0 kg, was trained beginning August 14, 2015 and tested 112 in the full task from Oct 28, 2015 to May 22, 2017. Thus, all three animals were tested in the full 113 task simultaneously from August 14, 2015 to Feb 8, 2017. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 114 This research has been ethically reviewed, approved, regulated and supervised by the 115 following individuals and institutions in the UK and at the University of Cambridge (UCam): the 116 Minister of State at the UK Home Office, the Animals in Science Regulation Unit (ASRU) of the 117 UK Home Office implementing the Animals (Scientific Procedures) Act 1986 with Amendment 118 Regulations 2012, the UK Animals in Science Committee (ASC), the UK Home Office Inspector 119 for our UCam laboratory, the UK National Centre for Replacement, Refinement and Reduction of 120 Animal Experiments (NC3Rs), the UCam Animal Welfare and Ethical Review Body (AWERB), 121 the UCam Governance and Strategy Committee, the Home Office Establishment License Holder of 122 the UCam Biomedical Service (UBS), the UBS Director for Governance and Welfare, the UBS 123 Named Information and Compliance Support Officer, the UBS Named Veterinary Surgeon (NVS), 124 and the UBS Named Animal Care and Welfare Officer (NACWO). 125 The animals were housed together with other adult male rhesus monkeys in home cages with 126 enrichment, as follows: Monkeys T and V were cage mates together with two other monkeys; 127 Monkey L was housed in a different holding room in a cage of his own together with two to three 128 other monkeys in the same room. Before behavioral testing, Monkeys T and L had a headpost that 129 had been surgically implanted for later neuronal recordings using general anesthesia and aseptic 130 procedures (the early implantation served for allowing gradual osteointegration); however, the 131 currently described behavioral tests were performed without head fixation. When seated in their 132 respective primate chairs, Monkey L dominated Monkey T who dominated Monkey V, as tested by 133 their tendency to grab food morsels offered by an experimenter; the dominance hierarchy appeared 134 to correlate with their respective body weights. In the common home cage of Monkeys T and V, 135 another monkey not used for the current experiments dominated Monkey T (who dominated 136 Monkey V). 137 For each daily session, the animal entered from their home cage into an individually 138 adjusted and comfortably fitting purpose-made primate chair (Crist Instruments); each animal had 139 its own primate chair for the duration of the experiment. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 When both animals had chosen within the four-second time window, they received specific numbers of 0.15 ml drops of blackcurrant juice depending on each choice option. A computer-controlled solenoid valve (SCB262C068; ASCO) delivered the juice at a 150 ms interval between the two animals from a spout in front of each animal's mouth. p To facilitate consideration of each option and prevent overtraining, a new set of two fractals was introduced after a given fractal set had been used for several trials (284 ± 185 trials per fractal set; mean ± standard deviation). Each fractal set was used only for one specific game. Thus, a new fractal set indicated either continuation of the same game or use of a new game. This procedure aimed to ensure exploration of both options before committing to a single option. All games were presented in pseudorandom order. 70 71 Fig. 1. Behavioral task and experimental design. Change of a single payoff mediates the transition from formal 72 coordination games to formal cooperation games (Prisoner's Dilemma, PD). (A) Task setup with two monkeys 73 facing each other across a horizontally mounted touchscreen. Each animal lifted one hand off a touch sensitive 74 holding key to touch one of two grey fractals indicating different amounts of reward juice. (B) Payoff matrix 75 defining the cooperation game. Payoffs are ranked as T (temptation) > R (reward) > P (punishment) > S (getting 76 suckered). D: defection; C: cooperation. (C) Payoff matrices of coordination games. Green circles indicate the 77 single payoff that distinguishes each game and varies across games (T, temptation). The second game from left is 78 also known as Stag Hunt. (D) Payoff matrices of formal cooperation games. The coordination game becomes a 79 coordination game when the temptation payoff (T) surpasses the reward payoff (T > R; green circles), making 80 defection (D) more valuable than cooperation (C). (E) Playing a coordination game. The players obtain maximal 81 payoff by both choosing the individually best option (coordination; circle). (F) Playing an iterated PD cooperation 82 game. Each player is tempted to choose the largest payoff (6 units). However, when both players do so, each 83 player receives only a low payoff (2 units; circle). 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 The chair with the animal inside was wheeled into the laboratory, where the animal was trained in three pair-wise combinations (dyads) with one of the other experimental monkeys to perform the behavioral task in which it received juice reward by contacting a touch key and subsequently touching a horizontally mounted touchscreen. After an initial habituation of several months to the primate chair, laboratory, touch key, touchscreen and juice spout, the animals were trained in the specific social task. Behavioral task. Two monkeys in any of the three possible dyads faced each other across the common horizontal touchscreen. Both animals performed in a binary choice task for juice reward (Fig. 1A). A trial started when both animals contacted a touch-sensitive key, upon which the same two fractal stimuli appeared on the touchscreen for each animal. The two fractals indicated the two choice options; they had grey color, equal size, normalized luminosity and were visible to both animals (unless otherwise stated). At 500 ms after fractal presentation, a small grey rectangle appeared next to each fractal to elicit each animal's choice. The animal released the touch key and December 25, 2022 5 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 5 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 selected the target of its choice. Each animal was free to choose at a self-determined moment but had to complete the trial within four seconds. Thus, there was neither a specific signal nor any other requirement that determined the sequence in which the animals chose. Although the limited response time encouraged rapid responses, the duration was long enough to allow an animal to observe the other animal's choice before making its own choice. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 (G) Unsatisfied, in the same cooperation game, one player may 84 instead choose the other option that would have provided a smaller payoff had both players chosen it (4 units) but 85 receives only 1 unit because of the other player’s choice (1 unit; circle). (H) In the same cooperation game, if the 86 other player also changes the choice, both players obtain the maximum common good (4 units for each player; 87 circle) 88 C D C D R T S P R S T P Prisoner’s Structure T > R > P > S C: cooperate D: defect Cooperation Games C D C D 4 5 1 2 4 1 5 2 Prisoner’s 1 C D C D 4 1 1 2 4 1 1 2 Coordination Games Pure coordination Stag Hunt C D C D 4 2 1 2 4 1 2 2 C D C D 4 3 1 2 4 1 3 2 C D C D 4 4 1 2 4 1 4 2 Temptation 1 Temptation 2 C D C D 4 6 1 2 4 1 6 2 Prisoner’s 2 A B C D 2 2 C D 4 6 C D 4 6 Try something else by paying a price: C D 4 6 C D 4 6 This may lead to cooperation: C D 4 6 C D 4 6 F G H 2 2 2 2 1 C D 4 1 C D 4 1 1 1 E 2 2 1 1 1 1 1 P iB C This may lead to cooperation: C D 4 6 C D 4 6 H 2 2 C D 4 1 C D 4 1 1 1 E 2 2 1 1 D E F Fig. 1. Behavioral task and experimental design. Change of a single payoff mediates the transition from formal 172 coordination games to formal cooperation games (Prisoner's Dilemma, PD). (A) Task setup with two monkeys 173 facing each other across a horizontally mounted touchscreen. Each animal lifted one hand off a touch sensitive 174 holding key to touch one of two grey fractals indicating different amounts of reward juice. (B) Payoff matrix 175 defining the cooperation game. Payoffs are ranked as T (temptation) > R (reward) > P (punishment) > S (getting 176 suckered). D: defection; C: cooperation. (C) Payoff matrices of coordination games. 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 The temptation 214 payoff T defined the difference between coordination games and cooperation games: payoff T in 215 coordination games was never higher than the payoff received when both players chose the same 216 option, whereas payoff T in cooperation games was the highest payoff and exceeded the payoff for 217 any same choice. Thus, the stepwise increase of the defining temptation payoff T defined the 218 transition from coordination games (Fig. 1C) to cooperation games when T became the highest 219 payoff (Fig. 1D). As the word indicates, the higher payoff for defecting on a cooperating player 220 constitutes a temptation that defines the PD. Thus, we were able to transition from coordination 221 games to cooperation (PD) games by varying a single payoff while holding all other payoffs 222 constant. 223 224 Analysis of choices. We recorded choices from the animals in all three dyads. We tested whether 225 choices by one animal were more likely based on the already known choices of the other animal, 226 using trials in which the animal had chosen second. We used the following logistic regression: 227 228 𝑃(𝐶ℎ𝑜𝑖𝑐𝑒𝑠𝑂𝑤𝑛) = ! !"#!(#$%#&∗()+,-./0)-1%2) Eq. 1 229 230 Analysis of strategies. Strategies can be summarised as sets of rules that determine the choice 231 given a set of circumstances. Trials were classed according to three strategies for each animal: 232 Games. The properties of economic games are determined by each game's specific payoff matrix. 189 The payoff from choosing a particular option depends on the own choice and, importantly, also on 190 the other player's choice. The PD game is special in paying the highest payoff for a deliberately 191 different choice than an opponent who is choosing an option that would lead to maximal common 192 payoff when both players were to choose the same option ('cooperation'). By contrast, in a 193 coordination game, both players receive the highest payoff when they both choose the best option 194 and, importantly, cannot get more payoff by choosing differently. All our games contained two 195 choice options that were identical for both animals and were labelled option C and option D 196 (alluding to 'cooperate' and 'defect' with PD). To simplify the task for the monkeys, we used only 197 positive outcomes, namely drops of rewarding fruit juice. 198 The characteristics of the PD game are as follows. 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 Games. The properties of economic games are determined by each game's specific payoff matrix. 189 The payoff from choosing a particular option depends on the own choice and, importantly, also on 190 the other player's choice. The PD game is special in paying the highest payoff for a deliberately 191 different choice than an opponent who is choosing an option that would lead to maximal common 192 payoff when both players were to choose the same option ('cooperation'). By contrast, in a 193 coordination game, both players receive the highest payoff when they both choose the best option 194 and, importantly, cannot get more payoff by choosing differently. All our games contained two 195 choice options that were identical for both animals and were labelled option C and option D 196 (alluding to 'cooperate' and 'defect' with PD). To simplify the task for the monkeys, we used only 197 positive outcomes, namely drops of rewarding fruit juice. 198 The characteristics of the PD game are as follows. When both players choose to cooperate 199 (C), they receive an intermediate payoff (R for reward). When both players choose to defect (D), 200 they receive a smaller intermediate payoff (P for punishment). When only one player chooses defect 201 (D) and the other player chooses C (cooperate), the defecting player receives the highest payoff (T 202 for temptation) and the cooperating player receives the lowest payoff (S for getting suckered). Thus, 203 the hierarchy of payoffs in PD is defined as (T = temptation) > R (= reward) > P (= punishment) > S 204 (= getting suckered) (Fig. 1B). 205 When playing a coordination game, both players receive the highest payoff when they both 206 choose the cooperation option C (for simplicity called ‘cooperation’ even for coordination games) 207 and the same or lower payoff when both choose the defection option D. When each player chooses 208 a different option, they receive a submaximal payoff that is smaller than when both players choose 209 option C. 210 We systematically advanced the animals’ training and testing from four coordination games 211 to two PD games by stepwise incrementing a single payoff specific for each game (Fig. 1C and D, 212 green circles). The defining payoff was labelled as T (temptation) in correspondence to PD. It was 213 paid out to the player choosing option D when the other player chooses option C. 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 Green circles indicate the 177 single payoff that distinguishes each game and varies across games (T, temptation). The second game from left is 178 also known as Stag Hunt. (D) Payoff matrices of formal cooperation games. The coordination game becomes a 179 coordination game when the temptation payoff (T) surpasses the reward payoff (T > R; green circles), making 180 defection (D) more valuable than cooperation (C). (E) Playing a coordination game. The players obtain maximal 181 payoff by both choosing the individually best option (coordination; circle). (F) Playing an iterated PD cooperation 182 game. Each player is tempted to choose the largest payoff (6 units). However, when both players do so, each 183 player receives only a low payoff (2 units; circle). (G) Unsatisfied, in the same cooperation game, one player may 184 instead choose the other option that would have provided a smaller payoff had both players chosen it (4 units) but 185 receives only 1 unit because of the other player’s choice (1 unit; circle). (H) In the same cooperation game, if the 186 other player also changes the choice, both players obtain the maximum common good (4 units for each player; 187 circle). 188 December 25, 2022 6 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 6 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 6 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 When both players choose to cooperate 9 (C), they receive an intermediate payoff (R for reward). When both players choose to defect (D), 0 they receive a smaller intermediate payoff (P for punishment). When only one player chooses defect 1 (D) and the other player chooses C (cooperate), the defecting player receives the highest payoff (T 2 for temptation) and the cooperating player receives the lowest payoff (S for getting suckered). Thus, 3 the hierarchy of payoffs in PD is defined as (T = temptation) > R (= reward) > P (= punishment) > S 4 (= getting suckered) (Fig. 1B). 5 When playing a coordination game, both players receive the highest payoff when they both choose the cooperation option C (for simplicity called ‘cooperation’ even for coordination games) and the same or lower payoff when both choose the defection option D. When each player chooses a different option, they receive a submaximal payoff that is smaller than when both players choose option C. We systematically advanced the animals’ training and testing from four coordination games to two PD games by stepwise incrementing a single payoff specific for each game (Fig. 1C and D, green circles). The defining payoff was labelled as T (temptation) in correspondence to PD. It was paid out to the player choosing option D when the other player chooses option C. The temptation payoff T defined the difference between coordination games and cooperation games: payoff T in coordination games was never higher than the payoff received when both players chose the same option, whereas payoff T in cooperation games was the highest payoff and exceeded the payoff for any same choice. Thus, the stepwise increase of the defining temptation payoff T defined the transition from coordination games (Fig. 1C) to cooperation games when T became the highest payoff (Fig. 1D). As the word indicates, the higher payoff for defecting on a cooperating player constitutes a temptation that defines the PD. Thus, we were able to transition from coordination games to cooperation (PD) games by varying a single payoff while holding all other payoffs constant. 240 241 242 T 243 s 244 a 245 246 247 248 249 250 251 252 S 253 254 R 255 256 P 257 t 258 m 259 i 260 d 261 d 262 g 263 s 264 o 265 b 266 p 267 268 g 269 f 270 1 271 b 272 w 273 A 274 ( 275 276 c 277 f 278 c 279 r 280 p 281 o 282 t 283 m 284 p 285 g 286 s 287 240 𝑊𝑆𝐿𝑆= 𝐹𝑖𝑟𝑠𝑡𝑇𝑟𝑖𝑎𝑙∗(𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 1) + (𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 𝐶ℎ𝑜𝑖𝑐𝑒𝑂𝑡ℎ𝑒𝑟𝑃𝑇) Eq. 2.3 241 242 The effect of strategies on cooperation was analysed by means of logistic regression. As the 243 strategies were too highly correlated to use them in one model, their effect on cooperation was 244 assessed separately using different models. 245 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗4567%2) Eq. 2.4 246 247 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗8494%2) Eq. 2.5 248 249 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗5:5%2) Eq. 2.6 250 251 Statistics were calculated with SPSS (IBM) and Excel (Microsoft). Matlab (Mathworks) and 252 SPSS (IBM) were used for all regression analyses. 253 254 Results 255 256 Patterns of coordination and cooperation. Using a total of three monkeys, we studied choices in 257 three pairs of two monkeys each (dyads) that sat opposite to each other across a horizontally 258 mounted computer touchscreen. The two animals chose near-simultaneously, without prescribed 259 inter-individual sequence, between two simultaneously presented options (C for cooperate, D for 260 defect); their payoffs were indicated by grey-scale fractal stimuli (Fig. 1A, B) (note that option C 261 denotes the coordination option in coordination games but the cooperation option in cooperation 262 games; for simplicity, we call C commonly ‘cooperation’ in this study). We inferred the animal's 263 stochastic preference from the probability of choosing one option over all other options of the same 264 option set (Luce 1959). However, in these social games, the rewards for each animal depended on 265 both the own and the other's choice, and the reward was not fully predictable from the choice of a 266 particular player alone. 267 The three monkeys were trained in six formal games in chronological sequence (although the 268 games were tested in pseudorandom alternation after full training). We changed the single payoff 269 for the temptation payoff (T) in steps of one unit, thus transitioning from coordination games (Fig. 270 1C, green circles) to cooperation games (Fig. 1D). With our settings, the formal coordination game 271 became a formal cooperation game when the temptation payoff exceeded the reward payoff (T > R), 272 which implemented the temptation that challenges the coordinated action as essence of cooperation. 273 According to general assumptions, the propensity of both players to choose the cooperation option 274 (C) increases with higher reward payoff (R) and lower temptation payoff (T) (Camerer 2002). 275 The difference in performing in these two classes of games is illustrated in Fig. 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 𝑊𝑆𝐿𝑆= 𝐹𝑖𝑟𝑠𝑡𝑇𝑟𝑖𝑎𝑙∗(𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 1) + (𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 𝐶ℎ𝑜𝑖𝑐𝑒𝑂𝑡ℎ𝑒𝑟𝑃𝑇) Eq. 2.3 𝑆𝐿𝑆= 𝐹𝑖𝑟𝑠𝑡𝑇𝑟𝑖𝑎𝑙∗(𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 1) + (𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 𝐶ℎ𝑜𝑖𝑐𝑒𝑂𝑡ℎ𝑒𝑟𝑃𝑇) Eq. 2.3 The effect of strategies on cooperation was analysed by means of logistic regression. As the 243 strategies were too highly correlated to use them in one model, their effect on cooperation was 244 assessed separately using different models. 245 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗4567%2) Eq. 2.4 246 247 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗8494%2) Eq. 2.5 248 249 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗5:5%2) Eq. 2.6 250 251 Statistics were calculated with SPSS (IBM) and Excel (Microsoft). Matlab (Mathworks) and 252 Statistics were calculated with SPSS (IBM) and Excel (Microsoft). Matlab (Mathworks) and SPSS (IBM) were used for all regression analyses. Statistics were calculated with SPSS (IBM) and Excel (Microsoft). Matlab (Mathworks) and SPSS (IBM) were used for all regression analyses. 256 Patterns of coordination and cooperation. Using a total of three monkeys, we studied choices in 257 three pairs of two monkeys each (dyads) that sat opposite to each other across a horizontally 258 mounted computer touchscreen. The two animals chose near-simultaneously, without prescribed 259 inter-individual sequence, between two simultaneously presented options (C for cooperate, D for 260 defect); their payoffs were indicated by grey-scale fractal stimuli (Fig. 1A, B) (note that option C 261 denotes the coordination option in coordination games but the cooperation option in cooperation 262 games; for simplicity, we call C commonly ‘cooperation’ in this study). We inferred the animal's 263 stochastic preference from the probability of choosing one option over all other options of the same 264 option set (Luce 1959). However, in these social games, the rewards for each animal depended on 265 both the own and the other's choice, and the reward was not fully predictable from the choice of a 266 particular player alone. 267 p p y The three monkeys were trained in six formal games in chronological sequence (although the 268 games were tested in pseudorandom alternation after full training). We changed the single payoff 269 for the temptation payoff (T) in steps of one unit, thus transitioning from coordination games (Fig. 270 1C, green circles) to cooperation games (Fig. 1D). With our settings, the formal coordination game 271 became a formal cooperation game when the temptation payoff exceeded the reward payoff (T > R), 272 which implemented the temptation that challenges the coordinated action as essence of cooperation. 240 241 242 T 243 s 244 a 245 246 247 248 249 250 251 252 S 253 254 R 255 256 P 257 t 258 m 259 i 260 d 261 d 262 g 263 s 264 o 265 b 266 p 267 268 g 269 f 270 1 271 b 272 w 273 A 274 ( 275 276 c 277 f 278 c 279 r 280 p 281 o 282 t 283 m 284 p 285 g 286 s 287 We inferred the animal's 263 stochastic preference from the probability of choosing one option over all other options of the same 264 option set (Luce 1959). However, in these social games, the rewards for each animal depended on 265 both the own and the other's choice, and the reward was not fully predictable from the choice of a 266 particular player alone. 267 The three monkeys were trained in six formal games in chronological sequence (although the 268 games were tested in pseudorandom alternation after full training). We changed the single payoff 269 for the temptation payoff (T) in steps of one unit, thus transitioning from coordination games (Fig. 270 1C, green circles) to cooperation games (Fig. 1D). With our settings, the formal coordination game 271 became a formal cooperation game when the temptation payoff exceeded the reward payoff (T > R), 272 which implemented the temptation that challenges the coordinated action as essence of cooperation. 273 According to general assumptions, the propensity of both players to choose the cooperation option 274 (C) increases with higher reward payoff (R) and lower temptation payoff (T) (Camerer 2002). 275 The difference in performing in these two classes of games is illustrated in Fig. 1E-H. In a 276 coordination game, reward is maximized when both players choose simply the best-paying option 277 for each player (Fig. 1E). This strategy contrasts with the choices in a typical iterated PD 278 cooperation game. When both players attempt to choose the largest payoff for themselves, they 279 receive only a suboptimal total payoff (Fig. 1F; circle: 2 units each). Alternatively, when the first 280 player chooses the largest payoff (6 units, red player in Fig. 1G), the other player may choose the 281 other, smaller payoff but receives only a very small payoff (circle: 1 unit); the total payoff for the 282 two players is still suboptimal (6 + 1 units). But the second player’s choice of the smaller payoff 283 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 Analysis of choices. We recorded choices from the animals in all three dyads. We tested whether choices by one animal were more likely based on the already known choices of the other animal, using trials in which the animal had chosen second. We used the following logistic regression: Eq. 1 𝑃(𝐶ℎ𝑜𝑖𝑐𝑒𝑠𝑂𝑤𝑛) = ! !"#!(#$%#&∗()+,-./0)-1%2) Analysis of strategies. Strategies can be summarised as sets of rules that determine the choice 1 given a set of circumstances. Trials were classed according to three strategies for each animal: 2 Perseverance (STAY), Win-stay Lose-Shift (WSLS) and Tit-for-Tat (TFT). All strategies depended 3 on combinations of current and previous trial (CT and PT) of own and other’s choices and payoffs. 4 We used the following definitions: 5 Eq. 2.1 December 25, 2022 7 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 7 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint 240 241 242 T 243 s 244 a 245 246 247 248 249 250 251 252 S 253 254 R 255 256 P 257 t 258 m 259 i 260 d 261 d 262 g 263 s 264 o 265 b 266 p 267 268 g 269 f 270 1 271 b 272 w 273 A 274 ( 275 276 c 277 f 278 c 279 r 280 p 281 o 282 t 283 m 284 p 285 g 286 s 287 1E-H. In a 276 coordination game, reward is maximized when both players choose simply the best-paying option 277 for each player (Fig. 1E). This strategy contrasts with the choices in a typical iterated PD 278 cooperation game. When both players attempt to choose the largest payoff for themselves, they 279 receive only a suboptimal total payoff (Fig. 1F; circle: 2 units each). Alternatively, when the first 280 player chooses the largest payoff (6 units, red player in Fig. 1G), the other player may choose the 281 other, smaller payoff but receives only a very small payoff (circle: 1 unit); the total payoff for the 282 two players is still suboptimal (6 + 1 units). But the second player’s choice of the smaller payoff 283 may prime the first player to also try that smaller payoff, which then leads to the highest total 284 payoff (Fig. 1H; circle: 4 + 4 units). Thus, instead of one player gaining high and the other player 285 gaining low both players obtain the highest total payoff by both choosing an option with 286 240 𝑊𝑆𝐿𝑆= 𝐹𝑖𝑟𝑠𝑡𝑇𝑟𝑖𝑎𝑙∗(𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 1) + (𝐶ℎ𝑜𝑖𝑐𝑒𝐶𝑇= 𝐶ℎ𝑜𝑖𝑐𝑒𝑂𝑡ℎ𝑒𝑟𝑃𝑇) Eq. 2.3 241 242 The effect of strategies on cooperation was analysed by means of logistic regression. As the 243 strategies were too highly correlated to use them in one model, their effect on cooperation was 244 assessed separately using different models. 245 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗4567%2) Eq. 2.4 246 247 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗8494%2) Eq. 2.5 248 249 𝑃(𝐶𝑜𝑜𝑝𝑒𝑟𝑎𝑡𝑖𝑜𝑛) = ! !"#!(#$%#&∗5:5%2) Eq. 2.6 250 251 Statistics were calculated with SPSS (IBM) and Excel (Microsoft). Matlab (Mathworks) and 252 SPSS (IBM) were used for all regression analyses. 253 254 Results 255 256 Patterns of coordination and cooperation. Using a total of three monkeys, we studied choices in 257 three pairs of two monkeys each (dyads) that sat opposite to each other across a horizontally 258 mounted computer touchscreen. The two animals chose near-simultaneously, without prescribed 259 inter-individual sequence, between two simultaneously presented options (C for cooperate, D for 260 defect); their payoffs were indicated by grey-scale fractal stimuli (Fig. 1A, B) (note that option C 261 denotes the coordination option in coordination games but the cooperation option in cooperation 262 games; for simplicity, we call C commonly ‘cooperation’ in this study). 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 8 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 December 25, 2022 g. 2. Patterns of coordination and cooperation. (A) Good performance in all coordination games tested acro ccessive trials within single sessions. After very brief exploration, the animals chose the optimal outcome mutual coordination). The payoff setting of option T (temptation to defect) defined the specific coordination ame, whereas all other payoffs were identical. In a coordination game, the payoff T for asymmetrical choice tween animals is never larger than the payoff R for same choices (reward R for coordination) between the t oosers (payoffs T < R). L, T and V refer to the three monkeys tested; L / V and V / T refer to two dyads. (B hallenging cooperation in Prisoner's Dilemma (PD). PD has higher payoff for temptation T compared to rew (T > R), making defection beneficial for the defector. Graphs show development of cooperation (increasing ope), alternation between cooperation and defection (approximately horizontal slopes) and defection (decre ope) across session trials, with payoff T of 5. 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 304 0 Trials 300 200 100 200 100 0 300 0 400 200 0 600 400 200 Coordination game (T = 1) Defect – Coordinate <== choices ==> 0 100 20 40 60 80 -20 100 0 Trials 60 80 20 40 T L 100 0 20 40 60 80 -20 0 100 200 50 150 Trials Cooperation game (T = 5 - 6) T L 0 100 200 50 150 Trials 0 100 20 40 60 80 -20 120 140 V T 200 0 100 50 150 -50 0 200 400 100 300 Trials V T C D 0 400 1200 Trials 800 200 -400 -300 -200 0 100 -500 -100 Defect – Cooperate <== choices ==> L T Before 0 100 200 50 150 250 Trials 100 0 20 40 60 80 -20 T L Defect – Cooperate <== choices ==> Several months later 0 200 400 100 300 500 Trials 160 0 40 80 120 -40 250 0 50 100 150 200 -50 0 100 200 300 Trials L V L V Before Several months later 0 200 400 100 300 500 Trials 200 0 50 100 150 -50 250 0 50 100 150 100 -50 0 200 400 100 300 500 Trials T V V T Before Several months later E F Coordinat Defect – Coordinate <== choices ==> 0 100 20 40 60 80 -20 100 0 Trials 60 80 20 40 T L C Coordination game (T = 1) Defect – Coordinate <== choices ==> 0 100 20 40 60 80 -20 100 0 Trials 60 80 20 40 T L 0 100 200 50 150 Trials 0 100 20 40 60 80 -20 120 140 V T C 100 0 20 40 60 80 -20 0 100 200 50 150 Trials Cooperat T L D 100 0 20 40 60 80 -20 0 100 200 50 150 Trials Cooperation game (T = 5 - 6) T L 200 0 100 50 150 -50 0 200 400 100 300 Trials V T D C ion game (T = 1) 0 100 200 50 150 Trials 0 100 20 40 60 80 -20 120 140 V T D Coordination game (T = 1) Cooperation game (T = 5 - 6) on game (T = 5 - 6) 200 0 100 50 150 -50 0 200 400 100 300 Trials V T 0 400 1200 Trials 800 200 -400 -300 -200 0 100 -500 -100 Defect – Cooperate <== choices ==> L T Before E E 0 200 400 100 300 500 Trials 200 0 50 100 150 -50 V T Before 160 0 40 80 120 -400 100 200 300 Trials L V Before F Trials 0 100 200 50 150 250 Trials 100 0 20 40 60 80 -20 T L Defect – Cooperate <== choices ==> Several months later F Several months later 250 0 50 100 150 100 -50 0 200 400 100 300 500 Trials T V Several months later 0 200 400 100 300 500 Trials 250 0 50 100 150 200 -50 L V Several months later 288 Fig. 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 Thus, the behavior can be described, from left to right, as leni d consistent offer to cooperate in the face of the other animal's defection, initial cooperation turning into 0 400 200 100 300 0 100 200 300 Temptation = 1 Defect – Coordinate <== choices ==> L 0 100 -100 200 300 0 0 100 -100 0 100 200 Temptation = 5 T = 5 T = 5 T = 5 Trials Defect – Cooperate <== choices ==> 0 40 80 120 300 200 100 200 100 0 300 0 400 200 0 600 400 200 T A-C L L T L T L V V 0 300 200 100 200 T = 2 0 100 T V 400 200 100 0 300 0 300 200 100 400 160 40 T = 3 T = 4 0 40 80 120 0 80 Trials L V L V A B Coordination game (T = 1) Defect – Coordinate <== choices ==> 0 100 20 40 60 80 -20 100 0 Trials 60 80 20 40 T L 100 0 20 40 60 80 -20 0 100 200 50 150 Trials Cooperation game (T = 5 - 6) T L 0 100 200 50 150 Trials 0 100 20 40 60 80 -20 120 140 V T 200 0 100 50 150 -50 0 200 400 100 300 Trials V T C D 0 400 1200 Trials 800 200 -400 -300 -200 0 100 -500 -100 Defect – Cooperate <== choices ==> L T Before 0 100 200 50 150 250 Trials 100 0 20 40 60 80 -20 T L Defect – Cooperate <== choices ==> Several months later 0 200 400 100 300 500 Trials 160 0 40 80 120 -40 250 0 50 100 150 200 -50 0 100 200 300 Trials L V L V Before Several months later 0 200 400 100 300 500 Trials 200 0 50 100 150 -50 250 0 50 100 150 100 -50 0 200 400 100 300 500 Trials T V V T Before Several months later E F 0 400 200 100 300 0 100 200 300 Temptation = 1 Defect – Coordinate <== choices ==> L V 0 300 200 100 200 T = 2 0 100 T V 400 200 100 0 300 0 300 200 100 400 160 40 T = 3 T = 4 0 40 80 120 0 80 Trials L V L V A B 0 400 200 100 300 0 100 200 300 Temptation = 1 Defect – Coordinate <== choices ==> L V 0 300 200 100 200 T = 2 0 100 T V A A V 0 300 200 100 200 T = 2 0 100 T V 0 300 200 100 400 160 T = 3 0 40 80 120 L V 400 200 100 0 300 40 T = 4 0 80 Trials L V 400 200 100 0 300 Trials B 0 100 -100 200 300 0 100 -100 Temptation = 5 T = 5 Defect – Cooperate <== choices ==> 0 400 200 0 600 400 200 T L L T B 0 0 100 200 T = 5 300 200 100 L T T = 5 Trials 0 40 80 120 200 100 0 300 A-C L V 288 Fig. 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 2. Patterns of coordination and cooperation. (A) Good performance in all coordination games tested across 289 successive trials within single sessions. After very brief exploration, the animals chose the optimal outcome 290 (mutual coordination). The payoff setting of option T (temptation to defect) defined the specific coordination 291 game, whereas all other payoffs were identical. In a coordination game, the payoff T for asymmetrical choices 292 between animals is never larger than the payoff R for same choices (reward R for coordination) between the two 293 choosers (payoffs T < R). L, T and V refer to the three monkeys tested; L / V and V / T refer to two dyads. (B) 294 Challenging cooperation in Prisoner's Dilemma (PD). PD has higher payoff for temptation T compared to reward 295 R (T > R), making defection beneficial for the defector. Graphs show development of cooperation (increasing 296 slope), alternation between cooperation and defection (approximately horizontal slopes) and defection (decreasing 297 slope) across session trials, with payoff T of 5. Thus, the behavior can be described, from left to right, as leniency 298 and consistent offer to cooperate in the face of the other animal's defection, initial cooperation turning into 299 defection as the other animal defected, unilateral offer to cooperate leading to mutual cooperation, and good 300 mutual cooperation. (C) Immediate and reliable coordination in two other dyads (T / L and V / T). (D) Slowly 301 developing cooperation in PD. (E - F) Development of cooperation in PD after several months of experience (T = 302 6) in all three dyads (L / T, L / V and T / V). The initial hopeless or variable cooperation (E) turns ultimately into 303 more consistent cooperation several months later in the same dyads (F). 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 273 According to general assumptions, the propensity of both players to choose the cooperation option 274 (C) increases with higher reward payoff (R) and lower temptation payoff (T) (Camerer 2002). 275 The difference in performing in these two classes of games is illustrated in Fig. 1E-H. In a 276 coordination game, reward is maximized when both players choose simply the best-paying option 277 for each player (Fig. 1E). This strategy contrasts with the choices in a typical iterated PD 278 cooperation game. When both players attempt to choose the largest payoff for themselves, they 279 receive only a suboptimal total payoff (Fig. 1F; circle: 2 units each). Alternatively, when the first 280 player chooses the largest payoff (6 units, red player in Fig. 1G), the other player may choose the 281 other, smaller payoff but receives only a very small payoff (circle: 1 unit); the total payoff for the 282 two players is still suboptimal (6 + 1 units). But the second player’s choice of the smaller payoff 283 may prime the first player to also try that smaller payoff, which then leads to the highest total 284 payoff (Fig. 1H; circle: 4 + 4 units). Thus, instead of one player gaining high and the other player 285 gaining low, both players obtain the highest total payoff by both choosing an option with 286 submaximal individual payoff. 287 p p y The three monkeys were trained in six formal games in chronological sequence (although the 268 games were tested in pseudorandom alternation after full training). We changed the single payoff 269 for the temptation payoff (T) in steps of one unit, thus transitioning from coordination games (Fig. 270 1C, green circles) to cooperation games (Fig. 1D). With our settings, the formal coordination game 271 became a formal cooperation game when the temptation payoff exceeded the reward payoff (T > R), 272 which implemented the temptation that challenges the coordinated action as essence of cooperation. 273 According to general assumptions, the propensity of both players to choose the cooperation option 274 (C) increases with higher reward payoff (R) and lower temptation payoff (T) (Camerer 2002). 275 D b 25 2022 8 . CC-BY 4.0 International license perpetuity. 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 2. Patterns of coordination and cooperation. (A) Good performance in all coordination games tested across 289 successive trials within single sessions. After very brief exploration, the animals chose the optimal outcome 290 (mutual coordination). The payoff setting of option T (temptation to defect) defined the specific coordination 291 game, whereas all other payoffs were identical. In a coordination game, the payoff T for asymmetrical choices 292 between animals is never larger than the payoff R for same choices (reward R for coordination) between the two 293 choosers (payoffs T < R). L, T and V refer to the three monkeys tested; L / V and V / T refer to two dyads. (B) 294 Challenging cooperation in Prisoner's Dilemma (PD). PD has higher payoff for temptation T compared to reward 295 R (T > R), making defection beneficial for the defector. Graphs show development of cooperation (increasing 296 slope), alternation between cooperation and defection (approximately horizontal slopes) and defection (decreasing 297 slope) across session trials, with payoff T of 5. Thus, the behavior can be described, from left to right, as leniency 298 and consistent offer to cooperate in the face of the other animal's defection, initial cooperation turning into 299 defection as the other animal defected, unilateral offer to cooperate leading to mutual cooperation, and good 300 mutual cooperation. (C) Immediate and reliable coordination in two other dyads (T / L and V / T). (D) Slowly 301 developing cooperation in PD. (E - F) Development of cooperation in PD after several months of experience (T = 302 6) in all three dyads (L / T, L / V and T / V). The initial hopeless or variable cooperation (E) turns ultimately into 303 more consistent cooperation several months later in the same dyads (F). 304 288 Fig. 2. Patterns of coordination and cooperation. (A) Good performance in all coordination games tested across 289 successive trials within single sessions. After very brief exploration, the animals chose the optimal outcome 290 (mutual coordination). The payoff setting of option T (temptation to defect) defined the specific coordination 291 game, whereas all other payoffs were identical. In a coordination game, the payoff T for asymmetrical choices 292 between animals is never larger than the payoff R for same choices (reward R for coordination) between the two 293 choosers (payoffs T < R). 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 The three monkeys performed a total of 40,903 dyad trials. In all four coordination games (T, temptation payoffs 1-4), the three animals learned rapidly to coordinate their actions and consistently chose together the more rewarded option C (Fig. 2A). By contrast, the choices varied considerably in the PD game (Fig. 2B). For example, with T set to payoff 5, Monkey L consistently failed to cooperate and defected on Monkey T across a whole daily session while Monkey T was lenient and stubbornly offered to cooperate (Fig. 2B, leftmost graph). In another session, Monkey L cooperated initially but came to defect as Monkey T defected almost consistently and only at session end started to cooperate (next graph); apparently Monkey L ran out of patience with the defecting Monkey T. When playing with Monkey T at temptation payoff T = 5, Monkey L cooperated consistently even though Monkey T defected initially (next graph); thus, the tolerant behavior of Monkey L may have led Monkey T to cooperate. In another session, both monkeys cooperated well with only short bouts of defection (rightmost graph). Further graphs confirm the rapid and consistent development of coordination (Fig. 2C) but the slower and more variable developing cooperation (Fig. 2D). Thus, the initial variable performance in the coordination games suggests that the animals explored the contingencies for a short period. The initial period was followed by a longer sustained period of stable cooperative choices. Nevertheless, compared to performance in coordination games, cooperative games involved more switching between choice options and larger chances of sustained defection, especially at first or early sessions. p g p y y Given the variability of performance in the cooperation (PD) games (T = 5 or T = 6), we 3 investigated whether extended experience might lead to better cooperation. All three dyads showed 4 initial difficulties in cooperation, with persistent defection even in the face of cooperation by the 5 opponent monkey (Fig. 2E left), considerable initial hesitation (Fig. 2E center), or only moderate 6 cooperation (Fig. 2E right). However, cooperation increased significantly with all three dyads over 7 3-5 months of testing on several days each week (Fig. 2F; from 10% to 50% of trials; P = 0.09 E- 8 30), whereas coordination was high early on and did not increase further (50 – 60% of trials; P = 9 0.086). Thus, cooperation developed more slowly than coordination but ultimately succeeded. 240 241 242 The e 243 strate 244 asses 245 246 247 248 249 250 251 252 SPSS 253 254 Resu 255 256 Patte 257 three 258 moun 259 inter- 260 defec 261 denot 262 game 263 stoch 264 optio 265 both t 266 partic 267 268 game 269 for th 270 1C, g 271 becam 272 which 273 Acco 274 (C) in 275 276 coord 277 for ea 278 coope 279 receiv 280 playe 281 other 282 two p 283 may p 284 payof 285 gainin 286 subm 287 L, T and V refer to the three monkeys tested; L / V and V / T refer to two dyads. (B) 294 Challenging cooperation in Prisoner's Dilemma (PD). PD has higher payoff for temptation T compared to reward 295 R (T > R), making defection beneficial for the defector. Graphs show development of cooperation (increasing 296 slope), alternation between cooperation and defection (approximately horizontal slopes) and defection (decreasing 297 slope) across session trials, with payoff T of 5. Thus, the behavior can be described, from left to right, as leniency 298 and consistent offer to cooperate in the face of the other animal's defection, initial cooperation turning into 299 defection as the other animal defected, unilateral offer to cooperate leading to mutual cooperation, and good 300 mutual cooperation. (C) Immediate and reliable coordination in two other dyads (T / L and V / T). (D) Slowly 301 developing cooperation in PD. (E - F) Development of cooperation in PD after several months of experience (T = 302 6) in all three dyads (L / T, L / V and T / V). The initial hopeless or variable cooperation (E) turns ultimately into 303 more consistent cooperation several months later in the same dyads (F). 304 December 25, 2022 9 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 9 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint D b 25 2022 10 . CC-BY 4.0 International license perpetuity. It is made available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 oi: December 25, 2022 December 25, 2022 December 25, 2022 10 354 Fig. 3. Experience with coordination leads to emergence of cooperation. (A) Heatmaps of choice probabilities for 355 each monkey of all three tested dyads. The single temptation payoff (T) defined the difference between the four 356 coordination games and two cooperation games. Rainbow colored dots concentrated at the top right corner of each 357 plot indicate good coordination and cooperation between the animals. (B) Summary of coordination and 358 cooperation levels, as indicated by the probability of choosing the cooperation (C) option over the defection (D) 359 option. In all dyads, performance was consistently high in all coordination games but decreased somewhat in 360 cooperation games in which choices still differed significantly from chance. T, temptation payoff. 361 (C) Wall control. Breakdown of cooperation when a wall between the two monkeys T and V prevented visual 362 inspection of each other's options and choice (while maintaining other visual contact between the animals). 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 363 Monkeys L - T Monkeys T - V Monkeys L - V P(Choice) in 20 trials Coordination game Temptation = 1 Cooperation game Temptation = 6 0 0.5 1 Coordination game Temptation = 2 Coordination game Temptation = 3 Coordination game Temptation = 4 Cooperation game Temptation = 5 Probability of common choices Monkeys L-V T-V L-T 0 0.25 0.5 0.75 1 Coordination T = 1 6 4 5 3 2 Cooperation <- chance level -> A B Defect – Cooperate <== choices ==> 0 800 Trials 400 600 200 200 0 100 300 0 800 Trials 400 200 600 200 0 100 Wall Wall T = 5 T = 6 T V T V 0 0.5 1 P(Cooperate) 1st monkey 0 0.5 1 P(Cooperate) 2nd monkey C Monkeys L - T Monkeys T - V Monkeys L - V P(Choice) in 20 trials Coordination game Temptation = 1 Cooperation game Temptation = 6 0 0.5 1 Coordination game Temptation = 2 Coordination game Temptation = 3 Coordination game Temptation = 4 Cooperation game Temptation = 5 A 0 0 5 1 P(Cooperate) 1st monkey 0 0.5 1 P(Cooperate) 2nd monkey A Probability of common choices Monkeys L-V T-V L-T 0 0.25 0.5 0.75 1 Coordination T = 1 6 4 5 3 2 Cooperation <- chance level -> B 0 0.5 1 P(Cooperate) 1st monkey B Coordination Cooperation Defect – Cooperate <== choices ==> 0 800 Trials 400 600 200 200 0 100 300 0 800 Trials 400 200 600 200 0 100 Wall Wall T = 5 T = 6 T V T V C C 354 Fig. 3. Experience with coordination leads to emergence of cooperation. (A) Heatmaps of choice probabilities for 355 each monkey of all three tested dyads. The single temptation payoff (T) defined the difference between the four 356 coordination games and two cooperation games. Rainbow colored dots concentrated at the top right corner of each 357 plot indicate good coordination and cooperation between the animals. (B) Summary of coordination and 358 cooperation levels, as indicated by the probability of choosing the cooperation (C) option over the defection (D) 359 option. In all dyads, performance was consistently high in all coordination games but decreased somewhat in 360 cooperation games in which choices still differed significantly from chance. T, temptation payoff. 361 (C) Wall control. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 0 Development of cooperation. The data presented so far demonstrate that monkeys can cooperate in 332 PD but that cooperation takes time to develop (Fig. 2E, F). As one of the aims of the current 333 experiments was to investigate more explicit ways to foster cooperation, we designed our gambles 334 such that a single payoff (T for temptation) defined each specific game and that the stepwise 335 increment of that payoff changed coordination games into cooperation (iterated PD) games (Fig. 336 1C, D). In all four coordination games, the animals showed good levels of commonly choosing the 337 coordination (C) option that maximized their reward. In Fig. 3A, the good coordination was 338 evidenced by high probabilities of each animal of each dyad consistently choosing the coordination 339 (C) option (close to top right corners in top four heatmap rows). When the payoff change for 340 temptation transformed the coordination games into cooperation games (from T = 1 - 4 to T = 5 and 341 6), both animals chose the cooperation option C less consistently, as indicated by more widely 342 scattered choice probabilities (Fig. 3A, two bottom rows). 343 p g Different quantification of the same data demonstrated high coordination levels in the four 344 coordination games (T = 1 – 4; probability of both animals choosing coordination between p = 0.55 345 and p = 0.70) that decreased somewhat to still significant, consistent and above-chance levels in 346 both cooperation games (T = 5 and 6; probability of both animals choosing cooperation between p = 347 0.28 and p = 0.68) in all three dyads (Fig. 3B) (p < 0.01). The dyad L – V showed lower 348 cooperation with the temptation payoff increasing from T = 4 to T = 5, which confirmed the notion 349 that cooperation decreases with increasing temptation payoff (T) (Camerer 2002), whereas the other 350 two dyads failed to show substantial changes. These data suggest overall excellent performance in 351 the coordination games, and slightly less but nevertheless significant cooperation in cooperation 352 games (iterated PD) despite the well-rewarded temptation to defect. 353 December 25, 2022 10 . CC-BY 4.0 International license perpetuity. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 ; https://doi.org/10.1101/2022.12.25.521899 oi: December 25, 2022 11 Offer and reciprocation. The monkeys were free to choose as soon as the two fractals for each 379 animal appeared. The unconstrained order of choice allowed separate analysis according to first or 380 second chooser. When Monkey L chose first in the coordination game, he chose the cooperation 381 option C significantly more frequently than the defection option D; the same was true for Monkey T 382 (Fig. 4A, blue to yellow). The tendency declined and became variable in the cooperation game 383 (orange and red). Only Monkey L chose cooperation more frequently than defection and thus 384 offered above-chance cooperation despite facing potential defection by the second chooser. By 385 contrast, Monkey T mostly chose defection as first mover. 386 contrast, Monkey T mostly chose defection as first mover. 386 387 388 Fig. 4. Offer and reciprocation. Monkeys were free to choose after the simultaneous appearance of th 389 options, which allowed post-hoc distinction between first and second chooser. (A) Probability of selec 390 cooperation option by first chooser. Error bars are 95% confidence intervals. T, temptation payoff. (B 391 of selecting the cooperation option by second chooser after first chooser had selected cooperation (lef 392 defection (right) in same trial. (C) Choice of cooperation option in previous trial lead to more coopera 393 current trial (temptation payoff T = 5 or T = 6). (D) Current cooperation induced future cooperation. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 394 0 0.25 0.5 0.75 1 P(Cooperate) Monkey L Coordination T = 1 6 4 3 2 Monkey T Coordination T = 1 4 3 2 Chance level 0 0.25 0.5 0.75 1 C (Given C) C (Given D) P(Cooperate) Monkey L After coord / coop offer After defect offer 1 6 4 5 3 2 1 6 4 5 3 2 C (Given C) C (Given D) Monkey T After coord / coop offer After defect offer 1 6 4 5 3 2 1 6 4 5 3 2 A B Both cooperated Other defected Self defected Both defected Monkey L Both cooperated Other defected Self defected Both defected Monkey T Both cooperated Other defected Self defected Both defected Monkey V P(cooperate \| previous-trial) 0 0.25 0.5 0.75 Current trial Previous trial C 5 5 6 Cooperation Cooperation 0 0.5 1 0 0.5 1 Next 20 trials Cooperation Current 20 trials Cooperation Temptation = 5 Temptation = 6 D 0 0.25 0.5 0.75 1 P(Cooperate) Monkey L Coordination T = 1 6 4 3 2 Monkey T Coordination T = 1 4 3 2 Chance level A 5 5 6 Cooperation Cooperation Monkey T Coordination T = 1 4 3 2 5 6 Cooperation 0 0.25 0.5 0.75 1 P(Cooperate) Monkey L Coordination T = 1 6 4 3 2 A 5 Cooperation Monkey T Monkey T A 0 0.25 0.5 0.75 1 C (Given C) C (Given D) P(Cooperate) Monkey L After coord / coop offer After defect offer 1 6 4 5 3 2 1 6 4 5 3 2 B B C (Given C) C (Given D) o ey After coord / coop offer After defect offer 1 6 4 5 3 2 1 6 4 5 3 2 387 388 Both cooperated Other defected Self defected Both defected Monkey L Both cooperated Other defected Self defected Both defected Monkey T Both cooperated Other defected Self defected Both defected Monkey V P(cooperate \| previous-trial) 0 0.25 0.5 0.75 Current trial Previous trial C 0 0.5 1 0 0.5 1 Next 20 trials Cooperation Current 20 trials Cooperation Temptation = 5 Temptation = 6 D P C D Fig. 4. Offer and reciprocation. Monkeys were free to choose after the simultaneous appearance of the two 389 options, which allowed post-hoc distinction between first and second chooser. (A) Probability of selecting the 390 cooperation option by first chooser. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 Breakdown of cooperation when a wall between the two monkeys T and V prevented visual 362 inspection of each other's options and choice (while maintaining other visual contact between the animals). 363 Importance of viewing each other's options and choice. To assess the influence of viewing each 365 others choices, we temporarily placed a 12 cm high wall in the center of the horizontal touchscreen 366 between the animals. The wall obstructed the view of the touchscreen and thus made it impossible 367 for each animal to view the other animals’ options and choices. The animals were still able to see 368 each other's hand on the touch key (but not the touchscreen) and the delivery of the reward after the 369 choice, as well as task-unrelated arm and face movements. The view of the remainder of the 370 laboratory, the behavioral setup, the behavioral task, the fractal stimuli predicting the payoffs, and 371 the payoffs themselves remained unchanged. Thus, the animal could only infer the other animal's 372 choice from seeing the reward the other animal received while taking into account his own choice. 373 Despite good performance in the cooperation game with both temptation values before wall 374 placement, cooperation broke down within about 100 trials after wall placement and was largely 375 replaced by defection in both animals of the tested dyad (Fig. 3C). Thus, viewing each other's 376 options and choice seemed to be crucial for mutual cooperation. 377 378 December 25, 2022 11 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25 2022 11 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint D b 25 2022 11 . CC-BY 4.0 International license perpetuity. It is made available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 y g p p Cooperation in PD depended in all three animals also on experience in the previous trials. 409 Cooperation of both animals in the previous trial resulted in high probability of choosing the 410 cooperation option C again (Fig. 4C, red). By contrast, choice of the cooperation option was less 411 likely in trials following any defection (grey and black). Correspondingly, the degree of mutual 412 cooperation in the current 20 trials correlated with the degree of mutual cooperation in the next 20 413 trials (Fig. 4D) (Monkey L: beta = 0.57, F(6,994,1) = 3484.8, p < 0.001; Monkey T: beta = 0.59, 414 F(6,304,1) = 3,304.4, p<0.001; Monkey V: beta = 0.68 , F(5,626,1) = 4,889.9, p < 0.001). Thus, 415 previous cooperation increased current cooperation, even when own defection could result in an 416 immediate higher payoff. It seemed that the benefits from experienced cooperation encouraged 417 further cooperation. 418 Consequences of coordination and cooperation. Reward gain increased with the increasing probability of mutual coordination and mutual cooperation in two of the three animals (Fig. 5B), both in current 20 trials (Monkey L: beta = 1.78, F(7,014,1) = 2,112.9, p < 0.001; Monkey T: beta = 1.26, F(6,324,1) = 697.35, p < 0.001; Monkey V: beta = -0.11, F(5,646,1) = 6.75, p = 0.009) and in future 20 trials (Monkey L: beta = 0.99, F(6,994,1) = 539.85, p < 0.001; Monkey T: beta= 1.09, F(6,304,1) = 503.44, p < 0.001; Monkey V: beta = -0.01, F(5,626,1) = 0.08, p = 0.8). Thus, both coordination and cooperation became more beneficial with more frequent reciprocation. p q p Closer analysis of reward gain demonstrated differential consequences between coordination 427 and cooperation games when choosing first. Current and future reward payoff increased with the 428 increasing probability of the first chooser offering coordination in the most simple coordination 429 game (T = 1) (Fig. 5B, dark and light blue). The result was confirmed by regression analysis of 20 430 current trials and future 20 trials. Thus, offering coordination was beneficial. 431 432 Fig. 5. Effects of offered and mutual coordination and cooperation on current and future reward gain. (A) Effects 433 of mutual choice. Higher probability of mutual coordination (T = 1) or cooperation (T = 6) increased reward gain 434 in current and future blocks of 20 trials. (B) Effects of first choice. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 Error bars are 95% confidence intervals. T, temptation payoff. (B) Probability 391 of selecting the cooperation option by second chooser after first chooser had selected cooperation (left) or 392 defection (right) in same trial. (C) Choice of cooperation option in previous trial lead to more cooperation in 393 current trial (temptation payoff T = 5 or T = 6). (D) Current cooperation induced future cooperation. 394 December 25, 2022 12 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint D b 25 2022 12 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 12 When analyzing the choices of the second-choosing animal, we found that both Monkeys L and T chose the cooperation option C in the four coordination games significantly more frequently than the defection option D when the first mover had also chosen cooperation (Fig. 4B, blue to yellow). The proportion of choices of coordination dropped significantly when the first mover had chosen defection (odds ratios for monkey L: 2.1, 6.8, 3.8 and 4.3 for temptation payoffs 1-4, respectively, all Wald Statistics significant with p < 0.01; odd ratios for monkey T: 3.4, 3.5, 2.6 and 3.4 for temptation payoffs 1-4, respectively, all Wald statistics significant with p < 0.01). A similar reduction was seen with the overall lower cooperation in the cooperation game. Cooperation by the second mover was significantly higher after the first mover had chosen the cooperation option, as compared with the first mover having chosen the defection option (orange and red) (odds ratios for monkey L: 1.6 and 1.5 for temptation payoffs 5 and 6, respectively, both Wald Statistics significant with p < 0.01; odd ratios for monkey T: 1.4 for both temptation payoffs 5 and 6, both Wald statistics significant with p < 0.05). Thus, the choice of the first-choosing monkey affected the other monkey's choice in all games. In particular, cooperation seemed infectious with both animals. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in py g p p g p p December 25, 2022 13 In contrast to the gain by coordination, reward payoff decreased with the increasing probability of the first chooser offering cooperation in the most tempting cooperation game (temptation T = 6) (Fig. 5B, red and green). The result was confirmed by regression analysis of 20 current trials (Monkey L: beta = -1.05, F(7,014,1) = 562.8, p < 0.001; Monkey T: beta = -1.09, F(6,324,1) = 543.7, p < 0.001; Monkey V: beta = -1.33, F(5,646,1) = 1391.2, p < 0.001) and future 20 trials (Monkey L: beta = -0.96, F(6,994,1) = 457.2, p < 0.001; Monkey T: beta = -0.4973, F(6,304,1) = 105.05, p < 0.001; Monkey V: beta = -0.8933, F(5,626,1) = 562.61, p < 0.001). Thus, unconditionally offering cooperation had a price; more frequently offered cooperation may lower reward gain. Strategies in coordination and cooperation games. We analyzed three strategies of choosing 449 from trial to trial: persistence of choosing the same option again (STAY), win-stay lose-shift 450 (WSLS) and tit-for-tat (TFT) (Eqs. 2.1 – 2.6). Choices under STAY and WSLS refer primarily to 451 own previous choice (with modulation by social choice with WSLS), whereas TFT refers only to 452 the other’s choice and thus is outright social. Overall, the animals used WSLS more often than the 453 other two strategies. Specifically, Monkey L consistently used STAY and WSLS more frequently 454 than TFT, and Monkey T used mostly WSLS (Fig. 6A). The transition from coordination to 455 cooperation games saw a moderate drop in using the non-social STAY and limited-social WSLS 456 strategies but a sharp drop in the outright-social TFT strategy (from 63% - 74% with temptation 457 values of 1 – 4 to 53% – 55% with temptation values of 5 and 6), although TFT performance still 458 significantly exceeded chance. Interestingly, the use of both WSLS and TFT consistently increased 459 cooperation more than STAY in both versions of the cooperation game (T = 5 and 6) in Monkeys L 460 and T, with TFT being the effective strategy in Monkey T (Fig. 6B). Thus, all three animals had 461 learned strategies that increased their cooperation. 462 g p 463 Fig. 6. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 Strategies used in coordination and cooperation games: persistence (STAY), win-stay lose-shift (WSLS) 464 and tit-for-tat (TFT). (A) Use of the different strategies by Monkeys L and T. Random choice would result in 50% 465 selection of trials. Monkey L used STAY and WSLS more often than TFT, whereas Monkey T used mostly 466 WSLS. Both animals maintained use of STAY and WSLS but decreased use of TFT substantially when passing 467 from coordination games (Temptation = 1 – 4; black) to cooperation games (T = 5 and 6; red). Error bars show 468 95% confidence intervals. (B) Increase of cooperation with specific strategies. WSLS and TFT consistently 469 increased cooperation more than STAY in the cooperation games. 470 A B Monkey L Odds ratio (cooperation) % trials 100 90 80 70 60 50 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Monkey L Monkey T Monkey T Monkey L Monkey L Monkey L Monkey L Monkey L Monkey L Monkey T Monkey T Monkey T Monkey T Monkey T Monkey T Temptation value (T) 1 2 3 4 5 6 5 6 Temptation value (T) STAY WSLS TFT 463 A B Monkey L Odds ratio (cooperation) % trials 100 90 80 70 60 50 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Monkey L Monkey T Monkey T Monkey L Monkey L Monkey L Monkey L Monkey L Monkey L Monkey T Monkey T Monkey T Monkey T Monkey T Monkey T Temptation value (T) 1 2 3 4 5 6 5 6 Temptation value (T) STAY WSLS TFT B Monkey L Odds ratio (cooperation) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Monkey L Monkey T Monkey T Temptation value (T) 5 6 Temptation value (T) 463 Fig. 6. Strategies used in coordination and cooperation games: persistence (STAY), win-stay lose-shift (WSLS) 464 and tit-for-tat (TFT). (A) Use of the different strategies by Monkeys L and T. Random choice would result in 50% 465 selection of trials. Monkey L used STAY and WSLS more often than TFT, whereas Monkey T used mostly 466 WSLS. Both animals maintained use of STAY and WSLS but decreased use of TFT substantially when passing 467 from coordination games (Temptation = 1 – 4; black) to cooperation games (T = 5 and 6; red). Error bars show 468 95% confidence intervals. (B) Increase of cooperation with specific strategies. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 5B, red and green). The result was confirmed by regression analysis of 20 441 current trials (Monkey L: beta = -1.05, F(7,014,1) = 562.8, p < 0.001; Monkey T: beta = -1.09, 442 F(6,324,1) = 543.7, p < 0.001; Monkey V: beta = -1.33, F(5,646,1) = 1391.2, p < 0.001) and future 443 20 trials (Monkey L: beta = -0.96, F(6,994,1) = 457.2, p < 0.001; Monkey T: beta = -0.4973, 444 F(6,304,1) = 105.05, p < 0.001; Monkey V: beta = -0.8933, F(5,626,1) = 562.61, p < 0.001). Thus, 445 unconditionally offering cooperation had a price; more frequently offered cooperation may lower 446 reward gain. 447 448 Strategies in coordination and cooperation games. We analyzed three strategies of choosing 449 from trial to trial: persistence of choosing the same option again (STAY), win-stay lose-shift 450 (WSLS) and tit-for-tat (TFT) (Eqs. 2.1 – 2.6). Choices under STAY and WSLS refer primarily to 451 own previous choice (with modulation by social choice with WSLS), whereas TFT refers only to 452 the other’s choice and thus is outright social. Overall, the animals used WSLS more often than the 453 other two strategies. Specifically, Monkey L consistently used STAY and WSLS more frequently 454 than TFT, and Monkey T used mostly WSLS (Fig. 6A). The transition from coordination to 455 cooperation games saw a moderate drop in using the non-social STAY and limited-social WSLS 456 strategies but a sharp drop in the outright-social TFT strategy (from 63% - 74% with temptation 457 values of 1 – 4 to 53% – 55% with temptation values of 5 and 6), although TFT performance still 458 significantly exceeded chance. Interestingly, the use of both WSLS and TFT consistently increased 459 cooperation more than STAY in both versions of the cooperation game (T = 5 and 6) in Monkeys L 460 and T, with TFT being the effective strategy in Monkey T (Fig. 6B). Thus, all three animals had 461 learned strategies that increased their cooperation. 462 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 13 In contrast to the gain by coordination, reward payoff decreased with the increasing 439 probability of the first chooser offering cooperation in the most tempting cooperation game 440 (temptation T = 6) (Fig. 5B, red and green). The result was confirmed by regression analysis of 20 441 current trials (Monkey L: beta = -1.05, F(7,014,1) = 562.8, p < 0.001; Monkey T: beta = -1.09, 442 F(6,324,1) = 543.7, p < 0.001; Monkey V: beta = -1.33, F(5,646,1) = 1391.2, p < 0.001) and future 443 20 trials (Monkey L: beta = -0.96, F(6,994,1) = 457.2, p < 0.001; Monkey T: beta = -0.4973, 444 F(6,304,1) = 105.05, p < 0.001; Monkey V: beta = -0.8933, F(5,626,1) = 562.61, p < 0.001). Thus, 445 unconditionally offering cooperation had a price; more frequently offered cooperation may lower 446 reward gain. 447 448 Strategies in coordination and cooperation games. We analyzed three strategies of choosing 449 from trial to trial: persistence of choosing the same option again (STAY), win-stay lose-shift 450 (WSLS) and tit-for-tat (TFT) (Eqs. 2.1 – 2.6). Choices under STAY and WSLS refer primarily to 451 own previous choice (with modulation by social choice with WSLS), whereas TFT refers only to 452 the other’s choice and thus is outright social. Overall, the animals used WSLS more often than the 453 other two strategies. Specifically, Monkey L consistently used STAY and WSLS more frequently 454 than TFT, and Monkey T used mostly WSLS (Fig. 6A). The transition from coordination to 455 cooperation games saw a moderate drop in using the non-social STAY and limited-social WSLS 456 strategies but a sharp drop in the outright-social TFT strategy (from 63% - 74% with temptation 457 values of 1 – 4 to 53% – 55% with temptation values of 5 and 6), although TFT performance still 458 significantly exceeded chance. Interestingly, the use of both WSLS and TFT consistently increased 459 cooperation more than STAY in both versions of the cooperation game (T = 5 and 6) in Monkeys L 460 and T, with TFT being the effective strategy in Monkey T (Fig. 6B). Thus, all three animals had 461 learned strategies that increased their cooperation. 462 . CC-BY 4.0 International license perpetuity. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 Higher probability of offering coordination by 435 first chooser increased reward gain in current and future blocks of 20 trials (dark and light blue; temptation payoff 436 T = 1), whereas higher probability of offering cooperation decreased reward payoff (red and green; T = 6). Thus, 437 offering to cooperate had a price. 438 0 1 2 3 4 5 0 0.5 1 P(choose cooperate) A B Coordination: current & future reward T = 1 Cooperation: current & future reward T = 6 0 1 2 3 4 5 0 0.5 1 P(choose cooperate) Reward (juice drops) A Coordina current & future rew T = 1 Cooperat current & future rew T = 6 0 1 2 3 4 5 0 0.5 1 P(choose cooperate) Reward (juice drops) 0 1 2 3 4 5 0 0.5 1 P(choose cooperate) B n: d : d A B 432 Fig. 5. Effects of offered and mutual coordination and cooperation on current and future reward gain. (A) Effects 433 of mutual choice. Higher probability of mutual coordination (T = 1) or cooperation (T = 6) increased reward gain 434 in current and future blocks of 20 trials. (B) Effects of first choice. Higher probability of offering coordination by 435 first chooser increased reward gain in current and future blocks of 20 trials (dark and light blue; temptation payoff 436 T = 1), whereas higher probability of offering cooperation decreased reward payoff (red and green; T = 6). Thus, 437 offering to cooperate had a price. 438 December 25, 2022 13 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25 2022 13 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 13 In contrast to the gain by coordination, reward payoff decreased with the increasing 439 probability of the first chooser offering cooperation in the most tempting cooperation game 440 (temptation T = 6) (Fig. Discussion We tested rhesus monkeys in a series of six games whose payoff matrices were identical except for 474 the temptation payoff that increased across the games (Fig. 1A-D). This temptation payoff defined 475 the transitioned from four coordination games to two cooperation games. The payoff matrix of 476 formal cooperation games (PD) is characterized by the temptation payoff that exceeds all other 477 payoffs and encourages defection that challenges cooperation (Fig. 1B). The animals showed strong 478 performance in the coordination games that lead to highest payoff by common choice; their 479 cooperation performance was somewhat lower but nevertheless substantial and stable despite the 480 challenging higher defection payoff (Fig. 2A-D). Cooperation developed gradually over several 481 weeks and months (Figs. 2E-F) but remained lower than performance in the coordination games 482 (Fig. 3A, B). The degraded cooperation with reduced visual contact between the animals 483 emphasized the social nature of the games (Fig. 3C). The animals’ choices seemed to follow 484 common intuition; in the cooperation games, the first-choosing animal chose the commonly 485 rewarded option less frequently than in the coordination games (Fig. 4A), which may reflect the low 486 non-cooperative payoff in cooperation games. Further, choice of the cooperation option by the first 487 player increased the chance of reciprocation by the second player (Fig. 4B, C). Correspondingly, 488 successful cooperation in the current trials increased cooperation in the next several trials, 489 suggesting that cooperation induced further cooperation and thus was behaviorally infectious (Fig. 490 4D). Reward accumulated more with mutual coordination compared to individually different 491 choices, which confirms the beneficial nature of working together; more interestingly, reward 492 payoff increased also with mutual cooperation despite the possibility of defection, thus 493 demonstrating gain from engaging in pro-social rather than selfish behavior (fig 5). This gain may 494 have been due to the acquisition of beneficial social strategies that increased cooperation (Fig. 6). 495 Thus, rhesus monkeys showed solid and beneficial cooperation behavior in formal economic games 496 following gradual, single-variable transition from coordination games. 497 demonstrating gain from engaging in pro-social rather than selfish behavior (fig 5). This gain may 494 have been due to the acquisition of beneficial social strategies that increased cooperation (Fig. 6). 495 Thus, rhesus monkeys showed solid and beneficial cooperation behavior in formal economic games 496 following gradual, single-variable transition from coordination games. 305 t 306 c 307 c 308 f 309 l 310 c 311 s 312 d 313 c 314 b 315 c 316 r 317 d 318 s 319 f 320 p 321 o 322 323 i 324 i 325 o 326 c 327 3 328 3 329 0 330 331 D 332 P 333 e 334 s 335 i 336 1 337 c 338 e 339 ( 340 t 341 6 342 s 343 344 c 345 a 346 b 347 0 348 c 349 t 350 t 351 t 352 g 353 WSLS and TFT consistently 469 increased cooperation more than STAY in the cooperation games. 470 D b 25 2022 14 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 December 25, 2022 14 14 D b 25 2022 14 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 experience of ultimately higher reward from mutual cooperation. The experience of the higher reward from cooperation would encourage a behavior associated with initially smaller payoff from unilateral choice. Thus, the challenge in cooperation, as represented in the formal PD, is to overcome the initial individually lower reward for the ultimately higher common gain. While ‘cooperate’ choices in the coordination games were robust irrespective of the temptation payoff, ‘cooperate’ choices decreased in cooperation games with increasing temptation payoff. Thus, the temptation payoff in the cooperation games constituted a challenging defection option; the less the animals cooperated unilaterally, the more they were rewarded. Thus, by choosing the more rewarded defection option, the animals acted ‘as if’ they were maximizing reward; the animals ‘knew what they were doing’, and their choices seemed meaningful. Nevertheless, their substantial, above-chance cooperation against selfish higher-paying defection indicated their understanding of the long-term gain of cooperation. General game behavior. The obtained cooperation results confirm the notion that cooperation is beneficial in iterated PD. By contrast, the single-shot PD has its Nash equilibrium in defection (nobody gains by using a different strategy). The difference in optimal behavior is surprising, has been long debated, and seems quite controversial. One explanation is related to the length of the iteration. With predetermined finite sequences with short and well known length, an agent can iteratively backtrack from the last choice via the preceding few choices to the first choice and behave as if it were a single-shot PD, thus choosing defection to maximize own reward. However, in sequences with unknown and extensive lengths, backtracking to the first choice is more difficult (Dixit & Skeath 2004). This characteristic may be a reason why cooperation can be the prime characteristic of iterated PD. The beneficial cooperation seen in the current experiments confirm the validity of this notion in rhesus monkeys. y y Our animals showed less frequent choice of the ‘cooperate’ option in the cooperation games 550 compared to the coordination games. This observation corresponds to a previously described 551 cooperation decline from two coordination games to the iterated PD, although the coordination 552 games differed from those tested presently (Smith et al. 2019). The similar pattern of cooperation 553 decline confirms empirically the intuitive notion that the the highest rewarded temptation payoff 554 defining PD games constitutes the main challenge to cooperation. Discussion 497 g g g g In sum, the studies by others and ourselves demonstrated variable behavior in cooperation games, in contrast to the more stable choices in coordination games devoid of attractive defection. Cooperation in our experiments likely benefitted from extensive experience with coordination games that familiarized the animals with the benefit from choosing the cooperative option and allowed them to develop mutually beneficial strategies. However, the low number of monkeys in all these studies would prevent us from drawing strong conclusions. Methodological aspects. The single difference between our coordination and cooperation games consisted in the temptation payoff (T) for choosing the defection option (D) when the other monkey chose the cooperation option (C). By definition, the temptation payoff in a cooperation game exceeds all other payoffs (along with the payoff hierarchy shown in Fig. 1B), whereas the temptation payoff in a coordination game does not exceed any other payoff (Fig. 1C). Despite this higher temptation payoff, the animals succeeded to cooperate above chance in the cooperation games. Nevertheless, cooperation decreased with increasing temptation payoff between the coordination and the cooperation games, a difference that was larger between coordination and cooperative games than it was within these two game types (Fig. 3B). The drop in cooperation with increasing temptation payoff suggests that the animals detected the transitions to the more costly cooperation. However, the animals still maintained significant cooperation in the cooperation games (Fig. 3B), which may reflect familiarity with the lower temptation payoffs in the coordination games that elicited less defection. The good performance in the cooperation games may be a result of having experienced the coordination games with closely related payoff matrices that might have made the animals resilient to temptation. Alternatively, the coordination games might have primed the animals to focus on the cooperation option whose common choice led to the highest payoff, analogous to the direct priming with maximum reward for common choice (Stephens et al. 2002). The ultimate gain in a cooperation game requires an initial loss. In our PD, unilateral defection by only one player paid always more reward (5 or 6 units) than unilateral cooperation (1 unit), whereas mutual defection (2 units) was less rewarding than mutual cooperation (4 units) (Fig. 1D). Thus, the initial higher reward with unilateral defection needs to be overcome by the December 25 2022 15 . CC-BY 4.0 International license perpetuity. Discussion It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 15 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 December 25, 2022 15 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 experience of ultimately higher reward from mutual cooperation. The experience of the higher reward from cooperation would encourage a behavior associated with initially smaller payoff from unilateral choice. Thus, the challenge in cooperation, as represented in the formal PD, is to overcome the initial individually lower reward for the ultimately higher common gain. While ‘cooperate’ choices in the coordination games were robust irrespective of the 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 First, the animals used STAY and 612 WSLS rather consistently in all games, whereas they used TFT much more rarely in both 613 cooperation games. Indeed, WSLS resulted in more cooperation than the other strategies (Fig. 6B). 614 An earlier study had shown substantial variability among individual monkeys to establish stable 615 strategies, but the animals expressing stable strategies did show reciprocal cooperation and 616 defection compatible with TFT (Smith et al. 2019). In another study, monkeys tended to continue 617 cooperating when the opponent had cooperated but tended to defect less when the opponent had 618 defected, suggesting some correspondence to both WSLS and TFT that significantly exceeded 619 random behavior (Haroush & Williams 2015). Their reciprocation depended on the social situation 620 by decreasing when one player was replaced by a computer or when performing in different rooms. 621 622 References 623 624 Báez-Mendoza R, Harris C, Schultz W. Activity of striatal neurons reflects social action and own 625 reward. Proc Natl Acad Sci (USA) 110: 16634-16639, 2013. 626 Báez-Mendoza R Mastrobattista EP Wang AJ Williams ZM Social agent identity cells in the 627 was observed when placing the opponent monkey in another room (from 35% to 14%; Haroush & Williams 2015). The replacement of a biological partner by a computer opponent and the animals’ placement in separate rooms reduce the social aspect more than the simple blocking of the view of each other’s options and choices achieved by our wall control. Nevertheless, irrespective of the degree of reduction of social interaction, the results demonstrate the importance of social aspects in these formal, and somewhat abstract and reductionist, economic tasks. was observed when placing the opponent monkey in another room (from 35% to 14%; Haroush & Williams 2015). The replacement of a biological partner by a computer opponent and the animals’ placement in separate rooms reduce the social aspect more than the simple blocking of the view of each other’s options and choices achieved by our wall control. Nevertheless, irrespective of the degree of reduction of social interaction, the results demonstrate the importance of social aspects in these formal, and somewhat abstract and reductionist, economic tasks. Strategies. Our monkeys cooperated even when they could see each other’s options and choice, which might have made them more vulnerable to exploitation and defection. 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 Despite frequent defection in the face of the opponent’s cooperation, the animals cooperated in most trials: when the first moving animal chose to cooperate, the other animal often chose also to cooperate. This result held for all animals and all games (Figs. 2, 4). Formal analyses of our animals’ choices revealed use of three major strategies (Fig. 6): 94 persistence (STAY), win-stay lose-shift (WSLS) and tit-for-tat (TFT). STAY consists of choosing 95 the same option as before irrespective of the outcome, whereas WSLS consists of choosing the 96 same option after receiving a good outcome but choosing the alternative after a lesser outcome. 97 Thus, players using STAY perseverate without taking the other player’s choice into account, and 98 players using WSLS respond to the outcome of their own choice that also depends on the other’s 99 choice. Thus, the two strategies are either not social at all (STAY) or social only to a limited extent 00 (WSLS). 01 By contrast, TFT consists of reciprocation and thus constitutes a fully social strategy. The 602 player replicates the opponent’s play, cooperating after a cooperation until the other player defects, 603 and defecting after a defection until the other player cooperates. As mutual defection results in 604 lower outcome than mutual cooperation, players should aim for mutual cooperation; they should 605 occasionally even offer to cooperate in the face of the other player’s defection. This behavior may 606 result in long periods of being unilaterally defected that is beneficial for the opponent but provides 607 suboptimal own outcome (see payoff matrix in Fig. 1D) that is only justified by the later gain from 608 mutual cooperation. In contrast to TFT, defection in WSLS would lead after only one trial to stable 609 cooperation. Thus, recovery of mutual cooperation after defection is more costly in TFT than 610 WSLS, which may be a reason why WSLS outperforms TFT in humans (Nowak & Sigmund 1993). 611 These strategies are also found in our monkeys (Fig. 6A). First, the animals used STAY and 12 WSLS rather consistently in all games, whereas they used TFT much more rarely in both 13 cooperation games. Indeed, WSLS resulted in more cooperation than the other strategies (Fig. 6B). Báez-Mendoza R, Harris C, Schultz W. Activity of striatal neurons reflects social action and own 625 reward. Proc Natl Acad Sci (USA) 110: 16634-16639, 2013. 626 Báez-Mendoza R, Mastrobattista EP, Wang AJ, Williams ZM. Social agent identity cells in the 627 prefrontal cortex of interacting groups of primates. Science 374: eabb4149, 2021. 628 Brosnan SF, Wilson, BJ, Beran MJ. Old World monkeys are more similar to humans than New 629 World monkeys when playing a coordination game. Proc Biol Sci 279 1522-1530, 2012. 630 Bullinger A, Wyman E, Melis A, Tomasello M. Coordination of chimpanzees (Pan troglodytes) in a 631 Stag Hunt Game. International Journal of Primatology 32: 1296-1310, 2011. 632 Camerer CF. Behavioral Game Theory: Experiments in Strategic Interaction. Russell-Sage 633 Foundation New York, NY. Princeton University Press, Princeton, NJ, 2003. 634 Chang SW, Fagan NA, Toda K, Utevsky AV, Pearson JM, Platt ML. Neural mechanisms of social 635 decision-making in the primate amygdala. Proc Natl Acad Sci (USA) 112: 16012-16017, 2015. 636 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 555 The robust cooperation in our iterated PDs surpassed the degree of cooperation seen in a previous study that did not implement a transition from coordination to cooperation games (Haroush & Williams 2015). These authors reported 17% common cooperation choices, which is below the 28 – 68% cooperation choices seen presently (Fig. 3B). Despite the experimental differences of such complex social studies between laboratories, one factor for the presently observed more frequent cooperation may be our priming of cooperation by extensive experience with coordination games. In particular, the experience with the cooperation games may have increased the animals’ preference of the ‘cooperate’ option to its ‘defect’ alternative. Use of social information. Our experimental setup allowed the monkeys to make choices while being able to see their opponent and its choice in every trial before making their own choice. Seeing the opponent’s choices that affect the outcome of the own choice emphasizes the social nature of the task; it allows the animals to learn the contingencies of the game and its outcomes and appreciate the consequences of their own choices in dependence on the opponent’s choices. And importantly, seeing the influence of the opponent’s choices on their own outcome should prevent the animals from assuming simple stochasticity of the outcomes; they learn that the other animal has an influence on the own outcome. Without such information, for example when replacing animals by computer opponents or placing them in separate rooms, the animals might assume that the choices are stochastic and then use the probabilities of outcomes as a measure to determine their choices. The visual interaction allowed each animal to respond to the choice of the opponent, thus minimising losses from unilateral cooperation. Indeed, a visual barrier between the two animals that prevented them to see the opponent’s options and choice resulted in rapid decline of cooperation (wall control; Fig. 3C). These results correspond to the previously reported drop in individual choice of the cooperation option when replacing the opponent monkey by a computer (from 35%% to 19%; Haroush & Williams 2015). A similar drop of individual choice of the cooperation option December 25 2022 16 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 16 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 16 was observed when placing the opponent monkey in another room (from 35% to 14%; Haroush & 582 Williams 2015). The replacement of a biological partner by a computer opponent and the animals’ 583 placement in separate rooms reduce the social aspect more than the simple blocking of the view of 584 each other’s options and choices achieved by our wall control. Nevertheless, irrespective of the 585 degree of reduction of social interaction, the results demonstrate the importance of social aspects in 586 these formal, and somewhat abstract and reductionist, economic tasks. 587 588 Strategies. Our monkeys cooperated even when they could see each other’s options and choice, 589 which might have made them more vulnerable to exploitation and defection. Despite frequent 590 defection in the face of the opponent’s cooperation, the animals cooperated in most trials: when the 591 first moving animal chose to cooperate, the other animal often chose also to cooperate. This result 592 held for all animals and all games (Figs. 2, 4). 593 Formal analyses of our animals’ choices revealed use of three major strategies (Fig. 6): 594 persistence (STAY), win-stay lose-shift (WSLS) and tit-for-tat (TFT). STAY consists of choosing 595 the same option as before irrespective of the outcome, whereas WSLS consists of choosing the 596 same option after receiving a good outcome but choosing the alternative after a lesser outcome. 597 Thus, players using STAY perseverate without taking the other player’s choice into account, and 598 players using WSLS respond to the outcome of their own choice that also depends on the other’s 599 choice. Thus, the two strategies are either not social at all (STAY) or social only to a limited extent 600 (WSLS). 601 By contrast, TFT consists of reciprocation and thus constitutes a fully social strategy. The 602 player replicates the opponent’s play, cooperating after a cooperation until the other player defects, 603 and defecting after a defection until the other player cooperates. 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 As mutual defection results in 604 lower outcome than mutual cooperation, players should aim for mutual cooperation; they should 605 occasionally even offer to cooperate in the face of the other player’s defection. This behavior may 606 result in long periods of being unilaterally defected that is beneficial for the opponent but provides 607 suboptimal own outcome (see payoff matrix in Fig. 1D) that is only justified by the later gain from 608 mutual cooperation. In contrast to TFT, defection in WSLS would lead after only one trial to stable 609 cooperation. Thus, recovery of mutual cooperation after defection is more costly in TFT than 610 WSLS, which may be a reason why WSLS outperforms TFT in humans (Nowak & Sigmund 1993). 611 These strategies are also found in our monkeys (Fig. 6A). First, the animals used STAY and 612 WSLS rather consistently in all games, whereas they used TFT much more rarely in both 613 cooperation games. Indeed, WSLS resulted in more cooperation than the other strategies (Fig. 6B). 614 An earlier study had shown substantial variability among individual monkeys to establish stable 615 strategies, but the animals expressing stable strategies did show reciprocal cooperation and 616 defection compatible with TFT (Smith et al. 2019). In another study, monkeys tended to continue 617 cooperating when the opponent had cooperated but tended to defect less when the opponent had 618 defected, suggesting some correspondence to both WSLS and TFT that significantly exceeded 619 random behavior (Haroush & Williams 2015). Their reciprocation depended on the social situation 620 by decreasing when one player was replaced by a computer or when performing in different rooms. 621 622 References 623 624 Báez-Mendoza R, Harris C, Schultz W. Activity of striatal neurons reflects social action and own 625 reward. Proc Natl Acad Sci (USA) 110: 16634-16639, 2013. 626 Báez-Mendoza R, Mastrobattista EP, Wang AJ, Williams ZM. Social agent identity cells in the 627 prefrontal cortex of interacting groups of primates. Science 374: eabb4149, 2021. 628 was observed when placing the opponent monkey in another room (from 35% to 14%; Haroush & 582 Williams 2015). The replacement of a biological partner by a computer opponent and the animals’ 583 placement in separate rooms reduce the social aspect more than the simple blocking of the view of 584 each other’s options and choices achieved by our wall control. 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 Nevertheless, irrespective of the 585 degree of reduction of social interaction, the results demonstrate the importance of social aspects in 586 these formal, and somewhat abstract and reductionist, economic tasks. 587 588 Strategies. Our monkeys cooperated even when they could see each other’s options and choice, 589 which might have made them more vulnerable to exploitation and defection. Despite frequent 590 defection in the face of the opponent’s cooperation, the animals cooperated in most trials: when the 591 first moving animal chose to cooperate, the other animal often chose also to cooperate. This result 592 held for all animals and all games (Figs. 2, 4). 593 Formal analyses of our animals’ choices revealed use of three major strategies (Fig. 6): 594 persistence (STAY), win-stay lose-shift (WSLS) and tit-for-tat (TFT). STAY consists of choosing 595 the same option as before irrespective of the outcome, whereas WSLS consists of choosing the 596 same option after receiving a good outcome but choosing the alternative after a lesser outcome. 597 Thus, players using STAY perseverate without taking the other player’s choice into account, and 598 players using WSLS respond to the outcome of their own choice that also depends on the other’s 599 choice. Thus, the two strategies are either not social at all (STAY) or social only to a limited extent 600 (WSLS). 601 By contrast, TFT consists of reciprocation and thus constitutes a fully social strategy. The 602 player replicates the opponent’s play, cooperating after a cooperation until the other player defects, 603 and defecting after a defection until the other player cooperates. As mutual defection results in 604 lower outcome than mutual cooperation, players should aim for mutual cooperation; they should 605 occasionally even offer to cooperate in the face of the other player’s defection. This behavior may 606 result in long periods of being unilaterally defected that is beneficial for the opponent but provides 607 suboptimal own outcome (see payoff matrix in Fig. 1D) that is only justified by the later gain from 608 mutual cooperation. In contrast to TFT, defection in WSLS would lead after only one trial to stable 609 cooperation. Thus, recovery of mutual cooperation after defection is more costly in TFT than 610 WSLS, which may be a reason why WSLS outperforms TFT in humans (Nowak & Sigmund 1993). 611 These strategies are also found in our monkeys (Fig. 6A). Báez-Mendoza R, Harris C, Schultz W. Activity of striatal neurons reflects social action and own 625 reward. Proc Natl Acad Sci (USA) 110: 16634-16639, 2013. 626 Bá M d R M t b tti t EP W AJ Willi ZM S i l t id tit ll i th 627 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 14 An earlier study had shown substantial variability among individual monkeys to establish stable 15 strategies, but the animals expressing stable strategies did show reciprocal cooperation and 16 defection compatible with TFT (Smith et al. 2019). In another study, monkeys tended to continue 17 cooperating when the opponent had cooperated but tended to defect less when the opponent had 18 defected, suggesting some correspondence to both WSLS and TFT that significantly exceeded 19 random behavior (Haroush & Williams 2015). Their reciprocation depended on the social situation 20 by decreasing when one player was replaced by a computer or when performing in different rooms. 21 22 y Chang SW, Fagan NA, Toda K, Utevsky AV, Pearson JM, Platt ML. Neural mechanisms of social 635 decision-making in the primate amygdala. Proc Natl Acad Sci (USA) 112: 16012-16017, 2015. 636 D b 25 2022 17 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint D b 25 2022 17 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 25, 2022. ; https://doi.org/10.1101/2022.12.25.521899 doi: bioRxiv preprint December 25, 2022 December 25, 2022 17 nder a Clements KC, Stephens DW. Testing models of non-kin cooperation: mutualism and the Prisoner’s 637 Dilemma. Anim Behav 50: 527–535, 1995. 638 Clements KC, Stephens DW. Testing models of non-kin cooperation: mutualis 637 Dilemma. Anim Behav 50: 527–535, 1995. 638 de Waal FB, Davis JM. 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Cell 160: 1-13, 2015. 649 g p Hosokawa T, Watanabe M. Prefrontal neurons represent winning and losing during competitive 650 video shooting games between monkeys. J Neurosci 32: 7662-7671, 2012. 651 g g y Luce RD. Individual Choice Behavior: A Theoretical Analysis. New York: Wiley, 195 y y Nowak MA, Five Rules for the Evolution of Cooperation. Science 314: 1560-1563, 200 Nowak M, Sigmund K. A strategy of win-stay, lose-shift that outperforms tit-for-tat in the 654 Prisoner's Dilemma game. Nature, 364: 56-58, 1993. 655 Rapoport A. Prisoner’s Dilemma — Recollections and observations. In A. Rapoport (Ed.), Game 656 Theory as a Theory of a Conflict Resolution (Vol. 2, pp. 17-34): Springer Netherlands, 1974. 657 Smith MF, Leverett KL, Wilson BJ, Brosnan SF. Capuchin monkeys (Sapajus [Cebus] apella) play 658 Nash equilibria in dynamic games, but their decisions are likely not influenced by oxytocin. 659 Am J Primatol 81: e22973, 2019. 660 Stephens DW, McLinn CM, Stevens JR. 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https://openalex.org/W4319602554	https://portal.findresearcher.sdu.dk/files/220998928/fbinf_03_1074212.pdf	English	null	Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae	Frontiers in bioinformatics	2,023	cc-by	7,601	Download date: 24. Oct. 2024 Citation for pulished version (APA): Nielsen, F. D., Møller-Jensen, J., & Jørgensen, M. G. (2023). Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae. Frontiers in Bioinformatics, 3, Article 1074212. https://doi.org/10.3389/fbinf.2023.1074212 OPEN ACCESS EDITED BY Vasco Ariston De Carvalho Azevedo, Federal University of Minas Gerais, Brazil REVIEWED BY Mamoon Rashid, (KAIMRC), Saudi Arabia Olli-Pekka Smolander, Tallinn University of Technology, Estonia Flemming Damgaard Nielsen1,2, Jakob Møller-Jensen1 and Mikkel Girke Jørgensen1* 1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 2Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark Introduction: Whole genome sequencing offers great opportunities for linking genotypes to phenotypes aiding in our understanding of human disease and bacterial pathogenicity. However, these analyses often overlook non-coding intergenic regions (IGRs). By disregarding the IGRs, crucial information is lost, as genes have little biological function without expression. CITATION Nielsen FD, Møller-Jensen J and Jørgensen MG (2023), Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Methods/Results: In this study, we present the ﬁrst complete pangenome of the important human pathogen Streptococcus pneumoniae (pneumococcus), spanning both the genes and IGRs. We show that the pneumococcus species retains a small core genome of IGRs that are present across all isolates. Gene expression is highly dependent on these core IGRs, and often several copies of these core IGRs are found across each genome. Core genes and core IGRs show a clear linkage as 81% of core genes are associated with core IGRs. Additionally, we identify a single IGR within the core genome that is always occupied by one of two highly distinct sequences, scattered across the phylogenetic tree. © 2023 Nielsen, Møller-Jensen and Jørgensen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Discussion: Their distribution indicates that this IGR is transferred between isolates through horizontal regulatory transfer independent of the ﬂanking genes and that each type likely serves different regulatory roles depending on their genetic context. genomics, pangenome, intergenic region, horizontal regulatory transfer, horizontal gene transfer, computational biology University of Southern Denmark Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae Nielsen, Flemming Damgaard; Møller-Jensen, Jakob; Jørgensen, Mikkel Girke Citation for pulished version (APA): Nielsen, F. D., Møller-Jensen, J., & Jørgensen, M. G. (2023). Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae. Frontiers in Bioinformatics, 3, Article 1074212. https://doi.org/10.3389/fbinf.2023.1074212 Citation for pulished version (APA): Nielsen, F. D., Møller-Jensen, J., & Jørgensen, M. G. (2023). Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae. Frontiers in Bioinformatics, 3, Article 1074212. https://doi.org/10.3389/fbinf.2023.1074212 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. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only y y p g • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. 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Please direct all enquiries to puresupport@bib.sdu.dk TYPE Original Research PUBLISHED 08 February 2023 DOI 10.3389/fbinf.2023.1074212 Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae OPEN ACCESS EDITED BY Vasco Ariston De Carvalho Azevedo, Federal University of Minas Gerais, Brazil REVIEWED BY Mamoon Rashid, (KAIMRC), Saudi Arabia Olli-Pekka Smolander, Tallinn University of Technology, Estonia *CORRESPONDENCE Mikkel Girke Jørgensen, mikkelj@bmb.sdu.dk SPECIALTY SECTION This article was submitted to Genomic Analysis, a section of the journal Frontiers in Bioinformatics RECEIVED 19 October 2022 ACCEPTED 24 January 2023 PUBLISHED 08 February 2023 CITATION Nielsen FD, Møller-Jensen J and Jørgensen MG (2023), Adding context to the pneumococcal core genes using bioinformatic analysis of the intergenic pangenome of Streptococcus pneumoniae. Front. Bioinform. 3:1074212. doi: 10.3389/fbinf.2023.1074212 frontiersin.org Many intergenic regions are universally conserved across all pneumococcal isolates Traditionally, pangenomes are limited to genes thereby excluding the non-coding intergenic regions (IGRs) (Page et al., 2015; Xiao et al., 2015). This focus on genes alone leaves out 15% of the genomes and ignores a signiﬁcant amount of crucial genomic information as IGRs contain several biologically relevant elements such as promoters, terminators, regulatory binding sites and non-coding RNAs (Koonin et al., 2001; Dagan et al., 2008; Peters et al., 2011; McCutcheon and Moran, 2012; Ochman and Caro-Quintero, 2016; Jørgensen et al., 2020). To effectively link genotypes to phenotypes through pangenomics, IGRs must be taken into consideration, as genes have little biological function without expression. We created a pangenome of 84 different pneumococcal isolates, spanning both genes and the non-coding IGRs. To put the nature of the pneumococcal IGR pangenome into perspective, we performed the same analysis for S. aureus. Both species may colonize the human upper respiratory tract, both are opportunistic pathogens and both possess open pangenomes, making them prime candidates for comparison (Laux et al., 2019). The analysis shows that the otherwise non-coding IGRs of both species are conserved in a similar manner to genes across the pangenome, although the number of unique genes outnumber the number of unique IGRs in both species (Figure 1). Recently, IGRs have attracted more attention as potential drivers of evolution (Molinaand VanNimwegen,2008;Orenetal.,2014;Thorpeetal., 2017). They persist through purifying selection, also known as negative selection, where unused or unwanted traits are removed. This persistence is true acrossseveraldiverse bacterial species,ina similar fashion to thatofcore genes, even when major regulatory elements are excluded (Molina and Van Nimwegen, 2008; Thorpe et al., 2017). Small variations in IGRs can lead to great phenotypical impact, for instance, the inversion of a single promoter element was demonstrated to turn a commensal bacterium pathogenic (Somvanshi et al., 2012). While the proportion of core genes roughly scales relative to the size of the genome (pneumococcus 2.1 Mbp/S. aureus 2.8 Mbp) the proportion of core IGRs relative to genome size is lower in pneumococcus (Figure 1). However, pneumococcus seemingly compensates for the lower number of unique IGRs by having multiple copies of several core IGRs in each genome. On average, each core IGR is present 1.23 times in each pneumococcal genome compared to 1.03 times in S. aureus. Each core gene is present 1.08 times in each pneumococcal genome and 1.02 times in S. Introduction Streptococcus pneumonia (pneumococcus) is the leading cause of sepsis, meningitis and bacterial pneumoniae in children worldwide (O’Brien et al., 2009). Widespread antibiotic resistance and the emergence of non-vaccine serotypes is making treatment increasingly difﬁcult. These threats have led the WHO to list pneumococcus as a “priority” pathogen (O’Brien et al., 2009; Weiser et al., 2018). This clinical relevance of pneumococcus has, in part, led to great scientiﬁc interest and the publication of several thousand sequenced genomes (National Center for Biotechnology Information, 2018). The availability of whole genome sequence (WGS) data has made it possible to study the entire pangenome of an organism rather than single isolates. A pangenome consists of the collective gene pool present in a group of organisms belonging to the same clade (Tettelin et al., 2005). The pangenome can Frontiers in Bioinformatics Frontiers in Bioinformatics 01 frontiersin.org Nielsen et al. 10.3389/fbinf.2023.1074212 be divided into a core genome, which constitutes genes present in all isolates and the accessory genome as the remaining genes (Tettelin et al., 2005). The pangenome of pneumococcus is considered at the extreme end of being open, that is, there is no deﬁned limit to its pangenome as new genes are acquired continuously (Donati et al., 2010). This openness is mainly due to new genes being acquired through horizontal gene transfer (HGT) mediated by pneumococcus’ natural competence (Vos, 2009; Chaguza et al., 2015). intergenic core genome is provided in Supplementary Appendix SA1. Additionally, we screen for any regulatory switching events present within the core genome. Many intergenic regions are universally conserved across all pneumococcal isolates aureus, this indicates that the high copy number of pneumococcal core IGRs is quite unusual. IGRs may also undergo genetic recombination, a term coined horizontal regulatory transfer (HRT) (Ragan and Beiko, 2009; Matus- Garcia et al., 2012). HRT can occur with the ﬂanking genes of the IGR, but in some cases, the IGRs are transferred independently of the genes they regulate (Oren et al., 2014). As much as 32% of the core regulatory regions in E. coli and 51% of the overall core IGRsare thought to have been acquired in this manner indicating that HRT is indeed common (Oren et al., 2014). Another aspect of HRT is regulatory switching where one IGR is replaced with another non-homologous IGR. This leads to two or more conserved IGRs occupying the same genomic space across different isolates of the same species (Ragan and Beiko, 2009; Matus-Garcia et al., 2012; Somvanshi et al., 2012; Oren et al., 2014; Thorpe et al., 2018). As much as 13% of the IGRs within the core genome of E. coli have undergone regulatory switching (Oren et al., 2014). Thus, IGRs seemingly contribute to greater variation in the core genome than genes themselves, thereby challenging the view of the bacterial core genome as being relatively stable (Oren et al., 2014; Caicedo- Montoya et al., 2021; Hyun et al., 2022). Core genes and IGRs constitute the majority of each genome The average pneumococcal genome has 79% of its genes as core genes and 66% of its IGRs as core IGRs. The average S. aureus genome is comparatively close to that observed in pneumococcus, here core genes constitute 79% and core IGRs 68% of each genome (Table 1). Despite pneumococcus having fewer unique core IGRs relative to genome size than S. aureus, as stated earlier, their copy number is higher in each genome, thus the percentage of core IGRs per genome is roughly equivalent in the two species (Table 1). The higher copy number of core IGRs in pneumococcus is also illustrated by the fact that 669 unique core IGRs exist in the pneumococcal core genome (Figure 1) but on average each genome has 817 core IGRs (Table 1). The average pneumococcal genome has 79% of its genes as core genes and 66% of its IGRs as core IGRs. The average S. aureus genome is comparatively close to that observed in pneumococcus, here core genes constitute 79% and core IGRs 68% of each genome (Table 1). Despite pneumococcus having fewer unique core IGRs relative to genome size than S. aureus, as stated earlier, their copy number is higher in each genome, thus the percentage of core IGRs per genome is roughly equivalent in the two species (Table 1). The higher copy number of core IGRs in pneumococcus is also illustrated by the fact that 669 unique core IGRs exist in the pneumococcal core genome (Figure 1) but on average each genome has 817 core IGRs (Table 1). In this study we map the complete core genome of pneumococcus and compare the nature of genesand IGRs against each other inthe pangenome. We ﬁnd a clear linkage between core genes and core IGRs, but core genes are associated with different IGRs, indicating that the pneumococcal core genome is less stable than previously thought. Additionally, we identify any potential regulatory switching events within this core genome. To our knowledge we are the ﬁrst to identify the complete core genome of pneumococcus, both coding and non-coding. IGRs are more likely to be unique to a few isolates than genes The number of unique IGRs in the pneumococcal pangenome increases with the number of isolates analyzed in a similar manner to the number of unique genes (Figure 2A). Overall, fewer unique IGRs are present in the pangenome than genes, part of this is due to the exclusion of IGRs of <30 bp in length, which are most often intraoperonic (Thorpe et al., 2018). Frontiers in Bioinformatics Results Most IGRs are either present in almost all pneumococcus isolates or unique to only a few, that is, they are either very common or very rare (Figure 2B). Pneumococcus genes show a similar distribution In this study, we map the ﬁrst complete pangenome of pneumococcus, spanning both genes and IGRs. The identiﬁed 02 frontiersin.org Nielsen et al. 10.3389/fbinf.2023.1074212 FIGURE 1 The pangenome of S. pneumoniae and S. aureus, spanning both intergenic regions (green) and genes (orange), illustrated by Venn diagrams. Both species possess a core genome of both IGRs and genes, deﬁned as being present in >95% of isolates. The pangenomes are constructed from 84 unique genomes of each species. S. pneumoniae has a core genome of 1,550 genes and 669 IGRs, while an accessory genome of 3,132 genes and 2683 IGRs. S. aureus has a core genome of 2096 genes and 1,142 IGRs, while an accessory genome of 3,846 genes and 3,322 IGRs. FIGURE 1 FIGURE 1 The pangenome of S. pneumoniae and S. aureus, spanning both intergenic regions (green) and genes (orange), illustrated by Venn diagrams. Both species possess a core genome of both IGRs and genes, deﬁned as being present in >95% of isolates. The pangenomes are constructed from 84 unique genomes of each species. S. pneumoniae has a core genome of 1,550 genes and 669 IGRs, while an accessory genome of 3,132 genes and 2683 IGRs. S. aureus has a core genome of 2096 genes and 1,142 IGRs, while an accessory genome of 3,846 genes and 3,322 IGRs. TABLE 1 The number of genes and IGRs in the core and accessory genome in selected genomes and across the collected pangenome, as well as the percentage of genes and IGRs that are core. Species/isolate Core genes Core IGRs Accessory genes Accessory IGRs Percentage core genes pr. genome (%) Percentage core IGRs pr. genome (%) S. pneumoniae (Species average) 1,670 817 448 425 78.93 65.83 S. pneumoniae 1,672 810 352 388 82.61 67.61 D39 S. pneumoniae 1,674 809 345 388 82.91 67.59 R6 S. pneumoniae 1703 820 447 447 79.21 64.72 Tigr4 S. aureus (Species average) 2131 1,170 570 544 78.98 68.28 TABLE 1 The number of genes and IGRs in the core and accessory genome in selected genomes and across the collected pangenome, as well as the percentage of genes and IGRs that are core. Results across the pangenome, though a larger proportion of IGRs are conﬁned to only a few isolates than genes. considered single regulatory (SR) and IGRs that are between two divergently transcribed genes are considered double regulatory (DR) (Figure 3). Pneumococcus retains more unique genes than IGRs within its pangenome (Figure 2A), and most unique IGRs are only found in single isolates, making them rare (Figure 2B). This scarcity of unique IGRs could indicate that IGRs experience a higher evolutionary selection threshold than genes, thereby lowering the likelihood of a newly acquired IGR of spreading to more isolates through HRT. Looking at the distribution of the IGR types across the pneumococcal pangenome, NR and DR regions are rare compared to SR IGRs (Figure 3). DR regions also constitute a greater relative proportion of the core IGRs than seen in the accessory genome. Double regulatory regions are more common in the core genome Next, we analyzed the degree of linkage between core IGRs and core genes, that is, how often a core IGR is directly upstream a core gene. IGRs and their ﬂanking genes were identiﬁed and any IGRs directly upstream the start codon of a gene was selected. The status of the IGR/gene pairs as accessory or core genome was then assessed and the ratio of each combination calculated. On average 81% of core genes IGRs can be categorized according to the orientation of their ﬂanking genes. IGRs that are downstream of two convergently transcribed genes are considered non-regulatory (NR), IGRs that are upstream one gene and downstream another gene are 03 Frontiers in Bioinformatics frontiersin.org 10.3389/fbinf.2023.1074212 Nielsen et al. FIGURE 2 Properties of the pneumococcal pangenome and its intergenic regions (IGRs) (A) Number of unique intergenic regions (green) and genes (orange) as a function of the number of isolates included in the pangenome. (B) Distribution of unique IGRs (green) and genes (orange) across the streptococcal pangenome, illustrated with a frequency histogram (number of IGRs/genes present in the given number of isolates). Most IGRs and genes are part of the core genome or conﬁned to a small fraction of the isolates. FIGURE 2 Properties of the pneumococcal pangenome and its intergenic regions (IGRs) (A) Number of unique intergenic regions (green) and genes (orange) as a function of the number of isolates included in the pangenome. (B) Distribution of unique IGRs (green) and genes (orange) across the streptococcal pangenome, illustrated with a frequency histogram (number of IGRs/genes present in the given number of isolates). Most IGRs and genes are part of the core FIGURE 2 Properties of the pneumococcal pangenome and its intergenic regions (IGRs) (A) Number of unique intergenic regions (green) and genes (orange) as a function of the number of isolates included in the pangenome. (B) Distribution of unique IGRs (green) and genes (orange) across the streptococcal pangenome, illustrated with a frequency histogram (number of IGRs/genes present in the given number of isolates). Most IGRs and genes are part of the core genome or conﬁned to a small fraction of the isolates. in S. pneumoniae are associated with a core IGR, whereas only 74% of accessory genes are linked to accessory IGRs (Table 2). For comparison, the linkage of core genes to core IGRs is greater in S. Double regulatory regions are more common in the core genome aureus at 86%, and accessory IGRs are ﬂanking accessory genes 82% of the time. even from a separate species. For this analysis, only switches where the IGRs share no signiﬁcant sequence homology with a BLASTN were included. even from a separate species. For this analysis, only switches where the IGRs share no signiﬁcant sequence homology with a BLASTN were included. We detected three switches within the pneumococcus pangenome and only one of these is ﬂanked by core genes. We designated the core switched IGR as csIGR (Table 3). While the two versions of the csIGR are highly conserved on their own, with both having a nucleotide identity of >99% amongst themselves, aligning the two versions with each other results in an insigniﬁcant nucleotide identity of 57%. These results were manually conﬁrmed with a blastn and conﬁrmed that all pneumococcal isolates always have one of these two csIGRs but only in a single copy and always between the same ﬂanking genes. We detected three switches within the pneumococcus pangenome and only one of these is ﬂanked by core genes. We designated the core switched IGR as csIGR (Table 3). While the two versions of the csIGR are highly conserved on their own, with both having a nucleotide identity of >99% amongst themselves, aligning the two versions with each other results in an insigniﬁcant nucleotide identity of 57%. These results were manually conﬁrmed with a blastn and conﬁrmed that all pneumococcal isolates always have one of these two csIGRs but only in a single copy and always between the same ﬂanking genes. None of the IGRs of the capsular polysaccharide synthesis (cps) operon were found to be core IGRs, however the highly conserved ﬂanking genes dexB and aliA were both associated with core IGRs (Appendix 1). A single core IGR shows sign of regulatory switching Listed are the percentage core and/or genes with a core and/or accessory TABLE 2 Genes and their upstream IGR was analyzed for their distribution in the pangenome. Listed are the percentage core and/or genes with a core and/or accessory IGR immediately upstream. BLE 2 Genes and their upstream IGR was analyzed for their distribution in the pangenome. Listed are the percentage core and/or genes wit R immediately upstream. TABLE 2 Genes and their upstream IGR was analyzed for their distribution in the pangenome. Listed are the percentage core and/or genes with a core and/or accessory IGR immediately upstream. Species/isolate Core gene: core IGR (%) Core gene: accessory IGR (%) Accessory gene: core IGR (%) Accessory gene: accessory IGR (%) S. pneumoniae (Species average) 80.92 19.08 26.45 73.55 S. pneumoniae 81.06 18.94 30.77 69.23 D39 S. pneumoniae 80.97 19.03 30.39 69.61 R6 S. pneumoniae 80.38 19.62 26.52 73.48 Tigr4 S.aureus (Species average) 86.19 13.81 17.62 82.38 TABLE 3 The two versions of the csIGR in-between the single copy core genes. csIGR1 is present in 32 of the isolates analyzed and is highly conserved across the genomes with an average nucleotide identity of 99.49%. csIGR2 is present in 52 of the strains analyzed and is likewise highly conserved with an average nucleotide identity of 99.28%. Both IGRs have roughly the same length in base pairs. Length SNPs Nuc_identity (%) Length_identity (%) No.isolates IGR immediately upstream. Species/isolate Core gene: core IGR (%) Core gene: accessory IGR (%) Accessory gene: core IGR (%) Accessory gene: accessory IGR (%) S. pneumoniae (Species average) 80.92 19.08 26.45 73.55 S. pneumoniae 81.06 18.94 30.77 69.23 D39 S. pneumoniae 80.97 19.03 30.39 69.61 R6 S. pneumoniae 80.38 19.62 26.52 73.48 Tigr4 S.aureus (Species average) 86.19 13.81 17.62 82.38 TABLE 3 The two versions of the csIGR in-between the single copy core genes. csIGR1 is present in 32 of the isolates analyzed and is highly conserved across the genomes with an average nucleotide identity of 99.49%. csIGR2 is present in 52 of the strains analyzed and is likewise highly conserved with an average nucleotide identity of 99.28%. Both IGRs have roughly the same length in base pairs. TABLE 3 The two versions of the csIGR in-between the single copy core genes. csIGR1 is present in 32 of the isolates analyzed and is highly conserved across the genomes with an average nucleotide identity of 99.49%. A single core IGR shows sign of regulatory switching The ﬂanking genes were both single copy core genes and were identiﬁed in the common lab strains S. pneumoniae D39 and TIGR4 (Figure 4). These two strains have distinct csIGR types, with D39 having csIGR1 and TIGR4 having csIGR2 (Figure 4). Interestingly, rather than ﬂanking an operon, the IGRs are predicted to sit in the middle of an operon. Little is known about the ﬂanking genes, other than their status as single copy core genes Next, we examined the IGR candidates for regulatory switching. Regulatory switching describes when one IGR is replaced by a different non-homologue IGR. The origin of these switched IGRs is not inferred in this analysis, thus they can both originate from within the isolate itself or 04 Frontiers in Bioinformatics frontiersin.org Nielsen et al. 10.3389/fbinf.2023.1074212 FIGURE 3 The types of intergenic regions (IGRs) and their distribution in the pneumococcal pangenome, illustrated with a raincloud plot. Each point is a unique IGR of that type plotted against the number of isolates in the pangenome it is present in. Cloud areas are scaled relative to the size of each dataset. Core IGRs are present in >95% strains (orange) and accessory IGRs are present in <95% of isolates (green). The IGRs are categorized according to the orientation of their ﬂanking genes. If the ﬂanking genes are pointing in the same direction the IGR is categorized as single regulatory (SR), if they face towards the IGR, it is categorized as non-regulatory (NR) and if they face away from the IGR, it is categorized as double regulatory (DR). FIGURE 3 The types of intergenic regions (IGRs) and their distribution in the pneumococcal pangenome, illustrated with a raincloud plot. Each point is a unique IGR of that type plotted against the number of isolates in the pangenome it is present in. Cloud areas are scaled relative to the size of each dataset. Core IGRs are present in >95% strains (orange) and accessory IGRs are present in <95% of isolates (green). The IGRs are categorized according to the orientation of their ﬂanking genes. If the ﬂanking genes are pointing in the same direction the IGR is categorized as single regulatory (SR), if they face towards the IGR, it is categorized as non-regulatory (NR) and if they face away from the IGR, it is categorized as double regulatory (DR). eam IGR was analyzed for their distribution in the pangenome. Frontiers in Bioinformatics A single core IGR shows sign of regulatory switching csIGR2 is present in 52 of the strains analyzed and is likewise highly conserved with an average nucleotide identity of 99.28%. Both IGRs have roughly the same length in base pairs. Length SNPs Nuc_identity (%) Length_identity (%) No.isolates csIGR1 215 1 99.49 99.53 32 csIGR2 214 1 99.28 99.05 52 05 frontiersin.org 10.3389/fbinf.2023.1074212 Nielsen et al. biogenesis of the 30 S ribosome subunit (Pek et al., 2007; Liu et al., 2017). The sequence of both csIGR types is provided in Supplementary Appendix SA2. Interestingly, neither of the csIGR types were conﬁned to a speciﬁc phylogenetic cluster of pneumococci (Figure 5). The fact that the csIGR types are spread across the phylogenetic tree indicates that their distribution is due to HRT. biogenesis of the 30 S ribosome subunit (Pek et al., 2007; Liu et al., 2017). The sequence of both csIGR types is provided in Supplementary Appendix SA2. Interestingly, neither of the csIGR types were conﬁned to a speciﬁc phylogenetic cluster of pneumococci (Figure 5). The fact that the csIGR types are spread across the phylogenetic tree indicates that their distribution is due to HRT. found in this study. SPD_1559/SP_1749 is considered essential in pneumococcus and is a homologue to ygeH, a gene involved in FIGURE 4 The core switched intergenic region (csIGR) in S. pneumoniae D39 and Tigr4. Each type of csIGR is represented in these strains, with D39 having csIGR1 (orange) and TIGR4 having csIGR2 (green). In D39, csIGR1 is ﬂanked by SPD_1558 and SPD_1559. In TIGR4, csIGR2 is ﬂanked by the genes SP_1748 and SP_1749. FIGURE 4 The core switched intergenic region (csIGR) in S. pneumoniae D39 and Tigr4. Each type of csIGR is represented in these strains, with D39 having csIGR1 (orange) and TIGR4 having csIGR2 (green). In D39, csIGR1 is ﬂanked by SPD_1558 and SPD_1559. In TIGR4, csIGR2 is ﬂanked by the genes SP_1748 and SP_1749. We performed a pangenome wide association study to see if any genes within the accessory genome were signiﬁcantly co-occurring with the csIGR alleles across the pangenome. However, no genes were exclusively associated with neither of the csIGR types. Both sequences were also screened for promoters, riboswitches and homology to known regulatory RNAs with no signiﬁcant hits. However, the translated RNA sequence of both sequences was predicted to form signiﬁcant secondary structures, the signiﬁcance of which remains to be elucidated. The predicted secondary structures are provided in Supplementary Appendix SA2. Pangenome creation Initially a pangenome of the coding sequences (CDS) was created using Roary (v3.13.0) (Page et al., 2015). Then a complementary pangenome of the IGRs was created using Piggy (v1.5), an intergenic pangenome analysis tool that emulates Roary (Thorpe et al., 2018). Some steps were taken to ensure comparability between the outputs of the software. Roary was set to cluster CDSs with -e -n (to perform alignments using MAFFT (Katoh et al., 2002)), -i 90 (90% sequence identity cut-off) and -s (to not split paralogs into separate clusters). The settings for running Piggy were set at the standard parameters of the software, except for -len_id 10 (the minimum percentage of length identity to form a cluster). The length identity was reduced for comparability with Roary, as gene clusters generated by Roary only require a sequence length identity of 120 bp for clustering CDSs, thus the len_id of 10 is recommended by the creators of Piggy for Roary consistency as IGRs are not erroneously placed into separate clusters (Koonin et al., 2001; Dagan et al., 2008; Molina and Van Nimwegen, 2008; Ragan and Beiko, 2009; Peters et al., 2011; Matus-Garcia et al., 2012; McCutcheon and Moran, 2012; Somvanshi et al., 2012; Oren et al., 2014; Page et al., 2015; Xiao et al., 2015; Ochman and Caro-Quintero, 2016; Thorpe et al., 2017; Thorpe et al., 2018; Jørgensen et al., 2020). The randomly assigned locus tags provided by Prokka were translated when necessary, by aligning the GFF ﬁles of the Genbank annotated ﬁles and Prokka output. Our analysis reveals that IGRs are highly conserved in pneumococcus. On average, 66% of IGRs in any isolate is shared with all other isolates and 79% of genes in any isolate is shared with all other isolates. A similar trend is seen in S. aureus, however, the overall number of unique IGRs is lower in pneumococcus relative to genome size. Instead, our analysis reveals that pneumococcus has several duplicates of some core IGRs across the genome, with core IGRs on average being present 1.25 times in each isolate. This trend is not seen with its core genes and is not observed in neither the core genes nor core IGRs of S. aureus. This suggests that pneumococcus is more rigid with its transcriptional proﬁle as the same regulatory regions might be repeated to a greater degree than observed in S. aureus. A single core IGR shows sign of regulatory switching FIGURE 4 The core switched intergenic region (csIGR) in S. pneumoniae D39 and Tigr4. Each type of csIGR is represented in these strains, with D39 having csIGR1 (orange) and TIGR4 having csIGR2 (green). In D39, csIGR1 is ﬂanked by SPD_1558 and SPD_1559. In TIGR4, csIGR2 is ﬂanked by the genes SP_1748 and SP_1749. FIGURE 4 The core switched intergenic region (csIGR) in S. pneumoniae D39 and Tigr4. Each type of csIGR is represented in these strains, with D39 having csIGR1 (orange) and TIGR4 having csIGR2 (green). In D39, csIGR1 is ﬂanked by SPD_1558 and SPD_1559. In TIGR4, csIGR2 is ﬂanked by the genes SP_1748 and SP_1749. found in this study. SPD_1559/SP_1749 is considered essential in pneumococcus and is a homologue to ygeH, a gene involved in found in this study. SPD_1559/SP_1749 is considered essential in pneumococcus and is a homologue to ygeH, a gene involved in FIGURE 5 Unrooted phylogenetic tree of the 84 S. pneumoniae strains used in this study. The tree is based on SNPs in the core genes. The shading of each label indicates the presence of csIGR1 (orange) or csIGR2 (green). The tree was created using Roary, Fasttree and iTol. The S. pneumoniae strain names are speciﬁed if applicable, if no clear strain name was given the sequence ID was used. FIGURE 5 Unrooted phylogenetic tree of the 84 S. pneumoniae strains used in this study. The tree is based on SNPs in the core genes. The shading of each label indicates the presence of csIGR1 (orange) or csIGR2 (green). The tree was created using Roary, Fasttree and iTol. The S. pneumoniae strain names are speciﬁed if applicable, if no clear strain name was given the sequence ID was used. 06 Frontiers in Bioinformatics frontiersin.org Nielsen et al. Nielsen et al. 10.3389/fbinf.2023.1074212 10.3389/fbinf.2023.1074212 Pangenome creation We identify a clear linkage between the core IGRs and core genes in pneumococcus, on average 81% of core IGRs are directly upstream of a core gene. This indicates that the transcriptional regulation of the core genome in pneumococcus is mostly conserved across all isolates, but to a lesser degree than seen in S. aureus. However, this leaves 19% of core genes being associated with accessory IGRs, indicating some plasticity to the core genome that is otherwise viewed as stable. The greatest difference seen between the two species in this regard is that core IGRs are more often associated with accessory genes in pneumococcus. This might be explained by pneumococcus retaining multiple copies of some core IGRs, making them associated with both core and accessory genes, though this remains to the investigated. Discussion future studies will beneﬁt from viewing the genes as a “package” with their upstream IGR, as even core genes maintain different regulatory regions within the pneumococcal species. Here we present the ﬁrst complete pangenome of pneumococcus, spanning both genes and the non-coding IGRs. A small but conserved IGR core genome in pneumococcus was identiﬁed. We ﬁnd that the pneumococcal core genome consists of 1,550 unique genes and 669 IGRs, whereas the accessory genome consists of 3,132 unique genes and 2683 IGRs. The number of unique genes surpasses that of unique IGR in both cases, this is unsurprising as most intraoperonic regions in pneumococcus are less than 30 bp in length and are therefore disregarded in the analysis. This also means that most IGRs identiﬁed are associated with the ﬂanking genes of operons i.e., the regulatory regions. Genomes All 84 complete S. pneumoniae genomes available from the National Center for Biotechnology Information, GenBank resource was downloaded in raw FASTA format. Additionally, 84 randomly selected S. aureus genomes were retrieved for comparison with S. pneumoniae (12/5/2021). Genomes were then annotated with Prokka (v 1.14.5), using the standard parameters of the software (Seemann, 2014). The genomes used are listed in Supplementary Appendix SA3. IGRs between two divergently transcribed genes are termed double regulatory (DR). These regions constituted a greater relative part of the core genome than the accessory genome. It is likely because meaningful regulation of two genes is harder to achieve than regulation of single genes, raising the selection threshold for the emergence of beneﬁcial divergence. This increased selection pressure has previously been observed as purifying selection has been shown to be more prominent in DR regions than the other types (Molina and Van Nimwegen, 2008). Frontiers in Bioinformatics Brynildsrud, O., Bohlin, J., Scheffer, L., and Eldholm, V. (2016). Rapid scoring of genes in microbial pan-genome-wide association studies with Scoary. Genome Biol. 17 (1), 238. doi:10.1186/s13059-016-1108-8 Author contributions FN, MJ and JM-J conceptualized the study. FN and MJ wrote the paper. Project supervised and funded by JM-J and MJ. Pangenome wide association study To identify whether any accessory genes were signiﬁcantly associated with any of the csIGR alleles, a pangenome-wide association study was performed using Scoary (v1.6.16) (Brynildsrud et al., 2016). A trait matrix was created as an input for Scoary, indicating which of the two csIGRs alleles were present in which genomes. Scoary then sorted the accessory genome provided by the gene_presence_absence ﬁle from Roary, scoring each accessory gene according to their co-occurrence with each csIGR. Core gene and core IGR linkage analysis Surprisingly, only three switches were detected in pneumococcus and only one of these was ﬂanked by core genes. In another study, the same analysis was done on a collection of E. coli genomes and 61 switches were detected (Thorpe et al., 2018). This indicates that regulatory switching does not play a major role in pneumococcal disease. It is possible that regulatory switching is more prominent in E. coli as it inhabits a great number of different niches compared to pneumococcus (Tenaillon et al., 2010; Weiser et al., 2018).Our results show that the pneumococcal core genome is less stable than previously thought. While there is indeed a stable reservoir of highly conserved core genes, their ﬂanking IGRs, which contain most of the regulatory regions responsible for controlling the transcription of these core genes show greater plasticity. We believe that The linkage of core genes and core IGRs was quantiﬁed using R (v. 4.1.0). The gene_presence_absence ﬁle from Roary and the IGR_ presence_absence ﬁle from Piggy was loaded as dataframes in R. For each gene and IGR cluster in the ﬁles their status as a core or accessory gene was identiﬁed and assigned. For each genome all IGRs were paired with their upstream gene. Thus, NR regions were removed from the dataset and both ﬂanking genes for DR regions were analyzed separately, if any of the two genes were core, the DR IGR was assigned as ﬂanking a core gene. The R code is provided in Supplementary Appendix SA4. Frontiers in Bioinformatics 07 frontiersin.org Nielsen et al. 10.3389/fbinf.2023.1074212 Nielsen et al. 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. SUPPLEMENTARY TABLE S1 SUPPLEMENTARY TABLE S1 Streptococcus pneumoniae intergenic core genome. SUPPLEMENTARY TABLE S1 SUPPLEMENTARY TABLE S1 Streptococcus pneumoniae intergenic core genome. SUPPLEMENTARY TABLE S2 Phylogenetic analysis A phylogenetic tree of the strains included in this study was created, based on single nucleotide polymorphisms (SNPs) in the core genes. Roary was run separately with the same settings as previously mentioned with the exception of -e (Core gene alignment with PRANK) (Page et al., 2015). This produced a highly accurate alignment of the core genes within the pangenome. FastTree (v2.1.11) was then run to infer an approximately-maximum- likelihood phylogenetic tree based on SNPs within the core genes (Price et al., 2009). The resulting newick ﬁle was then visualized using iTol (v5.7) and exported to Adobe Illustrator (Letunic and Bork, 2007). csIGR1 and csIGR2 analysis SUPPLEMENTARY TABLE S2 Sequences and predicted secondary structures of csIGR1 and csIGR2. SUPPLEMENTARY TABLE S2 Sequences and predicted secondary structures of csIGR1 and csIGR2. To assess homology to existing regulatory RNAs, a BLASTN was performed for each csIGR against the RefSeq RNA database. Both sequences were screened for potential riboswitches using Riboswitch Finder (Bengert and Dandekar, 2004). Any potential promoter or SUPPLEMENTARY TABLE S3 SUPPLEMENTARY TABLE S3 Genomes used in this study. Genomes used in this study. SUPPLEMENTARY TABLE S4 Code. Data availability statement Publicly available datasets were analyzed in this study. In total, 84 different pneumococcal genomes and 84 randomly selected S. aureus genomes were retrieved from the National Center for Biotechnology Information. Their accession numbers are stated in Supplementary Appendix SA3. The identiﬁed switch was validated manually with a BLASTN against all the genomes (data not shown). Switched intergenic regions analysis terminator regions were screened for using BPROM and FindTerm (Softberry) (Solovyev et al., 2011). Secondary structures were predicted for both sequences using the RNA structure package available through Mathews lab, at standard parameters (Reuter and Mathews, 2010). For identiﬁcation of switched IGRs, a separate analysis using Piggy was performed with -len_id 90 (the minimum percentage of length identity to form a cluster). This was done to perform a more strict analysis of the IGRs, as the higher threshold for forming a cluster ensured that homologue IGRs were not identiﬁed as switched IGRs (Thorpe et al., 2018). IGR switches were identiﬁed using the “gene- pair” method of Piggy, here two or more different IGR sequences that occupy the same space between a speciﬁc gene pair are analyzed. The candidate IGR sequences are then aligned with BLASTN with low complexity ﬁltering turned off and if there are no signiﬁcant matches between the IGR they are identiﬁed as “switched”. If there is a signiﬁcant match Piggy aligns the sequences using MAFFT and provides the nucleotide identity of the alignments. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbinf.2023.1074212/ full#supplementary-material References 1863, 194504. doi:10.1016/j.bbagrm.2020.194504 Reuter, J. S., and Mathews, D. H. (2010). RNAstructure: Software for RNA secondary structure prediction and analysis. BMC Bioinforma. 11, 129. doi:10.1186/1471-2105-11-129 Katoh, K., Misawa, K., Kuma, K. I., and Miyata, T. (2002). Mafft: A novel method for rapid multiple sequence alignment based on fast fourier transform. Nucleic Acids Res. 30, 3059–3066. doi:10.1093/nar/gkf436 Seemann, T. (2014). Prokka: Rapid prokaryotic genome annotation. 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https://openalex.org/W3164997644	https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/s13104-021-05689-3	English	null	The diversity of unique 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid coding common genes and Universal stress protein in Ectoine TRAP cluster (UspA) in 32 Halomonas species	BMC research notes	2,021	cc-by	5,104	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rekadwad et al. BMC Res Notes (2021) 14:296 https://doi.org/10.1186/s13104-021-05689-3 Rekadwad et al. BMC Res Notes (2021) 14:296 https://doi.org/10.1186/s13104-021-05689-3 BMC Research Notes Open Access Abstract Objectives: To decipher the diversity of unique ectoine-coding housekeeping genes in the genus Halomonas. Results: In Halomonas, 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid has a crucial role as a stress-tolerant chaperone, a compatible solute, a cell membrane stabilizer, and a reduction in cell damage under stressful conditions. Apart from the current 16S rRNA biomarker, it serves as a blueprint for identifying Halomonas species. Halomonas elongata 1H9 was found to have 11 ectoine-coding genes. The presence of a superfamily of conserved ectoine- coding among members of the genus Halomonas was discovered after genome annotations of 93 Halomonas spp. As a result of the inclusion of 11 single copy ectoine coding genes in 32 Halomonas spp., genome-wide evaluations of ectoine coding genes indicate that 32 Halomonas spp. have a very strong association with H. elongata 1H9, which has been proven evidence-based approach to elucidate phylogenetic relatedness of ectoine-coding child taxa in the genus Halomonas. Total 32 Halomonas species have a single copy number of 11 distinct ectoine-coding genes that help Halomonas spp., produce ectoine under stressful conditions. Furthermore, the existence of the Universal stress protein (UspA) gene suggests that Halomonas species developed directly from primitive bacteria, highlighting its role during the progression of microbial evolution. Keywords: Ectoine, Life under extreme conditions, Saline environments, Single-copy genes, Ancient bacteria and Archaea Bioactive compounds Keywords: Ectoine, Life under extreme conditions, Saline environments, Single-copy genes, Ancient bacteria and Archaea, Bioactive compounds The diversity of unique 1,4,5,6‑Tetrahydro‑ 2‑methyl‑4‑pyrimidinecarboxylic acid coding common genes and Universal stress protein in Ectoine TRAP cluster (UspA) in 32 Halomonas species Bhagwan Narayan Rekadwad1* , Wen‑Jun Li2 and P. D. Rekha1 Introduction function termed as ‘ectoine’ in general [6]. The moder- ately halophilic members of family Halomonadaceae displays osmoadaptation facilitated by pigments such as betaine and ectoine [3] and hydroxyectoine [12]. Fam- ily Halomonadaceae possess total 18 child taxa. Of these, names of 14 child taxa are validly published with their correct name. Other16 child taxa have their validly published name including synonyms under the Interna- tional Code of Nomenclature of Prokaryotes (ICNP). On similar note, currently Genus Halomonas represented by 114 type strains with 112 candidates having validity 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (C6H10N2O2, molecular weight 142.16) is a natural pig- ment produced within the cytoplasm of salt-loving bac- teria (e.g. genus Ectothiorhodospira; Halomonas). This pigment helps bacterium to perform osmoregulatory Correspondence: rekadwad@gmail.com 1 Yenepoya Research Centre, Yenepoya (Deemed to be University), University Road, Deralakatte, Mangalore, Karnataka 575018, India Full list of author information is available at the end of the article Correspondence: rekadwad@gmail.com 1 Yenepoya Research Centre, Yenepoya (Deemed to be University), University Road, Deralakatte, Mangalore, Karnataka 575018, India Full list of author information is available at the end of the article *Correspondence: rekadwad@gmail.com 1 Yenepoya Research Centre, Yenepoya (Deemed to be University), University Road, Deralakatte, Mangalore, Karnataka 575018, India Full list of author information is available at the end of the article Rekadwad et al. BMC Res Notes (2021) 14:296 Rekadwad et al. BMC Res Notes (2021) 14:296 Page 2 of 8 Page 2 of 8 published name and correct name and 10 candidates with synonyms. Also, three species have orthographic misspelled variants, and 18 invalidated species were not validated by ICNP [7]. Description of all Halomonas spe- cies is given on LPSN portal managed by Leibniz Insti- tute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany. Halomonas species are known producer of biotechnologically important ectoine. Being suspended in the cytoplasm, ectoine and hydrox- yectoine coded by Halomonas species has benefits to cell. It performs various activities in cell such as stress tolerant chaperones, as a compatible solute, stabilize of cell membrane and reduce cell damage [10]. Moreover, ectoine and hydro-ectoines are high-value chemicals and exploited for cosmetics, immune protection, sta- bilization of antibodies, anti-inflammatory and tissue protective agent, for co-production of bioplastic polyhy- droxybutyrate [8], as a skin aging and protectant agent against harsh environments viz. radiation and extreme temperatures. Whole cell and macromolecule under hos- tile conditions were protected by intracellular ectoine from freezing, drying, high salinity, heat stress, oxygen radicals, radiation and denaturing agents [10]. Various applications of ectione produced by Halomonas species reflect presence of diverse gene profiles and other con- served genes in their genomes. It is therefore important to evaluate indicative signatures genes that codes ectoine and governs vital biological function under extreme envi- ronmental conditions among the genus Halomonas. published name and correct name and 10 candidates with synonyms. Also, three species have orthographic misspelled variants, and 18 invalidated species were not validated by ICNP [7]. Description of all Halomonas spe- cies is given on LPSN portal managed by Leibniz Insti- tute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany. Halomonas species are known producer of biotechnologically important ectoine. Being suspended in the cytoplasm, ectoine and hydrox- yectoine coded by Halomonas species has benefits to cell. It performs various activities in cell such as stress tolerant chaperones, as a compatible solute, stabilize of cell membrane and reduce cell damage [10]. Moreover, ectoine and hydro-ectoines are high-value chemicals and exploited for cosmetics, immune protection, sta- bilization of antibodies, anti-inflammatory and tissue protective agent, for co-production of bioplastic polyhy- droxybutyrate [8], as a skin aging and protectant agent against harsh environments viz. radiation and extreme temperatures. Whole cell and macromolecule under hos- tile conditions were protected by intracellular ectoine from freezing, drying, high salinity, heat stress, oxygen radicals, radiation and denaturing agents [10]. Various applications of ectione produced by Halomonas species reflect presence of diverse gene profiles and other con- served genes in their genomes. It is therefore important to evaluate indicative signatures genes that codes ectoine and governs vital biological function under extreme envi- ronmental conditions among the genus Halomonas. Radar chart Halomonas spp., possesses multiple quantitative vari- ables (species in particular) i.e. variable genome length/ data points for visualization. Radar chart makes the way easy to compare the intra-species variable length to see similar values and find high or low scoring within outliers in the genus. Methods 128 type strains 16S rRNA genes and 94 Halomonas spp., genomes Phylogeny reconstruction and topology analysish Phylogeny reconstruction and topology analysis The evolutionary history of one hundred twenty- eight16S rRNA and 33 Halomonas single-copy genes were inferred using standalone tool MEGA X with 1000 bootstrap analysis followed by best scoring ML, NJ and ME tree. The Jukes-Cantor method and are in the units of the number of base substitutions per site. The clos- est child taxa of biotechnological important ectoine producing H. elongata 1H9 were deciphered. It helps for phylogenetic analysis and topology comparison to delineate nearest species and 1,4,5,6-Tetrahydro-2-me- thyl-4-pyrimidinecarboxylic acid gene coding species. Identification of protein families and single copy genes Identification of protein families and single copy genes Protein families and single-copy genes in 93 Halomonas spp., were identified using PATRIC 3.6.9 (https://www. patricbrc.org/). PLfams within the genus were computed with MCL inflation = 3.0 to obtain higher sequence simi- larity and better specificity for intra-genus/species close comparisons. Present study is a blue print of ectoine coding genes identified from H. elongata. Genome annotations of existing Halomonas spp., have uncovered existence of some common genes that codes ectoine (s) among mem- bers of the genus Halomonas. Thus, genome-wide eval- uations of ectoine coding genes were assessed. We also analyzed highly close 32 Halomonas spp., with Halo- monas elongata 1H9, which has phylogenetic related ectoine coding child taxa inferred using identified single copy genes. Selecting single copy number genes PLfams of 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidi- necarboxylic acid coding genes among 93 Halomonas spp., were extracted. Common genes coded by Halo- monas species were selected for analysis. The topology of the phylogenetic tree generated using concatenated sequences was compared with the topology of 16S rRNA based Halomonas spp., child taxa tree. RAST genome analysis Complete genome sequences of Ectothiorhodospira haloalkaliphila ATCC 51935 (CP007268), H. elongata 1H9 (NC_014532), Halorhodospira halochloris DSM 1059 (AP017372) and Halorhodospira halophila SL1 (CP000544) analyses done using RAST v2.0 (https://rast. nmpdr.org/) [11]. RAST server is a SEED-based National Microbial Pathogen Database Resource (NMPDR), prokaryotic genome annotation service, to predict sys- tem coverage, subsystem category distribution and sub- system feature count [2]. Complete genome sequences of Ectothiorhodospira haloalkaliphila ATCC 51935 (CP007268), H. elongata 1H9 (NC_014532), Halorhodospira halochloris DSM 1059 (AP017372) and Halorhodospira halophila SL1 (CP000544) analyses done using RAST v2.0 (https://rast. nmpdr.org/) [11]. RAST server is a SEED-based National Microbial Pathogen Database Resource (NMPDR), prokaryotic genome annotation service, to predict sys- tem coverage, subsystem category distribution and sub- system feature count [2]. Novel Universal stress protein in Ectoine TRAP cluster (UspA) and resistance mediated by UspA gene and resistance mediated by UspA gene Studies on genome sequence analyses and analysis of various ectoine coding in Halomonas spp., uncovered that type strains viz. H. aestuarii Hb2 (NZ_CP018139), H. anticariensis DSM 16096 (GCF_000409775), H. azer- baijanica TBZ202 (GCF_004551485), H. bachuensis DX6 (GCA_011742165), H. beimenensis NTU-111 (NZ_ CP021435), H. campisalis SS10-MC5 (NZ_CP065435), H. caseinilytica DSM 18067 (GCF_001662285), H. cerina CECT 7282 (GCF_014192215), H. cup- ida (GCF_900142755), H. daqingensis CGMCC 1.6443 (GCF_900108215), H. denitrificans DSM 18,045 (GCF_003056305), H. endophytica MC28 (GCF_002879615), H. eurihalina MS1 (GCF_008274785), H. gudaonensis (GCF_900100195), H. halmophila NBRC 15537 (GCF_006540005), H. heilongjiangensis 9-2 (GCF_003202165), H. huangheensis BJGMM-B45 (NZ_ CP013106), H. kenyensis DSM 17331 (GCF_013697085), H. korlensis CGMCC 1.6981 (GCF_900116705), H. lac- tosivorans KCTC 52281 (GCF_003254665), H. litope- naei SYSU ZJ2214 (GCF_003045775), H. niordiana ATF 5.4 (GCF_004798965), H. organivorans CECT 5995 (GCF_014192055), H. pacifica (GCF_007989625), H. qijiaojingensis KCTC 22228 (GCF_014651875), H. saliphila LCB169 (GCF_002930105), H. stenoph- ila CECT 7744 (GCF_014192275), H. taeanensis (GCF_900100755), H. urmiana TBZ3 (GCF_005780185), H. ventosae (GCF_004363555), H. xinjiangensis TRM 0175 (GCF_000759345) and H. zincidurans B6 (GCF_000731955) possess superfamily of conserved gene—UspA—suggests that the UspA gene/domain has been inherited from ancient protein family found in primitive bacteria. UspA protein helps Halomonas species provide support and assist Halomonas to function and produce ectoine in the saline environment under stressful conditions like high salt, low water activity and low tem- perature etc. Hence, UspA—stress protein—found in 32 species is a new report in the genus Halomonas. Phylogenetic analysis of 16S rRNA genes in the genus Halomonas H. elongata 1H9 is a bacterium that prefers saline envi- ronment and known for 1,4,5,6-Tetrahydro-2-methyl- 4-pyrimidinecarboxylic acid (ectoine) producer under extreme environmental condition. RAST genome analysis of the H. elongata 1H9 shows that various subsystem feature consists of various path- ways (Additional file 1: Figure S1) coded by bacterium. In addition, member of the genus Halomonas encodes and produce molecular variants of 1,4,5,6-Tetrahydro- 2-methyl-4-pyrimidinecarboxylic acid. Therefore, the diversity of ectoine coding Halomonas might form dis- tinct cluster with a similar kind of Halomonas species. Hence, phylogenetic analysis of 16S rRNA sequences of type strain amongst genus Halomonas revealed that type strains AJ261, 1H9, M8, 5-3, RS-16, AAD6, SS20, 11S, NTU-107, TBZ21, 5CR, F8-11, SL014B-69, TBZ202, KCTC 42685, Z-7009, SL014B-85, CIP 105456, 204, KMM 1376, 10-C-3, Hwa etc., (Additional file 2: Figure S2) formed a discrete clustered together from extracted sequences. This suggests that those spe- cies have a similar gene pool regardless of their genome length were grouped in one cluster. Variation in some branches may occur due to the use of single 16S rRNA genes for phylogenetic analysis. Hence, members of the genus Halomonas might possess similar single-copy ectoine coding genes reveals that apart from the 16S RNA gene. Results TBZ202, DX6, 9-2 and MC28) possessed by species were more or less similar kind of representative species similar to concatenated sequence of 32 Halomonas spe- cies (Fig. 1). It was observed that of the 93 annotated genome sequences, 31 + 1 (32) species have 11 ectoine coding genes (DoeA-DoeC-DoeX-EctC-EctD-EutB- EutC-TeaA-TeaB-TeaC-UspA) as single copy number genes (Additional file 5: Figure S5; Table 1). Heatmap of 11 ectoine coding genes shows a high degree of Pear- son correlation (Fig. 2) value lies between 0.50 and ± 1 (0 = no correlation, 1 = high degree correlation). 128 type strains 16S rRNA genes and 94 Halomonas spp., genomes One hundred twenty-eight 16S rRNA genes of type strains and 94 complete genomes and reference sequences of Halomonas spp., were obtained from LPSN and NCBI genome database deposited during 2006 to 2020. Rekadwad et al. BMC Res Notes (2021) 14:296 Page 3 of 8 Page 3 of 8 Rekadwad et al. BMC Res Notes Identification of protein families, single copy genes and Pearson correlation Whole-genome analyses and annotation have resolved the misery of unique genes distributed among the genus Halomonas spp. The radar chart shows that exist- ing genomic data of Halomonas spp., possesses com- plete genome sequences, reference genomes and some scaffolds (Additional file 3: Figure S3, Additional File 6: Table S1). Available genomic sequence data shows a similar gene pool and all ectoine-coding sequences from 93 type strains not having sets of genes. To resolve this issue and find relevant species in the genus Halo- monas, we, therefore, annotated all genomes and iden- tified the single-copy gene that codes ectoine. It was noticed that few Halomonas species that more than 11 single copy ectoine-coding genes. Therefore, inferred ML tree (Additional file 4: Figure S4) some type strains shows that ectoine biomarker (in 1H9, F9-6, AJ261, SP4, ACAM 71, 62, Hb3, DSM 15,911, N12, NTU- 107, G-16.1, ZJ2214, TBZ3, M29, 79, BJGMM-B45, LCB169, CFH 9008, AIR-2, DQD2-30, 4A, SL014B-69, Rekadwad et al. BMC Res Notes (2021) 14:296 Page 4 of 8 Fig. 1 Maximum-likelihood (ML) analysis of concatenated sequences of 11 genes (DoeA-DoeC-DoeX-EctC-EctD-EutB-EutC-TeaA-TeaB-TeaC-UspA) Fig. 1 Maximum-likelihood (ML) analysis of concatenated sequences of 11 genes (DoeA-DoeC-DoeX-EctC-EctD-EutB-EutC-TeaA-TeaB-TeaC-UspA) from 32 Halomonas species in MEGA X. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) Fig. 1 Maximum-likelihood (ML) analysis of concatenated sequences of 11 genes (DoeA-DoeC-DoeX-EctC-EctD-EutB-EutC-TeaA-TeaB-TeaC-UspA) from 32 Halomonas species in MEGA X. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) Page 5 of 8 Rekadwad et al. Identification of protein families, single copy genes and Pearson correlation BMC Res Notes (2021) 14:296 Table 1 Functions of ectoine-coding genes in the Genus Halomonas under different scenarios Gene symbol Gene description/accepted name EC number KEGG Id (KO) & name Gene name in patric server Comment/involved in pathways & KEGG KO Id DoeA Ectoine hydrolase DoeA 3.5.4.44 – Ectoine hydrolase The enzyme, found in some halophilic bacteria, is involved in the degradation of the compatible solute ectoine The enzyme, which belongs to peptidase family M24, only acts in the direction of ectoine hydrolysis It also produces smaller amounts of (2S)-4-acetamido-2-aminobutanoate, which is recycled back to ectoine by EC 4.2.1.108 DoeC Aspartate-semialdehyde dehydroge‑ nase 1.2.1.11 – Aspartate-semialdehyde dehydroge‑ nase DoeC in ectoine degradation Threonine and Homoserine Biosynthesis Lysine Biosynthesis DAP Pathway, GJO scratch Lysine Biosynthesis DAP Pathway DoeX DNA-binding protein DoeX, ectoine utilization regulator No EC recorded K15782 & Lrp/AsnC family transcrip‑ tional regulator, regulator of ectoine- degradation genes DNA-binding protein DoeX, ectoine utilization regulator Glycine, serine and threonine metabo‑ lism Monobactam biosynthesis Cysteine and methionine metabolism Lysine biosynthesis Metabolic pathways Biosynthesis of secondary metabolites Microbial metabolism in diverse environ‑ ments EctC Ectoine synthase 4.2.1.108 – l-Ectoine synthase (EC 4.2.1.108) Ectoine is an osmoprotectant that is found in halophilic eubacteria This enzyme is part of the ectoine bio‑ synthesis pathway and only acts in the direction of ectoine formation EctD Ectoine hydroxylase 1.14.11.55 – Ectoine hydroxylase The enzyme, found in bacteria, is specific for ectoine Glycine, serine and threonine metabo‑ lism Metabolic pathways EutB Ethanolamine ammonia-lyase large subunit 4.3.1.7 – Ectoine utilization protein EutB, threo‑ nine dehydratase-like Glycerophospholipid metabolism Metabolic pathways EutC Ethanolamine ammonia-lyase small subunit 4.3.1.7 – Ectoine utilization protein EutC, similar to ornithine cyclodeaminase Glycerophospholipid metabolism Metabolic pathways Rekadwad et al. Fig. 2 Heatmap of 11 ectoine coding genes in Halomonas spp., showing genome and protein-pairwise average linkage using Pearson correlation. Ectoine utilization protein EutC, similar to ornithine cyclodeaminase; ICNP: The International Code of Nomenclature of Prokaryotes; LPSN: List of Prokaryotic names with Standing in Nomenclature; MCL: The Markov Cluster Algorithm; ME: Minimum Evolution method; MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms version 10.0; MEGA X: Molecular Evolu‑ tionary Genetics Analysis, Version 10; ML: Maximum-Likelihood method; NCBI: National Center for Biotechnology Information; NJ: Neighbor-Joining method; NMPDR: National Microbial Pathogen Database Resource; PLfams: Genus- specific families; RAST: Rapid Annotation using Subsystem Technology; rRNA: Ribosomal RNA; spp.: Species; TeaA: Ectoine/hydroxyectoine TRAP transporter substrate-binding periplasmic protein TeaA; TeaB: Ectoine/hydroxyectoine TRAP transporter small permease protein TeaB; TeaC: Ectoine/hydroxyectoine TRAP transporter large permease protein TeaC; UspA: Universal stress protein UspA in Ectoine TRAP cluster. Moreover, ectoine or ectoine derivatives investigated by various groups worldwide for their biotechnologi- cal applications. For instance, few reports suggests that ectoine or ectoine derivatives were been in use for oral care, vulvovaginal conditions and in some in cosmetic formulations to protect cell damage and avoid microbial infections. For instance, reports suggest that ectoine and ectoin derivatives in combination with natural essential oil were employed as effective solution against patho- genic Pseudomonas aeruginosa [1] and antifungal resist- ant Candida strains causing candidiasis [4, 5]. Therefore, in biotechnological perspectives ectoine and derivatives of ectoines may have application against antimicrobial resistance and multi-drug resistant microorganisms. Conclusion Ectoine signatures can be found in 93 Halomonas genome sequences that are publicly available. 32 Halo- monas species have 11 separate ectoine genes in a single copy number in their genomes, which help Halomonas spp. produce ectoine under stressful conditions. Based on existing genomic data, it was discovered that H. elon- gata 1H9 has distinct ectoine-producing machinery from other Halomonas species. The existence of 11 dis- tinct genes in 32 species, including the UspA gene, sug- gests that Halomonas species evolved directly from their primitive ancestor, shedding light on their evolutionary significance. Additional file 2: Figure S2. The evolutionary history of Halomonas species was inferred using the Neighbor-Joining method. Analysis using 16S rRNA gene sequences were conducted in MEGA X. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The percentage of repli‑ cate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Additional file 3: Figure S3. RADAR Chart of Genus Halomonas spp. (see supplementary table F1 for names of the species). Yellow circle indicates average genome length of each species and differences in genome length. Additional file 4: Figure S4. Maximum-likelihood analysis among Halomonas species was inferred from 16S rRNA gene sequences in MEGA X. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13104-021-05689-3. The online version contains supplementary material available at https://doi. org/10.1186/s13104-021-05689-3. Additional file 1: Figure S1. RAST genome analysis of H. elongata 1H9 indicates subsystem coverage and distributed subsystem features in annotated genome. Each subsystem feature possesses pathways encoded by respective genes. Additional file 1: Figure S1. RAST genome analysis of H. elongata 1H9 indicates subsystem coverage and distributed subsystem features in annotated genome. Each subsystem feature possesses pathways encoded by respective genes. Additional file 5: Figure S5. Si Additional file 6: Table S1. Supplementary data for Radar Chart. Identification of protein families, single copy genes and Pearson correlation BMC Res Notes (2021) 14:296 Page 6 of 8 Table 1 (continued) Gene symbol Gene description/accepted name EC number KEGG Id (KO) & name Gene name in patric server Comment/involved in pathways & KEGG KO Id TeaA TRAP transporter substrate-binding protein No EC recorded K11688 & C4-dicarboxylate-binding protein (DctP) Ectoine/hydroxyectoine TRAP trans‑ porter substrate-binding periplasmic protein TeaA – TeaB Ectoine/hydroxyectoine TRAP trans‑ porter small permease protein No EC recorded – Ectoine/hydroxyectoine TRAP trans‑ porter small permease protein TeaB – TeaC Ectoine/hydroxyectoine TRAP trans‑ porter large permease protein No EC recorded K11690 C4-dicarboxylate transporter, DctM subunit Ectoine/hydroxyectoine TRAP trans‑ porter large permease protein TeaC – UspA Universal stress protein A No EC recorded Universal stress protein UspA in Ectoine TRAP cluster – Table represents accepted designations and codes ectoine coding genes along with their alternative names and applications/performed reactions in microorganisms Rekadwad et al. BMC Res Notes (2021) 14:296 Rekadwad et al. BMC Res Notes Page 7 of 8 g BR is thankful to editors and the reviewers for their comment to improve manuscript in present form. Limitations A possible restriction would be the presence of biomark- ers other than existing ectoine-coding genes responsible for Halomonas spp. producing 1,4,5,6-Tetrahydro-2-me- thyl-4-pyrimidinecarboxylic acid. Additional file 5: Figure S5. Single copy ectoine coding genes (DoeA- DoeC-DoeX-EctC-EctD-EutB-EutC-TeaA-TeaB-TeaC-UspA) in the genus Halomonas. Numbergiven above each bar indicates number of species coded respective gene. Abbreviations CDS: Coding sequence/the coding region of the gene; DoeA: Ectoine hydrolase; DoeC: Aspartate-semialdehyde dehydrogenase DoeC in ectoine degradation (EC 1.2.1.11); DoeX: DNA-binding protein DoeX, ectoine utiliza‑ tion regulator; EctC: l-Ectoine synthase (EC 4.2.1.108); EctD: Ectoine hydroxy‑ lase; EutB: Ectoine utilization protein EutB, threonine dehydratase-like; EutC: Availability of data and materials 3. Cánovas D, Vargas C, Csonka LN, Ventosa A, Nieto JJ. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline- betaine pathway. J Bacteriol. 1996;178:7221–6. https://doi.org/10.1128/jb. 178.24.7221-7226. 3. Cánovas D, Vargas C, Csonka LN, Ventosa A, Nieto JJ. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline- betaine pathway. J Bacteriol. 1996;178:7221–6. https://doi.org/10.1128/jb. 178.24.7221-7226. Data is available within this manuscript. Sequences were downloaded from NCBI database. Gene bank accession numbers: AB242910, AB680702, AB680891, AB681733, AB681766, AB971837, AF054286, AF211860, AF211861, AF212202, AF212204, AF212206, AF212218, AF251143, AF465604, AJ271864, AJ306893, AJ320530, AJ417388, AJ427627, AJ431369, AJ515365, AJ564880, AJ640133, AJ876733, AM229314, AM229315, AM229316, AM229317, AM238662, AM941388, AY245449, AY268080, AY382579, AY671975, AY858696, AY962236, AY962237, DQ131909, DQ421808, DQ645593, DQ834966, DQ836238, EF117909, EF121853, EF121854, EF144147, EF144148, EF144149, EF421176, EF442769, EF527873, EF613112, EF613113, EF622233, EU085033, EU135707, EU159469, EU218533, EU305728, EU305729, EU373088, EU447162, EU447163, EU541349, EU557315, EU822512, EU909458, FJ429198, FJ984862, GCA_011742165, GCF_000409775, GCF_000759345, GCF_001662285, GCF_002879615, GCF_002930105, GCF_003045775, GCF_003056305, GCF_003202165, GCF_003254665, GCF_004363555, GCF_004551485, GCF_004798965, GCF_005780185, GCF_006540005, GCF_007989625, GCF_008274785, GCF_013697085, GCF_014192055, GCF_014192215, GCF_014192275, GCF_014651875, GCF_900100195, GCF_900100755, GCF_900108215, GCF_900116705, GCF_900142755, GQ232738, GQ281062, GQ354374, GU726750, HE661586, HM026177, HM242216, JF766572, JN242765, JQ716246, JQ762286, JQ762289, JQ781698, JX870002, KC237714, KF010830, KF479230, KF963827, KM066108, KP259554, KP301091, KR024741, KT796562, KU221020, KU320882, KU886576, KX008964, KX090359, KX953854, KY034384, KY034386, KY034408, KY039330, LT223576, LT558840, M93354, M93355, M93358, MF782431, MF850257, MG030686, MH071180, MH071181, MH071182, MK138622, MK346303, MK347065, MK357745, MN099429, MN435603, MT180568, MT372904, MT759855, MT759856, MT759857, MT760065, MT760070, MT760104, MT760115, MT760136, NC_014532, NZ_CP013106, NZ_CP018139, NZ_CP021435, NZ_CP065435, SDSD01000014, X87217, X87218, X92150, X92417, X93493 and X93493 (National Center for Biotechnology Information, U.S. National Library of Medicine, https://www. ncbi.nlm.nih.gov/) [9]. 4. Donadu MG, Peralta-Ruiz Y, Usai D, Maggio F, Molina-Hernandez JB, Rizzo D, Bussu F, Rubino S, Zanetti S, Paparella A, Chaves-Lopez C. Colombian essen‑ tial oil of Ruta graveolens against nosocomial antifungal resistant Candida strains. J Fungi. 2021;7:383. https://doi.org/10.3390/jof7050383. 4. Donadu MG, Peralta-Ruiz Y, Usai D, Maggio F, Molina-Hernandez JB, Rizzo D, Bussu F, Rubino S, Zanetti S, Paparella A, Chaves-Lopez C. Colombian essen‑ tial oil of Ruta graveolens against nosocomial antifungal resistant Candida strains. J Fungi. 2021;7:383. https://doi.org/10.3390/jof7050383. 5. Donadu MG, Usai D, Marchetti M, Usai M, Mazzarello V, Molicotti P, Montesu MA, Delogu G, Zanetti S. Antifungal activity of oils macerates of North Sardinia plants against Candida species isolated from clinical patients with candidiasis. Nat Prod Res. 2019. https://doi.org/10.1080/14786419.2018. 1557175. 6. Galinski EA, Pfeiffer HP, Truper HG. 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidi‑ necarboxylic acid. Eur J Biochem. 1985;149:135–9. https://doi.org/10.1111/j. 1432-1033.1985.tb08903.x. 6. Funding This work was supported by Innovation Group Project Southern Marine Sci‑ ence and Engineering, Guangdong Laboratory (Zhihai), (No.311021006). Availability of data and materials Galinski EA, Pfeiffer HP, Truper HG. 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidi‑ necarboxylic acid. Eur J Biochem. 1985;149:135–9. https://doi.org/10.1111/j. 1432-1033.1985.tb08903.x. 7. LPSN-List of Prokaryotic names with Standing in Nomenclature. https://lpsn. dsmz.de/. Assessed 21 Apr 2021. 7. LPSN-List of Prokaryotic names with Standing in Nomenclature. https://lpsn. dsmz.de/. Assessed 21 Apr 2021. 8. Melmer G, Schwarz T. Ectoines: a new type of compatible solutes with great commercial potential. Extremophiles, vol II. Encyclopedia of Life Support Systems (EOLSS). ISBN: 978-1-905839-93-3 (eBook), ISBN: 978-1-84826-993-4 (Print); 2009. p. 382. 9. NCBI-National Center for Biotechnology Information. https://www.ncbi.nlm. nih.gov/. Assessed 21 Apr 2021. 10. Pastor JM, Salvador M, Argandoña M, Bernal V, Reina-Bueno M, Csonka LN, Iborra JL, Vargas C, Nieto JJ, Cánovas M. Ectoines in cell stress protection: uses and biotechnological production. Biotech Adv. 2010;28:782–801. https://doi.org/10.1016/j.biotechadv.2010.06.005. 10. Pastor JM, Salvador M, Argandoña M, Bernal V, Reina-Bueno M, Csonka LN, Iborra JL, Vargas C, Nieto JJ, Cánovas M. Ectoines in cell stress protection: uses and biotechnological production. Biotech Adv. 2010;28:782–801. https://doi.org/10.1016/j.biotechadv.2010.06.005. 11. RAST-Rapid Annotation using Subsystem Technology, v2.0. https://rast. nmpdr.org/. Assessed 15 Apr 2021. 12. Vargas C, Argandoña M, Reina-Bueno M, Rodríguez-Moya J, Fernández- Aunión C, Nieto JJ. Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance. Aqua Biosyst. 2008;4:14. https://doi.org/10.1186/ 1746-1448-4-14. Competing interests The author declares no competing interests. Declarations Ethics approval and consent to participate Not applicable. Acknowledgements BR i th kf l t dit Acknowledgements BR is thankful to editor BR is thankful to editors and the reviewers for their comment to improve manuscript in present form. Rekadwad et al. BMC Res Notes (2021) 14:296 Page 8 of 8 Page 8 of 8 Rekadwad et al. BMC Res Notes Authors’ contributions 1. Amorese V, Donadu M, Usai D, Sanna A, Milia F, Pisanu F, Molicotti P, Zanetti S, Doria C. In vitro activity of essential oils against Pseudomonas aeruginosa isolated from infected hip implants. J Infect Dev Ctries. 2018;12:996–1001. 1. Amorese V, Donadu M, Usai D, Sanna A, Milia F, Pisanu F, Molicotti P, Zanetti S, Doria C. In vitro activity of essential oils against Pseudomonas aeruginosa isolated from infected hip implants. J Infect Dev Ctries. 2018;12:996–1001. BR conceived the original idea, collected the data, analyses, prepared figures, writing of entire manuscript. BR, WJL and RPD have revised and critically evalu‑ ated the drafts. All authors read and approved the final manuscript. 2. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA 3rd, Stevens R, Vonstein V, Wattam AR, Xia F. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 2015;5:8365. https://doi.org/10.1038/srep0 8365. 2. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA 3rd, Stevens R, Vonstein V, Wattam AR, Xia F. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 2015;5:8365. https://doi.org/10.1038/srep0 8365. 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https://openalex.org/W2006612250	https://europepmc.org/articles/pmc3702353?pdf=render	English	null	IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa	Journal of health, population and nutrition	2,013	cc-by	6,860	IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa Ezekiel Green1,2, Lawrence C. Obi3, Anthony I. Okoh2, Maphoshane Nchabeleng4, Ba Villiers4, Tomas Letsoalo4, Anwar A. Hoosen5, Pascal O. Bessong1,6, Roland N. N Ezekiel Green1,2, Lawrence C. Obi3, Anthony I. Okoh2, Maphoshane Nchabeleng4, Babsie E. de Villiers4, Tomas Letsoalo4, Anwar A. Hoosen5, Pascal O. Bessong1,6, Roland N. Ndip2,7 1School of Mathematics and Natural Sciences, Department of Microbiology, University of Venda, Private Bag X5050, Thohoyandou 0960, South Africa; 2Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa; 3Division of Academic Affairs, University of Fort Hare, Alice 5700, South Africa; 4Department of Microbiological Pathology, NHLS/University of Limpopo, Medunsa campus, Pretoria, South Africa; 5Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria/NHLS, Pretoria, South Africa; 6AIDS Virus Research Laboratory, Department of Microbiology, University of Venda, South Africa; 7Department of Microbiology and Parasitology, University of Buea, Box 63, Buea, Cameroon Ezekiel Green1,2, Lawrence C. Obi3, Anthony I. Okoh2, Maphoshane Nchabeleng4, Babsie E. de Villiers4, Tomas Letsoalo4, Anwar A. Hoosen5, Pascal O. Bessong1,6, Roland N. Ndip2,7 1School of Mathematics and Natural Sciences, Department of Microbiology, University of Venda, Private Bag X5050, Thohoyandou 0960 South Africa; 2Department of Biochemistry and Microbiology Faculty of Science and Agriculture Ezekiel Green1,2, Lawrence C. Obi3, Anthony I. Okoh2, Maphoshane Nchabeleng4, Babsie E. de Villiers4, Tomas Letsoalo4, Anwar A. Hoosen5, Pascal O. Bessong1,6, Roland N. Ndip2,7 1School of Mathematics and Natural Sciences, Department of Microbiology, University of Venda, Private Bag X5050, Thohoyandou 0960, South Africa; 2Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa; 3Division of Academic Affairs, University of Fort Hare, Alice 5700, South Africa; 4Department of Microbiological Pathology, NHLS/University of Limpopo, Medunsa campus, Pretoria, South Africa; 5Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria/NHLS, Pretoria, South Africa; 6AIDS Virus Research Laboratory, Department of Microbiology, University of Venda, South Africa; 7Department of Microbiology and Parasitology, University of Buea, Box 63, Buea, Cameroon ABSTRACT Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cas- es, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tu- berculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological inde- pendence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22=30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa. Key words: Drug resistance; Epidemiology; IS6110; M. tuberculosis; PCR-RFLP; South Africa J HEALTH POPUL NUTR 2013 Mar;31(1):1-10 ISSN 1606-0997 \| $ 5.00+0.20 J HEALTH POPUL NUTR 2013 Mar;31(1):1-10 ISSN 1606-0997 \| $ 5.00+0.20 ©INTERNATIONAL CENTRE FOR DIARRHOEAL DISEASE RESEARCH, BANGLADESH INTRODUCTION Gauteng, and North West (20) province, similar in- formation has not been recorded for DR-TB isolates from Gauteng, Limpopo, and Mpumalanga prov- inces. There is, therefore, a paucity of information on epidemiological data based on molecular meth- ods addressing the transmission routes of DR-TB strains. This merits attention considering that the country has a high prevalence of HIV/AIDS, a con- founding factor for TB resurgence. Gauteng, and North West (20) province, similar in- formation has not been recorded for DR-TB isolates from Gauteng, Limpopo, and Mpumalanga prov- inces. There is, therefore, a paucity of information on epidemiological data based on molecular meth- ods addressing the transmission routes of DR-TB strains. This merits attention considering that the country has a high prevalence of HIV/AIDS, a con- founding factor for TB resurgence. The aim of this study was to determine the genetic diversity of resistant MTB isolates, using IS6110 to delineate the dissemination of major phylogenetic clades of the organism in Gauteng, Limpopo, and Mpumalanga regions of South Africa. INTRODUCTION (2). South Africa is a country with a high inci- dence of TB—600 cases per 100,000 population in 2005 (3), 550 cases per 100,000 population in 2003, and 718 cases per 100,000 population in 2004 (4,5). During the study period, the cases of drug resistance increased from 156 per 100,000 in 2004 to 177 per 100,000 in 2006 in Mpumalanga, and from 58 to 84 per 100,000 in Limpopo. However, Gauteng showed a high number of drug-resistant cases with an increase from 662 in 2004 to 794 per 100,000 in 2006 (3). Tuberculosis (TB) remains a deadly infectious dis- ease affecting millions of people worldwide; 95% of TB cases, with 98% of deaths, occurr in develop- ing countries (1). Approximately one-third of the world’s population is infected with tuberculosis, and 2 million people die of the disease every year Correspondence and reprint requests: Prof. R.N. Ndip Department of Biochemistry and Microbiology University of Fort Hare Private bag X1314, Alice 5700 South Africa Email: rndip@ufh.ac.za; ndip3@yahoo.com Fax: +27(0) 406 653 1730 World Health Organization recommends standard- ized TB treatment regimens based on short-course chemotherapy. The anti-TB drug regimen recom- mended for the treatment of new cases consists of Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. a two-month administration of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and etham- butol (EMB), followed by a continuation phase of INH/RIF and/or EMB for four months (2). Howev- er, this treatment is usually effective against MTB strains that have never been exposed to anti-TB drugs for more than 30 days (6) and against strains that do not possess drug-resistant mechanisms. For many years, Direct Observation of Treatment (DOT) has been promoted by the World Health Organization (WHO) as one of the five compo- nents of a wider strategy called Directly Observed Treatment, Short Course (DOTS) to tackle the re- surgence of TB throughout the world (7). Direct observation by health workers while patients take their tablets aims to improve adherence to therapy and completion of treatment. Specific therapy for patients with drug-resistant tuberculosis is included in DOTS-plus. A surge in drug-resistant tuberculosis in several parts of the world requires effective im- plementation of the DOTS-plus strategy to prevent the occurrence of new multidrug-resistant (MDR) TB cases and to reduce transmission of MTB. Patients’ characteristics Patients’ characteristics The samples used were routinely collected from patients by hospitals and sent to DGM Hospital for TB diagnosis. Data on the patients, concerning type of specimens and standard demographic in- formation (gender, age, and province), were regis- tered. The study was approved by the Research and Ethics Committee of the University of Venda, South Africa. Informed consent was not obtained from the subjects. Analysis of the spread and transmission of DR- TB strains, using molecular methods, has been reported (13). Techniques, such as restriction frag- ment length polymorphism (RFLP) and insertion sequence 6110 (IS6110), have been used in reliably differentiating M. tuberculosis isolates (14,15), and IS6110 has been suggested as a standard tool for characterization of isolates. IS6110 fingerprinting has also been used successfully to confirm labora- tory cross-contaminations and to trace small-scale outbreak of TB and DR-TB in a large variety of set- tings (16). Although research on MTB transmission, using molecular techniques, has been conducted in the Western Cape (17), Kwazulu-Natal (18,19), Study location and population The present study was conducted in the TB Labo- ratory of the DGM Hospital, Gauteng province of South Africa. This province showed a TB preva- lence rate of 500/100,000 population in 2006 (21). From January 2004 to December 2006, sputum specimens of all patients ((n=21,913) routinely sent for TB analysis to microbiology laboratory of DGM Hospital were included in the study. During the study period, we were aware of only three prov- inces that had TB diagnostic laboratories, which included Gauteng, Western Cape, and Kwazulu- Natal. We concentrated on DGM Hospital since it received specimens from other provinces, includ- ing Limpopo and Mpumalanga, without diagnos- tic centres. Resistance to antibiotics in MTB occurs due to ge- nomic mutations in certain genes, such as katG for INH resistance and rpoB for RIF resistance (8). Therefore, MTB will benefit from increased muta- tion rate. Unlike other pathogens with MDR patho- types, such as transposable elements, plasmid- mediated mechanisms of resistance have not been reported in MTB (9,10). In recent years, treatment of TB has become com- plicated by increasing emergence of drug-resistant M. tuberculosis (DR-TB) (11). The proportion of MDR-TB strains in South Africa rose from 1.1% in 2004 to 1.9% in 2006 (3). Extremely drug-resistant (XDR) TB strains have also been reported in the country (12). Extraction of DNA We restricted the analysis to the isolates with 5 or more bands because isolates with few or no copies of the IS6110 element cannot be reliably classified by this method (26). IS6110 fingerprints were ana- lyzed visually as described earlier (27). Recent trans- mission was considered likely if an isolate matched at least one other by identical or near-identical cri- teria. ‘Identical’ isolates were characterized by equal number of bands on gel electrophoresis, following digestion by restriction endonuclease; all such bands had to have matching molecular weights. ‘Near-identical’ isolates were characterized by dif- ference of a single band (addition or loss of a single band). RFLP patterns were grouped from 1 to 20 based on the number of IS6110 band. A cluster was defined as a group of two or more patients with drug-resistant MTB strains whose fingerprints were identical with respect to both number and the size of all bands. We used the n-1 method to estimate continuing transmission. The method is based on the assumption that one case per cluster is due to reactivation and that this ‘index’ infectious case gives rise to other cases in the cluster either by in- fecting them directly or infecting a secondary case that then infects other members of the cluster. It calculates the number of recently-transmitted cases by summing within clusters after reducing each cluster-size by one giving the transmission dynam- ics (28). It is calculated by the following formula: The MGIT 960 cultures were heat-inactivated at 80 °C for 1 hour before DNA extraction was per- formed in a biosafety level 2 laminar flow cabinet as previously described (22,24). Briefly, growth from the MGIT 960 system was suspended in 6 mL of DNA extraction buffer (5% monosodium gluta- mate, 50 mM Tris-HCl, pH 7.0 and 25 mM EDTA) in a sterile 50-mL polypropylene tube which con- tained approximately thirty 5-mm glass balls. The bacterial clumps were disrupted by vigorous shaking and vortexing. Five hundred microlitre of lysozyme (Amersham Biosciences, Greece) and 10 μL of RNAseA (Amersham Biosciences, Greece) were added to the tube. The contents of the tube were mixed by gentle inversion and then incubat- ed at 37 °C for 2 hours. After incubation, 600 μL of 10×Proteinase K buffer and 150 μL of Proteinase K (Amersham Biosciences, Greece) were added. The sample was gently mixed (inverting the tube a few times) and then incubated overnight at 45 °C. Bacterial isolates All specimens were processed and cultured in the BacT/Alert 3D (BioMérieux, Durham, NC, USA) system. Positive cultures were stained with Ziehl Neelsen and confirmed with the AccuProbe DNA hybridization assay (Gen-Probe, USA) according to the manufacturer’s instructions. Isolates were then advanced for susceptibility testing and se- quencing of katG and rpoB genes (22,23). The JHPN JHPN 2 Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. isolates of MTB analyzed in this study represent all available isolates obtained from patients of Limpopo, Mpumalanga, and Gauteng province, attending DGM Hospital between January 2004 and December 2006. The TB laboratory of DGM Hospital serves as one of the major TB laborato- ries situated in Gauteng province of South Africa. All the isolates were examined for their suscep- tibility to isoniazid (INH), ethambutol (EMB), streptomycin (SM), and rifampicin (RIF) follow- ing instructions of MGIT 960 system (MGITs; Becton Dickinson Microbiology systems, Sparks, MD, USA). Three hundred thirty-eight resistant strains of M. tuberculosis were obtained from 21,913 patients between January 2004 and De- cember 2006. Of all the resistant strains obtained, 97 (28.7%) were from Gauteng, 32 (9.5%) from Limpopo, and 209 (61.8%) from Mpumalanga province. The provinces share borders with one another, with Mpumalanga situated in the Southeast of Limpopo and East of Gauteng, and Limpopo in the North of Gauteng. Typing of M. tuberculosis strains, using IS6110 isolates of MTB analyzed in this study represent all available isolates obtained from patients of Limpopo, Mpumalanga, and Gauteng province, attending DGM Hospital between January 2004 and December 2006. The TB laboratory of DGM Hospital serves as one of the major TB laborato- ries situated in Gauteng province of South Africa. DNA was extracted using the phenol/chloroform method as described earlier (24,25). RFLP was per- formed using the standardized IS6110 technique as described previously (15). The extracted genomic DNA was restricted with Pvu II (20) in a reaction mix (final volume 30 mL) consisting of 3 μg of genomic DNA, and 15 units of Pvu II in 3 μL of the prescribed restriction buffer (Amersham biosci- ences, Greece). The restriction mix was incubated overnight (±16 h) at 37 °C. At the end of digestion, the reaction was incubated at 65 °C for 10 minutes to inactivate any remaining enzyme activity. Re- stricted products were resolved on a 0.8% agarose at constant voltage of 40 in 1X TBE buffer for 24 hours. Bacterial isolates The probe used for hybridization was gener- ated by PCR, using primers INS1 (5 CGT GAG GGC ATC GAG GTG GC 3) and INS2 (5/ GCG TAG GCG TCG GTG ACA AA 3/) labelled with horseradish peroxidase (ECLtm direct nucleic acid labelling and detection kit, Amersham, UK); hybridization and detection were carried out as per manufacturer’s instructions. All the isolates were examined for their suscep- tibility to isoniazid (INH), ethambutol (EMB), streptomycin (SM), and rifampicin (RIF) follow- ing instructions of MGIT 960 system (MGITs; Becton Dickinson Microbiology systems, Sparks, MD, USA). Three hundred thirty-eight resistant strains of M. tuberculosis were obtained from 21,913 patients between January 2004 and De- cember 2006. Of all the resistant strains obtained, 97 (28.7%) were from Gauteng, 32 (9.5%) from Limpopo, and 209 (61.8%) from Mpumalanga province. The provinces share borders with one another, with Mpumalanga situated in the Southeast of Limpopo and East of Gauteng, and Limpopo in the North of Gauteng. Total number of strains in clusters [Number of clusters]/ Total number of strains Extraction of DNA Proteins were removed by phenol/chloroform and chloroform/isoamyl-alcohol extraction meth- od (25). DNA was then precipitated with the addi- tion of 600 μL of 3 M sodium acetate (pH 5.5) and 7 mL of cold (-20 °C) isopropanol. The precipitated DNA was washed with 1 mL of 70% ethanol for approximately 1 minute. DNA was then air-dried and resuspended in 30 µL in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Volume 31 \| Number 1 \| March 2013 3 Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. Bacterial isolates Bacterial isolates Epidemiological investigation of fingerprint groups Epidemiological investigation of fingerprint groups A total of 1,648/21,913 (7.5%) MTB isolates were obtained from sputum specimens cultured during the study period. Furthermore, 338/1,648 (20.5%) resistant strains of MTB from the 1,648 culture- positive strains were obtained. Of these, 97/338 (28.7%) were from Gauteng, 32/338 (9.5%) from Limpopo, and 209/338 (61.8%) from Mpumalanga province. Of the isolates, 33 (63.5%) were MDR, 6 (11.5%) resistant to two drugs, 2 (3.8%) resistant to three drugs, and 11 (21.1%) resistant to one drug. The largest cluster comprised seven patients with drug-resistant TB strains, of which 4 were from Mpumalanga, 2 from Gauteng, and 1 from Lim- popo. Five of these patients were male, and two were female. The second cluster was made up of 4 DR-TB strains: two each were isolated from patients who were from Mpumalanga and Limpopo prov- ince. One cluster harboured three DR-TB strains isolated from male patients, of whom two were from Mpumalanga and one from Gauteng. The re- maining 19 clusters comprised two DR-TB strains isolated from patients who were from Gauteng, Mpumalanga and Limpopo province. The ages of the patients ranged from 10 to 69 years (Table). RESULTS Study location and patients’ characteristics made up of 52 (15.7%) strains were observed (Ta- ble). Thirty-two stains (61.5%) of the clustered 52 were from Mpumalanga, 16/52 (30.8%) from Gau- teng, and 4/52 (7.7%) from Limpopo province. The minimum estimate for the proportion of re- sistant TB that was due to transmission is 9.06% (52-22=30/331)x100 while the diversity was 90.9% (279+22/331)x100 as previously defined (29). DGM Hospital is a 1,200-bed tertiary referral hospi- tal attached to the Medunsa Campus of the Univer- sity of Limpopo. HIV status was confirmed for only 64 patients from the hospital-records. IS6110 RFLP clustering The copy number of IS6110 in each of the isolates was determined from the number of bands hybrid- izing with the probe, and the copies varied from 1 to 24. Seven of the 338 strains (2.07%) had less than 5 bands. Most strains (331/338, 97.9%) car- ried 5 or more copies of IS6110 (Figure 1 and 2). Of the 331 strains, 279 (84.3%) had unique RFLP patterns. Twenty-two clusters (designated A-V) Relationship of cluster and resistance The drug-resistance patterns of the strains clustered in different fingerprint groups were not similar. The Figure 1. Frequency of MTB isolates from male patients with different copy numbers of the IS6110 elements Gauteng Limpopo Mpumalanga 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Number of isolates Number of bands 1 2 3 4 5 6 7 8 9 101112131415161720212224 Figure 1. Frequency of MTB isolates from male patients with different copy numbers of the IS6110 elements Province JHPN 4 Gauteng Limpopo Mpumalanga 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Number of isolates Number of bands 1 2 3 4 5 6 7 8 9 101112131415161720212224 Figure 1. Frequency of MTB isolates from male patients with different copy numbers of the IS6110 elements Province Gauteng Limpopo Mpumalanga Province Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. Green E et al. Figure 2. Frequency of MTB isolates from female patients with different copy numbers of the IS6110 elements Gauteng Limpopo Mpumalanga Number of isolates Number of bands 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 4 5 6 7 8 9 10 11 12 13 14 15 16 17 21 22 25 Province Number of bands highest number of bands corresponding to 20 was noted for cluster D while the lowest made up of 5 bands occurred in strains from cluster O and P. However, band-sizes of 7, 9, 10, 11, 12, 14, 15, 18, and 19 were also noted. There were differences in the sizes of the individual bands (Figure 3). PCR and sequencing PCR and sequencing IS6110 RFLP clustering 315 in 90.1% (184/204) of the resistant isolates, of which 125/184 (67.9%) had a codon change from AGC to ACC, 38/184 (20.6%) had a change from AGC to AAC, and 21/184 (11.4%) changed from AGC to ACA. A mutation at codon 314 (ACC to CCC) of 20/204 isolates contributed 9.8% of INH resistance (Table). DISCUSSION Sequencing of the PCR products on the rpoB gene hotspot region showed nucleotide substitution at codon 516 (GAC to GTC) in 44.7% (72/161) and codon 526 causing a codon change at CAC to GTC in 50.3% (81/161) of 47.6% (161/338) RIF-resistant isolates studied. One isolate (0.6%, 1/161) showed the mutation in codon 512 AGC to ACC, 515 ATG to CAT, 516 GAC to GTC, 517 CAG to CCA, 518 AAC to GAA, 519 AAC to CAA, 525 ACC to CAC, 526 CAC to GAC, 529 CGA to CCC, 530 CTG to CCG, 531 TCG to TTG, 532 GCG to CGG, and 533 CTG to CGC in the rpoB region. Overall, 17 differ- ent mutations were observed: 29.4% (5/17) single, 64.7% (11/17) double, and 5.9% (1/17) triple muta- tions. IS6110-based DNA fingerprinting of MTB has prov- en to be highly effective in detecting the source of infection and route of transmission and in simpli- fying diagnosis of the outbreak (30). In this study, we utilized IS6110 to determine the genetic diver- sity of drug-resistant MTB isolates from the DGM Hospital in South Africa. Of the 338 resistant strains (209 from Mpuma- langa, 32 from Limpopo, and 97 from Gauteng), 7 (2.07%) showed fewer than 5 copies of IS6110. Similar results (2.39%) were obtained in isolates from Germany (31), 5% in isolates from Rio de Ja- neiro, Brazil (15), and 8% strains from East Azer- baijan, Iran (32). However, the number of strains with low-copy number of IS6110 in our study is lower (2.07%) than that observed in regions, such as Poland-12% (33), Delhi, India-18.3% (34), and In total, 60.3% (204/338) MTB isolates were resis- tant to INH. Sequencing of the INH-resistant iso- lates showed a nucleotide substitution at codon Volume 31 \| Number 1 \| March 2013 5 Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. Green E et al. JHPN Table. Fifty-two drug-resistant M. DISCUSSION tuberculosis JHPN Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. Green E et al. Table—contd. Group Gender Age Drug resistance pattern katG rpoB Province R Female Male 18 60 INH, RIF INH, RIF, EMB Thr315 Thr315 Asp530 Arg532 Gauteng Mpumalanga S Male Female 31 45 INH, SM INH Asn315 Thr315 His515 Val516, Gln518, Glu519 Mpumalanga Gauteng T Male Male 37 31 INH, RIF, SM INH, RIF Thr315 Asp315 Asp526, Arg532, Arg533 Val516, Asp526 Mpumalanga Mpumalanga U Male Male 29 37 INH, RIF, EMB, SM INH, SM Pro314 Thr315 Asp526, Gln518 Pro529 Gauteng Gauteng V Female Female 21 34 INH, RIF, EMB, SM INH, EMB Thr315 Thr315 Arg532 WT Mpumalanga Mpumalanga Figure 3. Representative examples of IS6110 RFLP patterns associated with 22 high-copy clusters (A-V). The number of strains in each cluster is shown at the bottom of each pattern. The last lane shows reference strain Figure 3. Representative examples of IS6110 RFLP patterns associated with 22 high-copy clusters (A-V). The number of strains in each cluster is shown at the bottom of each pattern. The last lane shows reference strain 7 4 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A B C D E F G H I J K L M N O P Q R S T U V STD Number of strains Cluster type 7 4 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A B C D E F G H I J K L M N O P Q R S T U V STD Number of strains Cluster type Germany-11.9% (29). Many reports on epidemio- logical studies of TB use a secondary typing to in- crease the accuracy of indicating epidemiological links. In the case of IS6110-RFLP patterns, this is considered necessary, mainly when dealing with MTB populations presenting a high proportion of strains with low-copy number (35). In light of these findings, IS6110 can be used without additional typing markers for this area. that tuberculosis transmission in this region may be described as a micro-epidemic. In the past two de- cades, the rare occurrence of drug-resistant strains was ascribed to the conviction that drug-resistant MTB strains were less virulent (37). DISCUSSION tuberculosis grouped into 22 IS6110 clusters Group Gender Age Drug resistance pattern katG rpoB Province A Male Male Female Female Male Male Male 33 25 35 26 47 29 30 INH, RIF INH INH, RIF, EMB, SM INH, RIF, EMB, SM EMB INH, RIF, EMB, SM INH, RIF, EMB Thr315 Thr315 Arg315 Arg315 WT Asn315 Thr315 Thr512, Val516 WT Gly516, Asp526 Gly516, Asp526, Tyr522 WT His525, Tyr522 His525 Mpumalanga Mpumalanga Mpumalanga Mpumalanga Gauteng Gauteng Limpopo B Male Male Male Female 53 26 51 34 SM INH, RIF, EMB, SM INH INH, RIF WT Thr315 Ile315 Ile315 WT Thr512, Asp526, Tyr522 WT Thr512, Asp526, Arg532 Mpumalanga Mpumalanga Limpopo Limpopo C Male Male Male 18 60 29 INH, RIF, EMB, SM INH, RIF, EMB INH, RIF, SM Thr315 Arg315 Thr315 Gln518 Asp526 Thr512, His515, Val516 Mpumalanga Mpumalanga Gauteng D Male Male 37 54 INH, RIF, SM INH, RIF, EMB, SM Arg315 Pro314,Thr315 Thr512, His515, Val516 Gly516 Mpumalanga Mpumalanga E Female Female 18 69 INH, RIF, SM SM Thr315 WT Thr512, Val516 WT Gauteng Limpopo F Male Male 41 44 INH, RIF, EMB, SM INH Arg315 Arg315 Thr512, Asp526, Arg532 WT Limpopo Mpumalanga G Male Female 35 37 SM INH, SM WT Thr315 WT WT Mpumalanga Gauteng H Male Male 44 33 INH, SM INH, RIF, EMB, SM Thr315 Pro314 WT Gly516, Arg532 Gauteng Mpumalanga I Female Male 22 29 INH, RIF, EMB INH Thr315 Thr315 Arg533 WT Gauteng Mpumalanga J Male Male 33 42 INH, RIF, EMB, SM INH, RIF Thr315 Pro314 Thr512, Asp526, Arg532, Gly516, Arg533 Mpumalanga Mpumalanga K Female Male 32 10 INH INH, RIF Pro314 Thr315 WT Pro529 Mpumalanga Gauteng L Male Male 50 31 INH INH, RIF Thr315 Pro314 WT Thr512, Arg532, Gln518 Mpumalanga Mpumalanga M Male Male 21 41 INH, RIF, EMB, SM INH, EMB, SM Asn315 Thr315 Glu519 Pro529, Glu519 Mpumalanga Mpumalanga N Male Male 34 26 EMB, SM INH, RIF, EMB, SM WT Arg315 WT Gln518, Cys531 Gauteng Mpumalanga O Male Female 33 50 INH, RIF, EMB, SM INH, RIF Thr315 Thr315 Pro530, Tyr522 Thr512, Pro529, Glu519 Mpumalanga Mpumalanga P Male Male 54 30 INH, RIF, SM INH, EMB, SM Pro314 Thr315 Pro529, Glu519 Gln518, Cys531 Mpumalanga Mpumalanga Q Male Male 33 28 INH, RIF, EMB, INH, RIF, EMB, SM Thr315 Thr315 Tyr522 Gly516, Cys531 Mpumalanga Mpumalanga Contd. Table. Fifty-two drug-resistant M. tuberculosis grouped into 22 IS6110 clusters Table. Fifty-two drug-resistant M. DISCUSSION However, stud- ies have shown that drug-resistant strains do not differ from drug-sensitive strains in their ability to create infection or disease, and that drug-resistant strains contribute substantially to the increase of tuberculosis (38-41). Recent population-based studies have shown that patients with MTB strains showing identical IS6110 fingerprint patterns are likely to have become in- fected recently (36). In our study, 22 fingerprint groups with identical patterns were found. Most of these patterns consisted of 2 patients, indicating Mpumalanga is one of the poorest provinces in South Africa, and people living in the province are of low socioeconomic status (42). MDR-TB cases of 1.99% were reported in the province in 2004 (3). The cases increased to 2.64% in 2006 and decreased 7 Volume 31 \| Number 1 \| March 2013 Typing of drug-resistant M. tuberculosis strains, using RFLP Green E et al. drastically to 1.17% in April 2007. This is highly worrisome as it may mean that many patients with MDR-TB are not being diagnosed. This region might be a risk-zone because most (65.4%) of the clustered isolates were obtained from this zone compared to the other investigated provinces. This corroborates previous works in which the region was defined as a risk-zone for tuberculosis clustering (43). However, clustering could have been overestimated as more samples analyzed (61.8%) were from Mpumalanga than from Gauteng and Limpopo while other prov- inces were not investigated. Gauteng is the richest province in South Africa (44). This province showed an incidence rate of 500/100,000 in 2006 compared to 722/100,000 for overall South Africa. which emanated from the clusters and the time it takes for MTB to grow (6-8 weeks); a study using a larger population should be performed to evaluate the real clustering rate. One of the risk factors identified in our study was the male gender. Of the 52 clustered strains, 39 (75%) were from the male gender. A study in Paris showed the same results (45). However, a study in Iran showed more clustering in females than in males (30). Although few epidemiological links were confirmed in this study, demographic and behavioural risk factors, such as body mass index, alcohol consumption, and smoking, have been identified as the causes of tuberculosis clustering elsewhere (46). Conclusions In our study, strains that clustered demonstrated resistance to the same antitubercular drug with or without resistance to additional drugs. The likeli- hood is that an additional resistance was acquired after transmission occurred. The close relationship of the strains clustered in different groups (e.g. A, O, and P) was further confirmed by the overall good association between IS6110 and data on drug resis- tance. This report of clustered strains further con- firms that drug-resistant MTB isolates can be trans- mitted. Furthermore, it should be noted that the study was performed using isolates obtained from one hospital and because of disease transmission Our results indicate that transmission of drug- resistant strains may contribute to the emergence of drug-resistant tuberculosis in South Africa—a finding with profound clinical and epidemiologi- cal significance. DISCUSSION This is understandable as smok- ing and alcohol consumption may be proxies for frequenting locations that puts one at risk of close contact with infectious individuals, such as neigh- borhood bar (46). In a study done in the same laboratory, Rampe et al. (20) reported a total number of clustering of 0.47, indicating a transmission rate of 47%. How- ever, that study focused on isolates from the loca- tions surrounding DGM Hospital (previously called Ga-Rankuwa Hospital), and the study had a lower sample-size. We observed a 52-22=30/331 (0.096) clustering among resistant M. tuberculosis isolates, indicating recent transmission rate of about 9%. We also observed that, besides failures of tuberculosis treatment, transmission of drug-resistant tubercu- losis contributes to the problem of drug resistance in South Africa. At least 91% of the problem of drug resistance in this population could be the product of acquired resistance. However, in the absence of clinical data (retreatment or primary resistance) on the patients from whom these strains were isolat- ed, it is difficult to conclude whether they repre- sent fresh cases or cases of reactivation. In a mixed population, the degree of DNA polymorphism is greater (15)—a situation common in South Africa. In South Africa, people also travel to other prov- inces looking for jobs, which may introduce new strains into otherwise unaffected areas, leading to high genetic diversity. Being a pro-drug that requires activation by cata- lase peroxidase enzyme produced by MTB, INH plays an important role in treating latent MTB infection (LTBI), for prevention of active dis- ease and the subsequent TB transmission. It is also a cornerstone of the modern short-course chemotherapy for tuberculosis. Mutation in the gene producing this enzyme will render the or- ganism resistant to INH. Codon 315 mutations were shown to be associated with high-level re- sistance to INH. Results obtained from this study suggest that serine substitution at codon 315 of the katG gene (57.7%) is a characteristic of local INH-resistant strains and, therefore, can serve as a genetic marker for INH-resistance in our region. The high proportion of katG 315Thr-resistant isolates in this study had mutation in the rpoB gene (46%), which was related to RIF resistance reported in South Africa (21). REFERENCES 1. World Health Organization. Guidelines for surveil- lance of drug resistance in tuberculosis. 4th ed. Gene- va: World Health Organization, 2009. 83 p. (WHO/ HTM/TB/2009.422). 14. Fandinho FC, Kritski AL, Hofer C, Júnior Conde H, Ferreira RM, Saad MH et al. RFLP patterns and risk factors for recent tuberculosis transmission among hospitalized tuberculosis patients in Rio de Janeiro, Brazil. Trans R Soc Trop Med Hyg 2000;94:271-5. 2. World Health Organization. Global tuberculosis con- trol: surveillance, planning, financing; WHO report 2007. 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Díaz R, Gómez RI, García N, Valdivia JA, van Soolin- gen D. Molecular epidemiological study on trans- mission of tuberculosis in a hospital for mentally handicapped patients in Havana, Cuba. J Hosp Infect 2001;49:30-6. 42. Verver S, Warren RM, Munch Z, Vinnycky E, van Helden PD, Richardson M et al. Transmission of tu- berculosis in a high incidence urban community in South Africa. Int J Epidemiol 2004;33:351-7. 31. Niemann S, Rüsch-Gerdes S, Richter E. REFERENCES Silva MSN, Senna SG, Ribeiro MO, Valim ARM, Telles MA, Kritski A et al. Mutations in katG, inhA, and ahpC genes of Brazilian isoniazid-resistant iso- lates of Mycobacterium tuberculosis. J Clin Microbiol 2003;41:4471-4. 20. Rampe AK, Hess A, Clay CK, Du Toit D. Character- ization of Mycobacterium tuberculosis strains isolated at Ga-Rankuwa Hospital by fingerprinting with in- sertion sequence IS6110. South Afr J Epidemiol Infect 2004;19:96-100. 9. 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https://openalex.org/W3042622432	https://orbit.dtu.dk/files/216851664/wevj_11_00048_published.pdf	English	null	Profitability of Frequency Regulation by Electric Vehicles in Denmark and Japan Considering Battery Degradation Costs	World electric vehicle journal	2,020	cc-by	8,316	Citation (APA): Calearo, L., & Marinelli, M. (2020). Profitability of Frequency Regulation by Electric Vehicles in Denmark and Japan Considering Battery Degradation Costs. World Electric Vehicle Journal, 11(3), Article 48. https://doi.org/10.3390/wevj11030048 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal 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. Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Profitability of Frequency Regulation by Electric Vehicles in Denmark and Japan Considering Battery Degradation Costs Calearo, Lisa; Marinelli, Mattia Published in: World Electric Vehicle Journal Document Version Publisher's PDF, also known as Version of record General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research Received: 15 May 2020; Accepted: 14 July 2020; Published: 16 July 2020 Abstract: This paper determines the proﬁtability of the primary frequency regulation (FR) service considering the wear of the electric vehicle (EV) battery as a cost. To evaluate the proﬁtability of the FR service, the cost of degradation from FR provision is separated from the degradation caused by driving usage. During FR, the power response is proportional to the frequency deviation with full activation power of 9.2 kW, when deviations are larger than 100 mHz. The degradation due to FR is found to be an additional 1–2% to the 7–12% capacity reduction of a 40 kWh Lithium-ion NMC battery pack over 5 years. The overall economic framework is applied in Denmark, both DK1 and DK2, and Japan, by considering historical frequencies. The DK2 FR market framework is taken as reference also for the Japanese and the DK1 cases. Electricity prices and charger efﬁciency are the two main parameters that affect the proﬁtability of the service. Indeed, with domestic prices there is no proﬁtability, whereas with industrial prices, despite differences between the frequencies, the service is similarly proﬁtable with approx. 3500¤ for a ﬁve-year period. Keywords: degradation; electric vehicle; frequency regulation; vehicle-to-grid World Electric Vehicle Journal 2020, 11, 48; doi:10.3390/wevj11030048 Link back to DTU Orbit Link back to DTU Orbit Citation (APA): Calearo, L., & Marinelli, M. (2020). Profitability of Frequency Regulation by Electric Vehicles in Denmark and Japan Considering Battery Degradation Costs. World Electric Vehicle Journal, 11(3), Article 48. https://doi.org/10.3390/wevj11030048 quency Regulation by Electric rk and Japan Considering Battery nelli , Department of Electrical Engineering, Technical University of Denmark, rk; matm@elektro.dtu.dk dk July 2020; Published: 16 July 2020 the proﬁtability of the primary frequency regulation (FR) service ic vehicle (EV) battery as a cost. To evaluate the proﬁtability of the n from FR provision is separated from the degradation caused by wer response is proportional to the frequency deviation with full n deviations are larger than 100 mHz. The degradation due to FR % to the 7–12% capacity reduction of a 40 kWh Lithium-ion NMC verall economic framework is applied in Denmark, both DK1 and historical frequencies. The DK2 FR market framework is taken as d the DK1 cases. Electricity prices and charger efﬁciency are the two roﬁtability of the service. Indeed, with domestic prices there is no rial prices, despite differences between the frequencies, the service x. 3500¤ for a ﬁve-year period. vehicle; frequency regulation; vehicle-to-grid (FR) service has been demonstrated to be technically feasible and studies worldwide [1–6]. In the Nordic grid the bi-directionality of proﬁt 17 times higher than the unidirectional case [7,8]. The revenue parameters: electricity, regulation market prices, plugin hours, [9]. In the Great Britain depending on the usage FR proﬁt varies In France EVs are found to have a revenue of approximately verage revenue is 200 ¤/year [12]. Most of the analyses, particularly om FR service as difference between the revenue and the charger tention to the wear the EV battery is exposed to. That is because the mical process with many uncertainties, non-linearities, and different mistry considered. Therefore, to account for inﬂuence of degradation, en derived in a semi-empirical way. Three of the most discussed L model based on NCA and LFP chemistries [13], Wang model 14] and MOBICUS model based on LMO/NMC chemistry [15]. ntation the Wang model was implemented, as the most complete try. Nevertheless, being the empirical models affected by different data, cycling deﬁnition and chemistry [3], the authors recommend Received: 15 May 2020; Accepted: 14 July 2020; Published: 16 July 2020 Proﬁtability of Frequency Regulation by Electric Vehicles in Denmark and Japan Considering Battery Degradation Costs Lisa Calearo * and Mattia Marinelli Center for Electric Power and Energy, Department of Electrical Engineering, Technical University of Denmark, Risø Campus, 4000 Roskilde, Denmark; matm@elektro.dtu.dk * Correspondence: lica@elektro.dtu.dk Lisa Calearo * and Mattia Marinelli Lisa Calearo * and Mattia Marinelli Center for Electric Power and Energy, Department of Electrical Engineering, Technical University of Denmark, Risø Campus, 4000 Roskilde, Denmark; matm@elektro.dtu.dk * Correspondence: lica@elektro.dtu.dk Lisa Calearo * and Mattia Marinelli Center for Electric Power and Energy, Department of Electrical Engineering, Technical University of Denmark, Risø Campus, 4000 Roskilde, Denmark; matm@elektro.dtu.dk * Correspondence: lica@elektro.dtu.dk 1. Introduction Primary frequency regulation (FR) service has been demonstrated to be technically feasible and economically proﬁtable in different studies worldwide [1–6]. In the Nordic grid the bi-directionality of the ±10 kW DC chargers can give a proﬁt 17 times higher than the unidirectional case [7,8]. The revenue from FR is function of different parameters: electricity, regulation market prices, plugin hours, power capacity of the charger etc. [9]. In the Great Britain depending on the usage FR proﬁt varies between 60 and 400 ¤/year [10]. In France EVs are found to have a revenue of approximately 100 ¤/year [11], in Germany the average revenue is 200 ¤/year [12]. Most of the analyses, particularly in the past, evaluated the proﬁt from FR service as difference between the revenue and the charger losses, without giving the proper attention to the wear the EV battery is exposed to. That is because the battery degradation is a physicochemical process with many uncertainties, non-linearities, and different peculiarities depending on the chemistry considered. Therefore, to account for inﬂuence of degradation, different types of models have been derived in a semi-empirical way. Three of the most discussed models in the literature are: NREL model based on NCA and LFP chemistries [13], Wang model based on LMO-NMC chemistry [14] and MOBICUS model based on LMO/NMC chemistry [15]. Therefore, in our battery implementation the Wang model was implemented, as the most complete and appropriate in terms of chemistry. Nevertheless, being the empirical models affected by different limitations due to time resolution, data, cycling deﬁnition and chemistry [3], the authors recommend readers to consider also new approaches for future investigations. World Electric Vehicle Journal 2020, 11, 48; doi:10.3390/wevj11030048 www.mdpi.com/journal/wevj 2 of 15 World Electric Vehicle Journal 2020, 11, 48 In this paper, the authors aim at evaluating the proﬁt of the service including the battery degradation (BD) loss. This loss is technically quantiﬁed considering a Simulink model previously developed by the authors [16] to determine the economic loss. The proﬁt from FR service is evaluated as difference between the revenue from FR and the sum of the costs of grid energy exchanged and BD. This paper is organized as follows. Section 2 describes the methodology for the proﬁt evaluation, with details on the revenue, costs of grid energy exchanged and BD loss. Section 3 presents the tested study case, and Section 4 provides the results of the different scenarios analyzed. 1. Introduction Section 5 presents a sensitivity analysis and Section 6 concludes the article with the main outcomes. Figure 1 provides the proﬁt evaluation ﬂowchart, from the technical (orange) to the economic (green) characterization which are described in Sections 3 and 2, respectively. Pbatt is the battery power output. Figure 1. Proﬁt evaluation ﬂowchart: orange for the technical analysis provided in Section 3, green for the economic analysis provided in Section 2. Figure 1. Proﬁt evaluation ﬂowchart: orange for the technical analysis provided in Section 3, green for the economic analysis provided in Section 2. 2. Proﬁt Evaluation In this section, the methodology to evaluate the proﬁt of the EV user is described in detailed. By deﬁnition, the proﬁt is the difference between the revenue and the costs, therefore the proﬁt from providing FR is determined as in (1): Pro f itFR = RevenueFR −CostFR Loss −CostFR BD (1) (1) where RevenueFR is the revenue from the FR service, CostFR Loss is the difference between the cost of the energy sold and the energy purchased with the grid, considering the charger loss, and CostFR BD is the cost of BD due to the FR service. The next Sections present how revenue and costs are evaluated. 2.1. Revenue Assessment In Denmark FR is a service which is paid per availability. This means that the price is based on the power capacity that is available for an hour (MW per h) and not on the actual energy that is provided. Thus, when providing FR service, the capacity provided (Pcap) is known and the revenue is quantiﬁed as in (2): t RevenueFR = tFR ∑ 0 Pcap ∗CP (2) (2) where tFR is the number of hours of service availability and CP is the hourly capacity payment. where tFR is the number of hours of service availability and CP is the hourly capacity payment. .2. Energy Cost Assessment During FR service, the power provided by the battery is derived from the frequency as follows [17] d Electric Vehicle Journal 2020, 11, 48 3 of 1 World Electric Vehicle Journal 2020, 11, 48 3 of 15 Pbatt =      Pcap −c, if ft < 49.9 Hz 50−ft 0.1 ∗Pcap −c, if 49.9 Hz ≤ft ≤50.1 Hz −Pcap −c, if ft > 50.1 Hz (3) (3) where c is the offset, needed to avoid that the vehicle gets too overcharged, Pcap is the power capacity of the charger and ft is the measured frequency at time t. In this analysis Pbatt is negative when the battery is charging, and positive when it is discharging. From the grid perspective, the power (Pgrid) provided is derived from the battery power and the charger efﬁciency (4): Pgrid =    Pbatt ∗ηd if Pbatt ≥0 Pbatt ηc if Pbatt < 0 (4) (4) where ηd and ηc are the discharging and charging efﬁciencies, which depend on the different power output, see Table 1. To evaluate the cost due to the energy exchanged with the grid, the energy for driving should be subtracted from the energy consumption as in (5): CostFR Loss = EdischFR ∗PElsale −EchFR ∗PElpurch (5) (5) EchFR is the energy from the grid (charging the EV) and EdischFR is the energy to the grid (discharging the EV) during the FR service. PElpurch is the purchase electricity price and PElsale is the sale electricity price. EchFR is the energy from the grid (charging the EV) and EdischFR is the energy to the grid (discharging the EV) during the FR service. PElpurch is the purchase electricity price and PElsale is the sale electricity price. 2.3. Degradation Cost Assessment As for the calendar loss, the cycle degradation is also function of the battery temperature, with the relation provided in Figure 2. 0 10 20 30 40 50 0 1.5 3 4.5 6 7.5 9 ·10−3 T[oC] Q∗ cycle/eqCycles[%] Figure 2. Cycle degradation dependency on battery temperature. Figure 2. Cycle degradation dependency on battery temperature. It is relevant to observe that being a semi-empirical model, it does not have absolute validity, as it is limited by time resolution, data limitation, parameters deﬁnition and chemistry of the cells. For further information regarding the battery model, please refer to the authors’ works [16,21]. 2.3. Degradation Cost Assessment The battery degradation consists of capacity fade and increase of the internal resistance. For the purpose of this article, only the capacity fade is considered, nevertheless it is in the authors’ interest to include the increase of internal resistance in future investigations [18]. The battery degradation is the sum of two effects: calendar aging and cycle degradation. In this manuscript the authors considered the battery degradation of the Wang model, which is based on a large set of testing data of 1.5 Ah 18650 LMO-NMC Sanyo technology [14]. The formulation was characterized and validated for the considered cells, nevertheless the authors are currently working on the validation of the formulation with the similar cells used in this manuscript [19]. In the Wang model, the calendar loss equation is derived from a combination of the Arrhenius equation and the ﬁt of the model parameters, and then the cycle degradation is calculated as difference between the measurements of the total capacity loss and the calendar ones. p y The calendar loss (Q∗ cal), when temperature and SOC are constant, is estimated through the model using the Arrhenius equation in (6) [14,20]: Q∗ cal = f ∗e−Ea RT ∗t0.5 (6) (6) where Q∗ cal is the percentage of capacity loss induced by calendar aging, f is the pre-exponential factor, a non-linear function of both SOC and temperature [16], equal to 14,876 day−1/2 for speciﬁc SOC and temperature, Ea is the activation energy equal to 24.5 kJ mol−1. R is the gas constant equal to 8.314 J mol−1 K−1. The calendar loss is function of the absolute temperature (T), temperature increases result on larger losses. he percentage cycle degradation is estimated as in (7) [14,20]: Q∗ cycle = B1 ∗eB2∗rate ∗eqCycles (7) (7) tric Vehicle Journal 2020, 11, 48 4 of 15 World Electric Vehicle Journal 2020, 11, 48 4 of 15 World Electric Vehicle Journal 2020, 11, 48 4 of 15 B1 = a ∗T2 + b ∗T + c (8) B2 = d ∗T + e (9) rate = \|I\| Cbatt (10) eqCycles = rate 2 ∗3600 (11) (10) (11) where a, b, c, d and e are 8.58 ∗10−6 Ah−1 K−2, −0.0051 Ah−1 K−1, 0.759 Ah−1, −0.0067 K−1 −(C − rate) and 2.35 (C −rate)−1, respectively. I is the current ﬂowing inside the battery pack and Cbatt is the initial capacity of the battery, expressed in Ah, considered in the investigation. 3. Test Case 3.1. Technical Characterization Battery A 40 kWh battery composed of Lithium-ion Nickel Manganese Cobalt pouch cells is taken in consideration in the analysis [16]. The Simulink model simulates the battery EV usage given as input the outside temperature and the battery power proﬁle. The outputs of the model are: current and voltage of the battery, temperature, state-of-charge (SOC) and degradation losses. 2.3.2. Frequency Regulation Assessment 2.3.2. Frequency Regulation Assessment r the evaluation of the BD costs caused by the FR service provision, two scenarios are deﬁned: For the evaluation of the BD costs caused by the FR service provision, two scenarios are deﬁned: iving (driv): the EV is only driven iving plus FR (driv + FR): the EV is driven and used for providing FR service. g y • driving plus FR (driv + FR): the EV is driven and used for providing FR service. • driving plus FR (driv + FR): the EV is driven and used for providing FR service. The lost capacity in the two scenarios is evaluated as in (13): The lost capacity in the two scenarios is evaluated as in (13): he lost capacity in the two scenarios is evaluated as in (13): Cdriv BD = Cbatt −Cdriv batt f Cdriv+FR BD = Cbatt −Cdriv+FR batt f (13) (13) where Cbatt is the initial battery capacity and Cdriv batt f , Cdriv+FR batt f are the battery capacities in the two scenarios, at the end of the simulation. where Cbatt is the initial battery capacity and Cdriv batt f , Cdriv+FR batt f are the battery capacities in the two scenarios, at the end of the simulation. Considering the 50% capacity as the full capacity of the battery, the cost of the BD in the two scenarios is evaluated as in (14): Costdriv BD = Cdriv BD 50% ∗PricebattkWh Costdriv+FR BD = Cdriv+FR BD 50% ∗PricebattkWh (14) (14) It is then possible to determine the costs due to FR provision as in (15): It is then possible to determine the costs due to FR provision as in (15): It is then possible to determine the costs due to FR provision as in (15): It is then possible to determine the costs due to FR provision as in (15): CostFR BD = Costdriv+FR BD −Costdriv BD (15) CostFR BD = Costdriv+FR BD −Costdriv BD (15) 2.3.1. General Assessment For EV uses the battery end-of-life is usually deﬁned by a 20% reduction of the initial capacity. Nevertheless, a Li-ion battery can still be used when the state-of-health (SOH) is equal to 80% for second-hand uses. The end-of-life of second-hand batteries is set at approx. 50% SOH as shown in Figure 3. Figure 3. Battery end-of-life evaluation. Figure 3. Battery end-of-life evaluation. Figure 3. Battery end-of-life evaluation. Figure 3. Battery end-of-life evaluation. Thus, considering the 50% capacity as usable capacity of the battery, the cost of the BD is evaluated as in (12): C Thus, considering the 50% capacity as usable capacity of the battery, the cost of the BD is evaluated as in (12): C hus, considering the 50% capacity as usable capacity of the battery, the cost of the BD is evaluated 2): CostBD = CBD 50% ∗PricebattkWh (12) (12) where CBD is the capacity loss due to degradation and PricebattkWh is the price of the battery per k where CBD is the capacity loss due to degradation and PricebattkWh is the price of the battery per kWh. World Electric Vehicle Journal 2020, 11, 48 5 of 15 2.3.2. Frequency Regulation Assessment 2.3.2. Frequency Regulation Assessment requency Regulation Assessment 3.1.1. Input: Power In the driv scenario the EV is considered to be driven 40 km, 20 km in the morning and 20 in the afternoon, and it can charge from 6:00 to 8:00 (for how long, it depends on the driving, approximately 40 min), until when it reaches the requested SOC [22]. In the driving + FR scenario the EV is driving 40 km and performing FR service from 17:15 to 6:00 of the day after (approx. 13 h) and then it can charge from 6:00 to 8:00 to the required SOC. Since the battery degradation is function of the SOC, two simulations are performed: in the ﬁrst one the average SOC of the simulation is equal to 55% and in the second one it is equal to 75%. To keep the average SOC at these levels, avoiding that the battery gets too charged or discharged, the model is constrained between a maximum and a minimum SOC limits of 20 and 65% in the case with 55% average SOC, and 30 and 85% in the second case. The battery power during the FR service provision is derived from the frequency values, considering a charger power capacity equal to 9.2 kW and an offset equal to 0.8 kW. From (3) the droop is derived and shown in Figure 4. In this paper, the Danish frequencies, both DK1 and DK2 [23] (DK1 is the western Denmark part of the European power system, and DK2 is the eastern Denmark part of the Scandinavian countries. The two areas are electrically connected through the DC Great Belt Power Line), and the Japanese (JP) ones are considered. Figure 5 shows the 13 h of frequency both as Gaussian distribution and as frequency versus time. 6 of 15 World Electric Vehicle Journal 2020, 11, 48 49.8 49.9 50 50.1 50.2 −12 −10 −8 −6 −4 −2 0 2 4 6 8 10 Frequency [Hz] Pbatt [kW] Figure 4. Droop characteristic. 3.1.1. Input: Power 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 Std: 0.0175 Mean: 50.0001 Frequency [Hz] Counts DK1 0 4 8 12 16 20 24 49.8 49.9 50 50.1 50.2 Time [h] Frequency [Hz] 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 25,000 Std: 0.043 Mean: 50.0037 Frequency [Hz] DK2 0 4 8 12 16 20 24 49.8 49.9 50 50.1 50.2 Time [h] 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 25,000 Std: 0.0261 Mean: 50.0007 Frequency [Hz] JP 0 4 8 12 16 20 24 49.8 49.9 50 50.1 50.2 Time [h] Figure 5. Frequency histogram ﬁtted in Gaussian distribution and frequency trend during the 13 h of FR service provision in DK1, DK2 and Japan. 49.8 49.9 50 50.1 50.2 −12 −10 −8 −6 −4 −2 0 2 4 6 8 10 Frequency [Hz] Pbatt [kW] 49.8 49.9 50 50.1 50.2 −12 −10 −8 −6 −4 −2 0 2 4 6 8 10 Frequency [Hz] Pbatt [kW] Figure 4. Droop characteristic. .1 50.2 01 ] 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 25,000 Std: 0.043 Mean: 50.0037 Frequency [Hz] DK2 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 25,000 Std: 0.0261 Mean: 50.0007 Frequency [Hz] JP 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 25,000 Std: 0.043 Mean: 50.0037 Frequency [Hz] DK2 49.8 49.9 50 50.1 50.2 0 5,000 10,000 15,000 20,000 Std: 0.0175 Mean: 50.0001 Frequency [Hz] Counts DK1 JP q y [ ] 0 4 8 12 16 20 24 49.8 49.9 50 50.1 50.2 Time [h] q y [ ] 0 4 8 12 16 20 24 49.8 49.9 50 50.1 50.2 Time [h] 0 4 8 12 16 20 24 49.8 49.9 50 50.1 50.2 Time [h] Frequency [Hz] Figure 5. Frequency histogram ﬁtted in Gaussian distribution and frequency trend during the 13 h of FR service provision in DK1, DK2 and Japan. Given the frequency, the battery and grid powers are derived as in (3) and (4), considering the efﬁciency of the ±10 kW bidirectional charger as in Table 1. The battery power (Pbatt) for one-day period of the driv scenario and the driv + FR scenario for the three areas DK1, DK2 and JP are provided in Figure 6. 3.1.2. Input: Temperature Figure 7 shows the outside temperature during year 2017 in Denmark, which is considered during the simulations for DK1, DK2 and JP. Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec −20 −10 0 10 20 30 Tout [oC] Figure 7. Outside temperature year 2017 in Denmark. Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec −20 −10 0 10 20 30 Tout [oC] Figure 7. Outside temperature year 2017 in Denmark. 3.2. Economic Characterization 3.2. Economic Characterization 3.1.1. Input: Power When providing FR, even though the battery could be charged from 6:00 to 8:00, in all three cases the charging time is very short in comparison to the driving scenario. This is due to the fact that the battery is charged more than what it is discharged during the FR hours, thus there is less need for charging to the required SOC level. This is in accordance with the frequency mean values provided in Figure 5, which are slightly higher than 50 Hz in all the three areas. Table 1. Bidirectional 10 kW DC charger efﬁciency [8]. Pgch 0 −0.78 −0.91 −1.15 −1.49 −1.99 −2.63 −3.67 −4.68 −5.94 −6.9 −7.97 −9.09 −10 ηch 0.01 0.11 0.27 0.42 0.54 0.66 0.73 0.79 0.82 0.85 0.86 0.87 0.87 0.87 Pgdisch 0 0.17 0.33 0.59 0.92 1.29 1.87 2.42 3.27 4.25 5.27 6.08 7.49 8.71 ηdisch 0.01 0.14 0.3 0.45 0.57 0.67 0.74 0.78 0.82 0.84 0.85 0.86 0.86 0.88 Table 1. Bidirectional 10 kW DC charger efﬁciency [8]. 7 of 15 World Electric Vehicle Journal 2020, 11, 48 Figure 6. Battery power of the three scenarios during one-day period: driving, driving + FR in DK1, DK2 and JP. Figure 6. Battery power of the three scenarios during one-day period: driving, driving + FR in DK1, DK2 and JP. 3.2.1. Frequency Regulation Price The FR market framework in DK2 is taken as reference for all cases, due to the fact that JP does not have a market yet, and in DK1 the prices over the last 10 years are similar to the DK2 ones [24]. The average hourly price of frequency containment reserves in DK1 from 2015 to 2019 is provided in Figure 8. In 2015 the prices were exceptionally low due to high rainfall in the Nordic region, where hydro plants lead the provision of FR decreasing the prices [8,25]. By contrast, 2018 was a “dry” year, where the less FR from hydro plants increased the FR provided by the conventional units, and so did the prices. 8 of 15 World Electric Vehicle Journal 2020, 11, 48 0 2 4 6 8 10 12 14 16 18 20 22 24 0 20 40 60 Time [h] Price [¤/MW] 2015 2016 2017 2018 2019 Figure 8. Average hourly prices of FR in DK2 [24,26]. Figure 8. Average hourly prices of FR in DK2 [24,26]. 3.2.2. Electricity Price 3.2.2. Electricity Price In 2018–2019 the electricity price for the Danish domestic consumers was 0.31 ¤/kWh, included taxes, whereas the industrial one (mid-size industry) was 0.08 ¤/kWh, excluded taxes as they are not applied to industrial prices. For what concerns the sale electricity price, over the last 10 years the average price was approx. 0.04 ¤/kWh. 3.2.3. Battery Price For the evaluation of the BD costs, the value of 180 ¤/kWh is considered to be battery pack price per kWh [27]. Nevertheless, this is a conservative assumption since batteries are becoming cheaper and the price is expected to drop to less than 100 ¤/kWh by 2030 [28,29]. 4. Results During the following analysis, the battery model is considered to receive as input the same outside temperature and battery power for the 5 years of simulation. The scenario driv is run with both SOC equal to 55 and 75%, and it is the same for all the three areas. The scenario driv + FR is run for all the three areas—DK1, DK2 and JP—with both SOC equal to 55 and 75%. 4.1. Technical Results 4.2. Economic Results In this section, the proﬁtability of the FR service is quantiﬁed. Revenue, costs and proﬁts are determined as described in Section 2. Table 2 provides the RevenueFR, which is independent on the SOC, electricity price and area. Table 2. RevenueFR for the 5 years of frequency regulation and sum of the 5 years. Table 2. RevenueFR for the 5 years of frequency regulation and sum of the 5 years. Year 2015 2016 2017 2018 2019 mean RevenueFR [¤/y] 667 1324 1226 1925 1466 1322 RevenueFR [¤/5 y] 6608 Year 2015 2016 2017 2018 2019 mean RevenueFR [¤/y] 667 1324 1226 1925 1466 1322 RevenueFR [¤/5 y] 6608 Table 3 shows the costs due to energy exchanged with the grid and charger loss, comparing the case with domestic and industrial electricity prices and the three considered areas. The domestic and industrial electricity prices in DK2 are considered for all three areas. Since the power proﬁle of the battery is the same for the 5 years, the cost for 5 years is the CostFR loss for one year multiplied by 5. It is relevant to observe that with domestic electricity prices the CostFR loss in DK2 is higher than in DK1 and JP, which is not the case when the industrial electricity prices are considered. This is due to two main reasons: ﬁrst the frequency in DK2 has a standard deviation of 0.043, larger than in DK1 and JP, meaning that the power request during the regulation is larger. With larger power, both for charging and discharging, losses are lower, because the charger efﬁciency is closer to the optimal. Second, the domestic electricity price during purchase is higher than the electricity price for sale, making the purchasing component large in comparison to the selling one. The combination of these two shows that during FR the purchase/sale of electricity is causing costs, which are much larger in the domestic case. Table 3. CostFR loss for 5-year period of frequency regulation, due to the charging and discharging charger loss. Electricity Price Domestic Industrial Country DK1 DK2 JP DK1 DK2 JP CostFR loss [¤/5 y] 12,065 13,260 12,815 2970 2860 3000 Table 3. CostFR loss for 5-year period of frequency regulation, due to the charging and discharging charger loss. 3. 4.1. Technical Results Figure 9 provides the SOH of the battery over the ﬁve years in the different scenarios: 1 2 3 4 5 90 95 100 SOH [%] Mean SOC ~ 55% Driv Driv+FRDK1 Driv+FRDK2 Driv+FRJP 1 2 3 4 5 90 95 100 Year SOH [%] Mean SOC ~ 75% Figure 9. SOH comparison of the driv and driv + FR scenarios over ﬁve years in DK1, DK2 and JP: subplot 1 with average SOC 55% and subplot 2 with average SOC 75%. Mean SOC ~ 55% Figure 9. SOH comparison of the driv and driv + FR scenarios over ﬁve years in DK1, DK2 and JP: subplot 1 with average SOC 55% and subplot 2 with average SOC 75%. 9 of 15 World Electric Vehicle Journal 2020, 11, 48 4.2. Economic Results CostFR loss for 5-year period of frequency regulation, due to the charging and discharging l The difference between the battery power in the three areas is graphically provided in Figure 10 as battery power in function of the gradient of the battery power. Figure 10. Battery power as function of the gradient of the battery power for DK1, DK2 and JP. Figure 10. Battery power as function of the gradient of the battery power for DK1, DK2 and JP. Table 4 provides the costs due to battery degradation when just driving, driving plus FR and just FR. The ﬁrst two costs are given to underline that the degradation is a relevant cost during the lifetime of the battery, nevertheless the FR provision is not adding a large component to the driv scenario. Furthermore, a difference can be observed between DK2 and the other two areas: in DK2 CostFR BD represents approximately 16–20% of the total Costdriv+FR BD , depending on the SOC level. Differently 10 of 15 World Electric Vehicle Journal 2020, 11, 48 in DK1 and JP the FR represents approx. the 10% of the total costs. It is important to highlight that, in contrast to the CostFR loss, the costs due to BD are not the same for all the 5 years. This is due to the calendar and cycling degradation, which have an exponential behavior during the ﬁrst years of the battery lifetime, see Figure 9. Table 4. Costdriv BD , Costdriv+FR BD and CostFR BD for 5 years period of battery usage. Mean SOC 55% 75% Country DK1 DK2 JP DK1 DK2 JP Costdriv BD [¤/5 y] 876 1314 Costdriv+FR BD [¤/5 y] 968 1081 1019 1418 1565 1438 CostFR BD [¤/5 y] 92 205 143 104 251 124 Table 4. Costdriv BD , Costdriv+FR BD and CostFR BD for 5 years period of battery usage. Figure 11 summarizes in the ﬁrst subplot revenues and costs of the different cases whereas the second subplot compares the proﬁts during the ﬁve years period, as calculated with (1). The difference between the SOC, with both domestic and industrial electricity prices, does not have a large impact on the ﬁnal proﬁtability of the service. By contrast, the energy exchanged with the grid is substantial, causing economic loss when the domestic prices are considered. 4.2. Economic Results For what concerns the industrial electricity prices, the cost of the purchase is one fourth of the domestic one and thus the service is proﬁtable with approx. 3500¤ for the considered ﬁve years and in the different scenarios. Figure 11. Comparison of domestic and industrial cases, in black for SOC equal to 55% and in red for SOC equal to 75%, in DK1, DK2 and JP; revenues and costs in the ﬁrst subplot, proﬁts in the second subplot. Figure 11. Comparison of domestic and industrial cases, in black for SOC equal to 55% and in red for SOC equal to 75%, in DK1, DK2 and JP; revenues and costs in the ﬁrst subplot, proﬁts in the second subplot. 5. Sensitivity Analysis In this section, three parameters are further investigated, as they provide further highlights for the evaluation of the BD losses and relative costs. In this section, three parameters are further investigated, as they provide further highlights for the evaluation of the BD losses and relative costs. In this section, three parameters are further investigated, as they provide further highlights for the evaluation of the BD losses and relative costs. 11 of 15 World Electric Vehicle Journal 2020, 11, 48 11 of 15 5.1. Outside Temperature The outside temperature is a relevant parameter during the calculation of the battery degradation, because it inﬂuences both the calendar and the cycle processes, as shown in (6) and (7). In this regards, the authors selected the JP case with SOC equal to 55% and investigated the battery degradation when the temperature is increased of 5, 10 and 15 ◦C throughout the entire year. Figure 12 compares the results of the different cases, ﬁrst for the calendar and the cycle degradation and then for the SOH. Results show that despite the temperature increase of 5, 10 and 15 ◦C, the maximum SOH difference is 3%, which would result on a contained increase of the BD costs. Furthermore, the calendar losses are observed to be predominant in the total loss and their increase goes with the increase of the temperature. By contrast, the cycle degradation for the 10 and 15 ◦C temperature increase is almost the same. This is explained by the relation between the cycle degradation and the temperature provided in Figure 2, which shows that around 25 ◦C there is a minimum, and then the degradation starts to increase again. 1 2 3 4 5 0 2 4 6 8 10 Qcal [%] Base case + 5oC + 10oC + 15oC 1 2 3 4 5 0 0.5 1 1.5 2 2.5 Qcycle [%] 1 2 3 4 5 90 95 100 Year SOH [%] Figure 12. Comparison between the JP base case with SOC equal to 55% and the cases with outside temperature increase of 5, 10 and 15 ◦C: subplot 1 calendar degradation, subplot 2 cycle degradation and subplot 3 SOH. Figure 12. Comparison between the JP base case with SOC equal to 55% and the cases with outside temperature increase of 5, 10 and 15 ◦C: subplot 1 calendar degradation, subplot 2 cycle degradation and subplot 3 SOH. 5.3. Battery Price To evaluate the BD costs, the battery price per kWh has been considered equal to 180¤. However, the price of NMC batteries is decreasing year-by-year and forecasts show that the price could become lower than 90 ¤/kWh by 2030 [29]. Furthermore, to account for price variations among automakers and the price difference between the cost for the automaker and the costumer, prices higher than 180 ¤/kWh are also considered. In this regard, Figure 13 compares the CostFR BD evaluated in Table 4, with the BD cost for FR when the battery price per kWh is equal to 240, 210, 180, 150, 120 and 90¤. The BD cost due to FR are shown to decrease to less than 100¤ for DK1 and JP, meaning that in the future their weight on the ﬁnal proﬁt can become even lower than what shown in Figure 11. 240 210 180 150 120 90 0 100 200 300 400 Battery Price [¤/kWh] CostFR BD [¤] DK1 DK2 JP Figure 13. Comparison of CostFR BD with battery prices equal to 240, 210, 180, 150, 120 and 90 ¤/kWh. In black the cases with SOC = 75% and in red the ones with SOC = 55%. Figure 13. Comparison of CostFR BD with battery prices equal to 240, 210, 180, 150, 120 and 90 ¤/kWh. In black the cases with SOC = 75% and in red the ones with SOC = 55%. 5.2. Used Capacity The second parameter is the remaining capacity of the battery. As shown in Figure 3, the battery end-of-life is set to SOH of 50%, after the battery has been used in the vehicle and for a second use. Nevertheless, as presently, not all batteries used in EVs are then used in second life applications. To consider this case the 50% remaining battery capacity in (12) is compared with the 20% used capacity, when the battery end-of-life coincides with the EV end-of-life (ﬁnal SOH = 80%). The CostFR BD of the initial case (CostFR BD50%) is compared with the CostFR BD20% in Table 5: 12 of 15 World Electric Vehicle Journal 2020, 11, 48 Table 5. CostFR BD for 5 years period of battery usage considering the used capacity equal to 20%, 50%. Mean SOC 55% 75% Country DK1 DK2 JP DK1 DK2 JP CostFR BD20% [¤/5 y] 230 513 358 260 628 310 CostFR BD50% [¤/5 y] 92 205 143 104 251 124 Table 5. CostFR BD for 5 years period of battery usage considering the used capacity equal to 20%, 50%. Mean SOC 55% 75% Table 5. CostFR BD for 5 years period of battery usage considering the used capacity equal to 20%, 50%. The results show that the battery degradation is max 5% of the total cost when considering the domestic charger loss present in Table 3. When considering the industrial prices, the battery degradation could represent up to 20% of the cost, resulting on lower proﬁt, but still positive. 6. Conclusions In this manuscript the proﬁtability of primary frequency regulation provided by EVs, taking into account the battery degradation costs has been quantiﬁed based on the frequency measured in Denmark, both DK1 and DK2, and Japan. First, the battery power proﬁles have been derived from the driving pattern and the frequency measurements. Second, the battery model has been simulated to derive the degradation loss and to calculate the related costs the user would encounter while providing frequency regulation. Afterwards the energy exchanged with the grid has been used to determine the economic loss due to the higher purchase electricity price in comparison to the sale one. Finally, revenue and costs were combined to determine the proﬁt. It was noticed that the frequency in DK2 was more spread between the 49.8 and 50.2 Hz causing higher costs due to degradation and energy exchanged with the grid. By contrast, for what concerns the battery degradation, the losses are larger when the frequency is more spread, because the battery is exposed to more cycles during its lifetime. Nevertheless, the battery degradation cost is approx. 1% of the total costs when considering domestic electricity prices, and 3–8% when considering industrial prices, both with 55 and 75% SOC. 13 of 15 13 of 15 World Electric Vehicle Journal 2020, 11, 48 Finally, it was observed that the electricity price and the charger efﬁciency are two main parameters that affect the proﬁtability of the service. Indeed, with domestic prices the charger losses are too large and there are only economic losses in the service provision. Further considerations and analysis can be done regarding the losses: • charger efﬁciency: the charger losses of this analysis are based on the efﬁciency of a three years old charger. It is interesting to compare the charger efﬁciency of this charger with new and future chargers, which could have higher efﬁciency and thus lower costs [30]. • battery capacity: the considered battery has a 40 kWh capacity. Considering the same charging/discharging power proﬁle, larger is the battery, lower is the amount of cycles the battery is exposed to. Thus, for smaller batteries the battery degradation is expected to be a larger component of the total amount of loss. For larger batteries, which are expected in the future, the degradation can be even lower, decreasing further the degradation costs. 6. Conclusions This would also give more ﬂexibility on the charging power that can be used during the frequency regulation provision. Even though DK1 (belonging to the continental Europe synchronous areas) has been considered during the analyses, to generalize the results to the all Europe the following aspects should be carefully considered: • Cost of electricity: in Europe it ranges around 0.21 €/kWh whereas in Denmark it is approx. 0.31 €/kWh, resulting on lower cost of electricity in most European countries. • Vehicle driving pattern and annual distance: European countries have similar driving patterns, nevertheless distances ranges between 40 and 80 km/day depending on the considered country [31]. • Ambient temperature: European countries are characterized by wide variety of climate zones, meaning that the temperature behavior throughout the year can vary greatly. Similar aspects must be considered when generalizing the analyses of DK2 to the Scandinavian countries. Further studies of the authors will include the different considerations for the evaluation of the proﬁtability of frequency regulation. Author Contributions: Conceptualization, L.C. and M.M.; data curation, L.C.; investigation, L.C. and M.M.; methodology, L.C. and M.M.; writing—original draft preparation, L.C.; writing—review and editing, L.C. and M.M. All authors have read and agreed to the published version of the manuscript. Funding: The work in this paper has been supported by the research projects ACES (EUDP grant EUDP17-I-12499) and CAR (EU-Interreg grant nr: STHB.03.01.00-SE-S112/17). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Kempton, W.; Tomi´c, J. Vehicle-to-grid power fundamentals: Calculating capacity and net revenue. J. Power Sources 2005, 144, 268–279. [CrossRef] 2. Andersen, P.B.; Sousa, T.; Thingvad, A.; Berthou, L.S.; Kulahci, M. Added Value of Individual Flexibility Proﬁles of Electric Vehicle Users for Ancillary Services. 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https://openalex.org/W4252122266	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-020-6671-4	English	null	Comprehensive Analysis of mRNAs and miRNAs in the Ovarian Follicles of Uniparous and Multiple Goats at Estrus Phase	Research Square (Research Square)	2,019	cc-by	13,935	Zou et al. BMC Genomics (2020) 21:267 https://doi.org/10.1186/s12864-020-6671-4 Zou et al. BMC Genomics (2020) 21:267 https://doi.org/10.1186/s12864-020-6671-4 Open Access Comprehensive analysis of mRNAs and miRNAs in the ovarian follicles of uniparous and multiple goats at estrus phase Xian Zou1,2†, Tingting Lu1†, Zhifeng Zhao1, Guangbin Liu1, Zhiquan Lian1, Yongqing Guo1, Baoli Sun1, Dewu Liu1 and Yaokun Li1* © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Fertility is an important economic trait in the production of meat goat, and follicular development plays an important role in fertility. Although many mRNAs and microRNAs (miRNAs) have been found to play critical roles in ovarian biological processes, the interaction between mRNAs and miRNAs in follicular development is not yet completely understood. In addition, less attention has been given to the study of single follicle (dominant or atretic follicle) in goats. This study aimed to identify mRNAs, miRNAs, and signaling pathways as well as their interaction networks in the ovarian follicles (large follicles and small follicles) of uniparous and multiple Chuanzhong black goats at estrus phase using RNA- sequencing (RNA-seq) technique. Results: The results showed that there was a significant difference in the number of large follicles between uniparous and multiple goats (P < 0.05), but no difference in the number of small follicles was observed (P > 0.05). For the small follicles of uniparous and multiple goats at estrus phase, 289 differentially expressed mRNAs (DEmRNAs) and 16 DEmiRNAs were identified; and for the large follicles, 195 DEmRNAs and 7 DEmiRNAs were identified. The functional enrichment analysis showed that DE genes in small follicles were significantly enriched in ovarian steroidogenesis and steroid hormone biosynthesis, while in large follicles were significantly enriched in ABC transporters and steroid hormone biosynthesis. The results of quantitative real-time polymerase chain reaction were consistent with those of RNA-seq. Analysis of the mRNA-miRNA interaction network suggested that CD36 (miR-122, miR-200a, miR-141), TNFAIP6 (miR-141, miR-200a, miR-182), CYP11A1 (miR-122), SERPINA5 (miR-1, miR-206, miR-133a-3p, miR-133b), and PTGFR (miR-182, miR-122) might be related to fertility, but requires further research on follicular somatic cells. Conclusions: This study was used for the first time to reveal the DEmRNAs and DEmiRNAs as well as their interaction in the follicles of uniparous and multiple goats at estrus phase using RNA-seq technology. Our findings provide new clues to uncover the molecular mechanisms and signaling networks of goat reproduction that could be potentially used to increase ovulation rate and kidding rate in goat. Keywords: Goat, Follicular development, Kidding rate, RNA-seq * Correspondence: liyaokun1986@163.com †Xian Zou and Tingting Lu contributed equally to this work. 1College of Animal Science, South China Agricultural University, Wushan Rd., Tianhe Dist, Guangzhou 510642, Guangdong Province, China Full list of author information is available at the end of the article * Correspondence: liyaokun1986@163.com †Xian Zou and Tingting Lu contributed equally to this work. 1College of Animal Science, South China Agricultural University, Wushan Rd., Tianhe Dist, Guangzhou 510642, Guangdong Province, China Full list of author information is available at the end of the article Background In goat, the number of small follicles [S, diameter (d) < 3 mm] far exceeding the number of mid-follicles (d > 3 mm) is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. Hence, it is important to study the contribution of ovarian follicular compartments (follicular fluid, oocyte, CC, GC, and TC) of small follicles and large follicles to the regula- tion of ovulation number and the timing of ovulation. Chuanzhong (CZ) black goat is an excellent local goat resource in China. The resources are abundant in China as well as Southeast Asia, and play an important role in herb- ivorous livestock [43]. After long-term natural selection and artificial cultivation, CZ black goat has gradually formed local meat goat breeds with high genetic stability [44]. However, low fecundity remains a key bottleneck lim- iting the development of goat industry. To better understand the role and importance of follicles in kidding rate, we performed transcriptome profiling of small follicles (S, d < 3 mm) and large follicles (L, d > 10 mm) from uniparous and multiple CZ black goats at the es- trus phase to identify DEmRNAs and DEmiRNAs, respect- ively. Furthermore, the interaction networks of DEmRNAs and DEmiRNAs were constructed, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out for DEmR- NAs and target genes of DEmiRNAs. In addition, we ex- plored the role of ovarian follicular mRNAs and miRNAs in goat reproduction. Collectively, our findings provide a theoretical basis for improving ovulation and kidding rates in the future. g In mammals, studies on follicles have mainly been con- ducted in mice [17], human [18], pig [19], bovine [20], rat [21], and sheep [22]. These studies revealed the effects of GCs and theca cells on follicular development, follicular atresia, and luteal development, and further demonstrated the mechanism of genes and signaling pathways. However, little is known about goat follicles. In goat, previous studies have identified the key genes involved in the regulation of ovulation rate and kidding rate by transcriptome analysis of goat ovaries, as well as the signaling pathways that affect ovulation and fertility [1, 4, 23–29]. Studies on litter size of goats have shown that PDGFRB, MARCH1, KDM6A, CSN1S1, SIRT3, KITLG, GHR, ATBF1, INHA, GNRH1, and GDF9 might be candidate genes for goat reproductive traits [26, 30–39]. Background with the high fecundity of goats [41]. In addition, many studies have suggested that microRNAs (miRNAs) influ- ence ovarian biological processes in goat, and several differ- entially expressed miRNAs (DEmiRNAs), such as miR-21, miR-99a, miRNA-143, let-7f, miR-493, and miR-200b have been identified and comparatively analyzed in the ovaries of prolific and non-prolifc goats [1, 29, 42]. However, the major genes and miRNAs related to ovulation rate and lit- ter size have not yet been identified in goats through tran- scriptome sequencing of the ovary as a whole. Hence, since the follicle is a unique microenvironment within which the oocyte can develop and mature into a fertilizable gamete, it is important to individually study single follicles to explore factors that affect ovulation rate and kidding rate in goats. g Ovulation rate is a key factor affecting the kidding rate, which is one of the most important economic traits for goat production [1–3]. However, the genetic mechanism of kid- ding rate associated with ovulation rate is poorly under- stood, which largely limits the improvement of kidding rate through genetic selection. The major function of the ovary is to produce oocytes for fertilization and secrete steroid hormones for regulating follicular development during the estrus cycle in goat [4]. Oocytes develop and mature in the ovarian follicle, and acquire their developmental compe- tence in follicle through tight bidirectional communication with follicular somatic cells [5, 6]. The follicular somatic cells include epithelial-like granulosa cells (GC), mesenchymal-like theca cells (TC) and cumulus cells (CC), with each type secrets specific regulation factors [7, 8]. GC and TC face each other across a basement membrane. CC are the differentiated GC, which are tightly connected and metabolically coupled with an oocyte via gap junctions and form the cumulus oocyte complex (COC) [9]. The pre- antral follicle is filled with a fluid that is rich in proteins, steroids, and lipids, coming from the blood and secretory activity of follicular somatic cells [10, 11]. The microenvir- onment of the oocyte has a crucial impact on the acquisi- tion of oocyte developmental competence and possesses molecular factors that are predictive of oocyte developmen- tal potential. However, only 1% of follicles reach ovulation, more than 99% of follicles undergo atresia in mammals [12–16]. Background Growth hormones and members of the insulin-like growth factor (IGF) system (IGF-I and IGF-II) may play a key role in follicular development and atresia [2, 40], and the genes FER1 L4 and SRD5A2 may be associated © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zou et al. BMC Genomics (2020) 21:267 Zou et al. BMC Genomics (2020) 21:267 Page 2 of 15 Comparison of follicles between uniparous and multiple CZ black goats The follicles around the large follicles were sacrificed dur- ing follicle separation, including small follicles (d < 3 mm) and mid-follicles (3 < d < 10 mm). Sometimes the nearby large follicle (d > 10 mm) had to be sacrificed, too. Unfortu- nately, some small or large follicles were broken up during follicle separation. Finally, eight to fifteen small follicles were isolated from each goat, one to two large follicles were isolated from each uniparous goat, and one to three large follicles each multiple goat. Among the separated fol- licles used for sequencing, the large follicles (d > 10 mm) were larger in size than the small follicles (d < 3 mm) (Fig. 1a). After separation of follicles, the number of folli- cles in uniparous and multiple goats was counted and ana- lyzed (Table 1). Multiple goats showed a greater number of Zou et al. BMC Genomics (2020) 21:267 Page 3 of 15 Fig. 1 Images of ovaries and follicles. a Large follicles (black arrows) and small follicles (yellow arrows) in the ovaries before follicle separation. b Large follicles after separation. c Small follicles after separation Fig. 1 Images of ovaries and follicles. a Large follicles (black arrows) and small follicles (yellow arrows) in the ovaries before follicle separation. b Large follicles after separation. c Small follicles after separation Fig. 1 Images of ovaries and follicles. a Large follicles (black arrows) and small follicles (yellow arrows) in the ovaries before follicle separation. b Large follicles after separation. c Small follicles after separation follicles than uniparous goats (P < 0.05). Furthermore, there was a significant difference in the number of large follicles between uniparous and multiple goats (P < 0.05), but no difference in the number of small follicles was observed (P > 0.05). mapped ration, and Q30 of sequencing data are dis- played in Table 2. The data showed that the sequencing results met the requirements and could be used for fur- ther analysis. The expression of 21,343 mRNAs and 436 miRNAs was detected by RNA-sequencing (RNA-seq). Finally, samples were divided into four groups for fur- ther analysis: uniparous-small follicles vs multiple-small follicles (Uni-S vs Mul-S) and uniparous-large follicles vs multiple-large follicles (Uni-L vs Mul-L). Unsupervised hierarchical clustering of mRNAs and miRNAs showed that each group clustered together, despite inter- individual variation (Figure S1). Transcriptome sequencing analysis and mapping We collected twelve large follicles from four uniparous and four multiple goats, and nine small follicular pools from four uniparous and five multiple goats. The large follicle with few or no blood vessels on the surface were not used to RNA-seq because they considered to be atretic follicles [45, 46]. Eight to ten small follicles were pooled from each goat for a replicate, and only one sin- gle large follicle with clear follicular fluid and abundant blood vessels on the surface from each goat for a repli- cate (Fig. 1b, c). Next, we sequenced the RNA libraries of these seventeen follicular samples (eight large follicles and nine small follicular pools). Clean read counts, p Means within a row with no common superscript letter differ significantly (P < 0.05). Identification of DEmRNAs and DEmiRNAs BMC Genomics (2020) 21:267 Page 4 of 15 Table 2 Q30, clean read counts and mapping ratio of sequencing results sample mRNA miRNA (%) Q30 (%) Clean Reads (%) Mapped Ration(%) Q30 (%) Clean Reads (%) Mapped Ration (%) 1 L 94.23 104,768,836 (99.62) 88.85 96.66 23,562,383 (92.40) 74.06 1S 94.26 103,870,160 (99.62) 86.00 96.48 23,729,111 (88.44) 49.21 2 L 94.85 100,396,168 (99.70) 89.57 93.56 18,638,711 (73.47) 40.21 2S 94.88 102,014,414 (99.69) 84.48 93.04 12,918,467 (36.29) 47.62 3 L 94.15 104,879,676 (99.48) 91.70 92.23 22,367,901 (51.67) 67.31 3S 94.24 102,993,890 (99.45) 90.72 91.14 10,392,354 (31.81) 50.17 4 L 92.66 105,470,926 (99.40) 89.70 92.32 12,456,243 (43.15) 49.26 4S 92.27 103,362,624 (99.45) 86.12 94.29 21,982,767 (82.43) 58.40 5 L 92.57 102,209,432 (99.37) 89.67 94.68 21,272,821 (84.74) 67.20 5S 94.11 106,653,668 (99.77) 88.98 93.69 20,242,442 (60.17) 53.04 6 L 93.86 100,346,902 (99.75) 90.32 95.03 18,734,838 (73.61) 48.23 6S 91.52 105,756,846 (99.19) 83.74 94.48 23,270,301 (84.46) 41.44 7 L 92.26 101,449,700 (99.50) 86.32 94.57 20,398,068 (77.25) 35.00 7S 93.79 103,645,956 (99.58) 88.33 95.17 20,963,044 (83.72) 47.44 8 L 91.91 102,150,920 (99.54) 86.68 95.01 22,371,142 (80.09) 47.07 8S 92.38 106,529,902 (99.45) 83.59 94.96 18,992,453 (75.81) 43.75 9S 91.87 105,002,632 (99.51) 86.16 95.2 20,407,498 (87.38) 53.85 L large follicles; S small follicles Table 2 Q30, clean read counts and mapping ratio of sequencing results in Uni-S vs Mul-S (Fig. 2a and Table 3a). For large folli- cles, a total of 195 DEmRNAs (120 upregulated and 75 downregulated in multiple goats) and 16 DEmiRNAs (4 upregulated and 12 downregulated in multiple goats) were identified in Uni-L vs Mul-L (Fig. 2b and Table 3b). For better analysis, the fragments per kilobase mil- lion (FPKM) values > 1 of at least three samples per group was used to further quantify the mRNA expres- sion levels. We identified 119 and 37 DEmRNAs from Uni-S vs Mul-S and Uni-L vs Mul-L, respectively, and the top 10 DEmRNAs and DEmiRNAs are shown in Table 3. The venn diagrams of shared DEmRNAs and DEmiRNAs are shown in Fig. 2c, d. Two shared DEmR- NAs (AMDHD1 and LOC102190765) and five shared DEmiRNAs (miR-141, miR451-5p, miR-122, miR-182, and miR-206) were identified in both Uni-S vs Mul-S and Uni-L vs Mul-L. periphery, plasma membrane, animal organ development, embryo development, and anion channel activity (Table 4, Additional file 3). in Uni-S vs Mul-S (Fig. 2a and Table 3a). Identification of DEmRNAs and DEmiRNAs For large folli- cles, a total of 195 DEmRNAs (120 upregulated and 75 downregulated in multiple goats) and 16 DEmiRNAs (4 upregulated and 12 downregulated in multiple goats) were identified in Uni-L vs Mul-L (Fig. 2b and Table 3b). For better analysis, the fragments per kilobase mil- lion (FPKM) values > 1 of at least three samples per group was used to further quantify the mRNA expres- sion levels. We identified 119 and 37 DEmRNAs from Uni-S vs Mul-S and Uni-L vs Mul-L, respectively, and the top 10 DEmRNAs and DEmiRNAs are shown in Table 3. The venn diagrams of shared DEmRNAs and DEmiRNAs are shown in Fig. 2c, d. Two shared DEmR- NAs (AMDHD1 and LOC102190765) and five shared DEmiRNAs (miR-141, miR451-5p, miR-122, miR-182, and miR-206) were identified in both Uni-S vs Mul-S and Uni-L vs Mul-L. DEmRNAs were also plotted to KEGG reference path- ways (https://www.kegg.jp/kegg/pathway.html). The sig- nificantly enriched KEGG pathways (P < 0.05) were listed in Additional file 4. Of these KEGG pathways, ovarian ste- roidogenesis, cortisol synthesis and secretion, cytokine- cytokine receptor interaction, steroid hormone biosyn- thesis, and metabolism of xenobiotics by cytochrome P450 were significantly enriched between the Uni-S and Mul-S groups (Fig. 2e), and ABC transporters, retinol metabol- ism, steroid hormone biosynthesis, drug metabolism- cytochrome P450, and metabolism of xenobiotics by cyto- chrome P450 were significantly enriched between the Uni-L and Mul-L groups (Fig. 2f). Identification of DEmRNAs and DEmiRNAs DEmRNAs and DEmiRNAs were initially identified by P < 0.05 (Additional files 1 and 2). For small follicles, a total of 289 DEmRNAs (131 upregulated and 158 down- regulated in multiple goats) and seven DEmiRNAs (seven downregulated in multiple goats) were identified Table 1 Comparison of follicles between uniparous and multiple CZ black goats Groups The number of follicles Small follicles (d < 3 mm) Large follicles (d > 10 mm) Total number1 Uniparous 30.29 ± 4.36 a 2.71 ± 0.36 b 39.57 ± 3.41 b Multiple 45.83 ± 7.01 a 4.83 ± 0.7 a 69.17 ± 7.13 a 1 The total number of follicles were close to the sum of small (d < 3 mm), mid- (3 < d < 10 mm) and large follicles (d > 10 mm) Values are expressed as the means ± standard error. Means within a row with no common superscript letter differ significantly (P < 0.05). Table 1 Comparison of follicles between uniparous and multiple CZ black goats p Means within a row with no common superscript letter differ significantly (P < 0.05). Zou et al. Functional annotation of DEmRNAs For small follicles (Uni-S vs Mul-S), the miRNA–mRNA tar- get prediction analyses identified 76 miRNA–mRNA target pairs, including only seven significant miRNAs, chi-miR- 200a (degree = 20, degree means the number of DEmiR- NAs’target genes), chi-miR-141 (degree = 20), chi-miR-182 (degree = 16), chi-miR-206 (degree = 10), chi-miR-122 (de- gree = 7), chi-miR-184 (degree = 2) and chi-miR-145-5p (de- gree = 1) (Fig. 3a). For large follicles (Uni-L vs Mul-L), a total of 153 possible significant miRNA–mRNA interaction pairs were obtained; chi-miR-141 (degree = 17), chi-miR-182 (degree = 13), chi-miR-122 (degree = 12) and chi-miR-154b- DEmRNAs were enriched in biological process, cellular component, and molecular function categories by GO analysis (http://www.geneontology.org/). GO terms with P < 0.05 were considered significantly enriched in DEmR- NAs. For small follicles, 455 GO terms were significantly enriched, including cell periphery, plasma membrane, steroid biosynthetic process, steroid hydroxylase activity, and receptor binding between the Uni-S and Mul-S groups (Table 4, Additional file 3). For large follicles, 322 GO terms were significantly enriched, including cell Zou et al. BMC Genomics (2020) 21:267 Page 5 of 15 Fig. 2 RNA-seq data of DEmRNA expression in large and small follicles from uniparous and multiple goats. a Unsupervised clustering analysis showing the expression profiles of DEmRNAs between Uni-S and Mul-S groups. b Unsupervised clustering analysis showing the expression profiles of DEmRNAs between Uni-L and Mul-L groups. c Venn diagrams demonstrating the distribution of shared DEmRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L groups. d Venn diagrams demonstrating the distribution of shared DEmiRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L groups, respectively. e Top 20 KEGG pathways of DEmRNAs in Uni-S vs Mul-S. f Top 20 KEGG pathways of DEmRNAs in Uni-L vs Mul-L. Fig. 2 RNA-seq data of DEmRNA expression in large and small follicles from uniparous and multiple goats. a Unsupervised clustering analysis showing the expression profiles of DEmRNAs between Uni-S and Mul-S groups. b Unsupervised clustering analysis showing the expression profiles of DEmRNAs between Uni-L and Mul-L groups. c Venn diagrams demonstrating the distribution of shared DEmRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L groups. d Venn diagrams demonstrating the distribution of shared DEmiRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L groups, respectively. e Top 20 KEGG pathways of DEmRNAs in Uni-S vs Mul-S. f Top 20 KEGG pathways of DEmRNAs in Uni-L vs Mul-L. Fig. Validation by quantitative real-time polymerase chain reaction (qRT-PCR) and miRNAs related to ovulation rate and kidding rate have not been identified in goats. In addition, the interaction between mRNAs and miRNAs in follicular development is not yet completely understood. Hence, we compared DEmRNAs and DEmiRNAs from different size follicles be- tween uniparous and multiple CZ black goats at estrus phase using RNA-seq. The results showed that the number of large follicles in multiple goats was significantly higher than that in uniparous goats (P < 0.05), while no difference in the number of small follicles was observed between unip- arous and multiple goats, verifying that the greater number of large follicles was related to higher ovulation rate [39]. Based on the RNA-seq data, we identified 119 and 37 DEmRNAs by comparing Uni-S with Mul-S and Uni-L with Mul-L, respectively (FPKM> 1 of at least three samples per group). These DEmRNAs were found to be involved in various ovarian development-related pathways, such as ovarian steroidogenesis, steroid hormone biosynthesis, and metabolism of xenobiotics by cytochrome P450, etc. (Fig. 2e, f). Approximately 37% of the DEgenes in the Uni-S vs Mul-S group and 41% in the Uni-L vs Mul-L group were reported to be associated with mammalian reproduction, such as TNFAIP6, MMP9, INSL3, LEPR, 3BHSD, LHCGR, ARL4C, CD36, CYP11A1, AMDHD1, SPOCK2, AMDHD1, MFAP5, CCL21, PTGFR, and SERPINA5 [23, 47–60]. Of these genes, TNFAIP6, CYP11A1, CD36, PTGFR, and SER- PINA5 were found to be associated with the ovulation rate. (q ) A total of 6 DEmRNAs and 5 DEmiRNAs were selected for verification by qRT-PCR. Based on the RNA-seq re- sults, 3BHSD and STAR expression were upregulated and LEPR expression was downregulated in small follicles of multiple goat, and CCL21, RARRES1, and DPT expression were downregulated in large follicles of multiple goat. Notably, genes 3BHSD and STAR are both involved in the four significant pathways (ovarian steroidogenesis, cush- ing’s syndrome, cortisol synthesis and secretion and aldos- terone synthesis and secretion), LEPR gene is involved in the two significant pathways (cytokine-cytokine receptor interaction and neuroactive ligand-receptor interaction). Five DEmiRNAs were downregulated in multiple large fol- licles. Based on the qRT-PCR results, expression of these 6 DEmRNAs and 5 DEmiRNAs were consistent with that in the RNA-seq results (Fig. 4). Functional annotation of DEmRNAs 2 RNA-seq data of DEmRNA expression in large and small follicles from uniparous and multiple goats. a Unsupervised clustering analysis showing the expression profiles of DEmRNAs between Uni-S and Mul-S groups. b Unsupervised clustering analysis showing the expression profiles of DEmRNAs between Uni-L and Mul-L groups. c Venn diagrams demonstrating the distribution of shared DEmRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L groups. d Venn diagrams demonstrating the distribution of shared DEmiRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L groups, respectively. e Top 20 KEGG pathways of DEmRNAs in Uni-S vs Mul-S. f Top 20 KEGG pathways of DEmRNAs in Uni-L vs Mul-L vs Mul-L, respectively (Fig. 3c, d). Among them, in Uni-S vs Mul-S, TNFAIP6 (degree = 3) was upregulated in multiple goats; CD36 (degree = 3), BTK (degree = 2) and AKAP4 (degree = 2) were downregulated in multiple goats. In Uni-L vs Mul-L, SERPINA5 (degree = 5) was upregulated in multiple goats; ENSCHIG00000017462 (degree = 6) and PTGFR (degree = 4) were downregulated in multiple goats. 3p (degree = 12) were the top DEmiRNAs that had most tar- get genes (Fig. 3b). In order to further narrow the scope of genes and obtain more meaningful candidate genes, DEmRNAs were screened according to FPKM > 1 of at least three samples per group. Then, 19 pairs and 19 pairs of DEmRNAs- DEmiRNAs were obtained from Uni-S vs Mul-S and Uni-L 3p (degree = 12) were the top DEmiRNAs that had most tar- get genes (Fig. 3b). In order to further narrow the scope of genes and obtain more meaningful candidate genes, DEmRNAs were screened according to FPKM > 1 of at least three samples per group. Then, 19 pairs and 19 pairs of DEmRNAs- DEmiRNAs were obtained from Uni-S vs Mul-S and Uni-L Validation by quantitative real-time polymerase chain reaction (qRT-PCR) A total of 6 DEmRNAs and 5 DEmiRNAs were selected for verification by qRT-PCR. Based on the RNA-seq re- sults, 3BHSD and STAR expression were upregulated and LEPR expression was downregulated in small follicles of multiple goat, and CCL21, RARRES1, and DPT expression were downregulated in large follicles of multiple goat. Notably, genes 3BHSD and STAR are both involved in the four significant pathways (ovarian steroidogenesis, cush- ing’s syndrome, cortisol synthesis and secretion and aldos- terone synthesis and secretion), LEPR gene is involved in the two significant pathways (cytokine-cytokine receptor interaction and neuroactive ligand-receptor interaction). Functional annotation of DEmRNAs Five DEmiRNAs were downregulated in multiple large fol- licles. Based on the qRT-PCR results, expression of these 6 DEmRNAs and 5 DEmiRNAs were consistent with that in the RNA-seq results (Fig. 4). Discussion Exploring the genetic mechanism associated with ovulation rate is important to improve kidding rate, which is funda- mental to goat production. The greater number of large fol- licles that stimulate ovulation is believed to be the primary reason for higher ovulation rate [39]. Although several mRNAs and miRNAs have been found to play critical roles in ovarian biological processes, the majority of the genes and miRNAs related to ovulation rate and kidding rate have not been identified in goats. In addition, the interaction between mRNAs and miRNAs in follicular development is not yet completely understood. Hence, we compared DEmRNAs and DEmiRNAs from different size follicles be- tween uniparous and multiple CZ black goats at estrus phase using RNA-seq. The results showed that the number of large follicles in multiple goats was significantly higher than that in uniparous goats (P < 0.05), while no difference in the number of small follicles was observed between unip- arous and multiple goats, verifying that the greater number of large follicles was related to higher ovulation rate [39]. Based on the RNA-seq data, we identified 119 and 37 DEmRNAs by comparing Uni-S with Mul-S and Uni-L with Mul-L, respectively (FPKM> 1 of at least three samples per group). These DEmRNAs were found to be involved in various ovarian development-related pathways, such as ovarian steroidogenesis, steroid hormone biosynthesis, and metabolism of xenobiotics by cytochrome P450, etc. (Fig. 2e, f). Approximately 37% of the DEgenes in the Uni-S vs Mul-S group and 41% in the Uni-L vs Mul-L group were reported to be associated with mammalian reproduction, such as TNFAIP6, MMP9, INSL3, LEPR, 3BHSD, LHCGR, ARL4C, CD36, CYP11A1, AMDHD1, SPOCK2, AMDHD1, MFAP5, CCL21, PTGFR, and SERPINA5 [23, 47–60]. Of these genes, TNFAIP6, CYP11A1, CD36, PTGFR, and SER- PINA5 were found to be associated with the ovulation rate. Functional annotation of DEmRNAs Table 3 The top 10 DEmRNAs and DEmiRNAs in Uni-S vs Mul-S (a) and Uni-L vs Mul-L (b) DEmiRNAs log2fold change P-value DEmRNAs log2fold change P-value a chi-miR-122 −12.44 1.24E-03 ARL4C −1.59 7.98E-07 chi-miR-451-5p −9.01 9.45E-03 WNT5B −1.65 2.25E-06 chi-miR-206 −11.91 1.07E-02 MMP9 −2.55 3.15E-06 chi-miR-141 −7.94 1.18E-02 TGFBI −1.55 1.64E-05 chi-miR-182 −10.62 1.39E-02 S100A12 −3.75 5.93E-05 chi-miR-200a −7.77 3.18E-02 INSL3 2.39 8.34E-05 chi-miR-184 −11.45 3.83E-02 MAP7D2 1.32 8.68E-05 LFNG −1.35 8.70E-05 SDC1 −1.14 9.74E-05 NT5E 1.03 1.16E-04 b chi-miR-182 −10.83 1.61E-03 BRINP3 −1.52 3.59E-04 chi-miR-122 −10.73 2.17E-03 DPT −1.81 8.07E-04 chi-miR-133b −9.62 3.12E-03 SPOCK2 −1.37 1.09E-03 chi-miR-206 −12.91 5.17E-03 AMDHD1 1.19 3.19E-03 chi-miR-141 −6.46 5.90E-03 XG −1.22 5.27E-03 chi-miR-34b-5p −9.82 6.78E-03 COL6A6 1.77 6.14E-03 chi-miR-451-5p −7.05 6.81E-03 MFAP5 −1.67 6.36E-03 chi-miR-1 −7.02 1.25E-02 RNASE6 −1.33 7.43E-03 chi-miR-496-5p 4.43 2.06E-02 CCL21 −1.01 9.18E-03 chi-miR-34c-5p −7.86 2.22E-02 ADAM33 −1.04 1.01E-02 Zou et al. BMC Genomics (2020) 21:267 Page 6 of 15 Zou et al. BMC Genomics (2020) 21:267 Page 6 of 15 Table 3 The top 10 DEmRNAs and DEmiRNAs in Uni-S vs Mul-S (a) and Uni-L vs Mul-L (b) DEmiRNAs log2fold change P-value DEmRNAs log2fold change P-value a chi-miR-122 −12.44 1.24E-03 ARL4C −1.59 7.98E-07 chi-miR-451-5p −9.01 9.45E-03 WNT5B −1.65 2.25E-06 chi-miR-206 −11.91 1.07E-02 MMP9 −2.55 3.15E-06 chi-miR-141 −7.94 1.18E-02 TGFBI −1.55 1.64E-05 chi-miR-182 −10.62 1.39E-02 S100A12 −3.75 5.93E-05 chi-miR-200a −7.77 3.18E-02 INSL3 2.39 8.34E-05 chi-miR-184 −11.45 3.83E-02 MAP7D2 1.32 8.68E-05 LFNG −1.35 8.70E-05 SDC1 −1.14 9.74E-05 NT5E 1.03 1.16E-04 b chi-miR-182 −10.83 1.61E-03 BRINP3 −1.52 3.59E-04 chi-miR-122 −10.73 2.17E-03 DPT −1.81 8.07E-04 chi-miR-133b −9.62 3.12E-03 SPOCK2 −1.37 1.09E-03 chi-miR-206 −12.91 5.17E-03 AMDHD1 1.19 3.19E-03 chi-miR-141 −6.46 5.90E-03 XG −1.22 5.27E-03 chi-miR-34b-5p −9.82 6.78E-03 COL6A6 1.77 6.14E-03 chi-miR-451-5p −7.05 6.81E-03 MFAP5 −1.67 6.36E-03 chi-miR-1 −7.02 1.25E-02 RNASE6 −1.33 7.43E-03 chi-miR-496-5p 4.43 2.06E-02 CCL21 −1.01 9.18E-03 chi-miR-34c-5p −7.86 2.22E-02 ADAM33 −1.04 1.01E-02 Validation by quantitative real-time polymerase chain reaction (qRT-PCR) Validation by quantitative real-time polymerase chain reaction (qRT-PCR) Discussion Accordingly, TNFAIP6 and CYP11A1 expres- sion was upregulated in the small follicles of multiple goats, while CD36 expression was downregulated, indicating that there were more small follicles that could grow into domin- ant follicles in multiple goats. Thus, these genes might play a key role in the ovulation rate or kidding rate in goats. CD36 knockdown has been shown to increase proliferation and expression of survival and angiogenic markers in GCs [70]. In this study, CD36 expression was shown to be downregulated in multiple goats and it was associated with specific hematopoietic cell lineages, suggesting that the low expression of CD36 expression in multiple individuals may promote follicular maturation by stimulating GCs prolifera- tion or angiogenesis, which then increases the kidding rate. Overall, TNFAIP6 and CYP11A1 played a positive role in the regulation of ovulation, while CD36 contributed to fol- licular atresia. Accordingly, TNFAIP6 and CYP11A1 expres- sion was upregulated in the small follicles of multiple goats, while CD36 expression was downregulated, indicating that there were more small follicles that could grow into domin- ant follicles in multiple goats. Thus, these genes might play a key role in the ovulation rate or kidding rate in goats. TNFAIP6 is a secretory protein of the hyaluronan- binding protein family that played a role in CC stabilization and expansion, and it was upregulated in bovine GCs dur- ing ovulation [47, 61–63]. TNFAIP6-deficient female mice were sterile [44]. The present study reported that TNFAIP6 expression was 8-fold higher in Mul-S than in Uni-S, sug- gesting a possible role of TNFAIP6 in CC expansion in the small follicle of multiple goats during the estrus phase. CYP11A1 played a key role in the regulation of steroid- producing pathways in GCs [64]. The first step in steroid biosynthesis was the conversion of cholesterol into preg- nenolone through the action of CYP11A1 in the mitochon- dria, and then pregnenolone acted as a substrate for progesterone synthesis through the mediation of 3b-HSD expression [65, 66]. In this study, CYP11A1 expression was found to be upregulated in the small follicle of multiple goats, which was consistent with previous reports [67]; this observation demonstrates that CYP11A1 might play multiple roles in goat ovarian development. CD36 was a multifunctional receptor-binding autocrine growth factor that could regulate angiogenesis, cell growth, and adhesion. Discussion Exploring the genetic mechanism associated with ovulation rate is important to improve kidding rate, which is funda- mental to goat production. The greater number of large fol- licles that stimulate ovulation is believed to be the primary reason for higher ovulation rate [39]. Although several mRNAs and miRNAs have been found to play critical roles in ovarian biological processes, the majority of the genes Zou et al. BMC Genomics (2020) 21:267 Page 7 of 15 Table 4 Top 5 GO terms of DEmRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L Term gene count P-value Term gene count P-value Uni-S vs Mul-S Uni-L vs Mul-L Biological process Immune system process 45 3.10E-07 Anterior/posterior pattern specification 11 1.20E-07 Immune response 24 6.90E-05 Definitive hemopoiesis 4 9.30E-06 Steroid biosynthetic process 7 1.20E-04 Regionalization 11 1.10E-05 Organic hydroxy Compound biosynthetic process 8 2.40E-04 Pattern specification process 12 1.10E-05 Superoxide anion generation 4 3.00E-04 Skeletal system morphogenesis 9 1.80E-05 Cellular component Cell surface 18 4.40E-05 Extracellular region part 13 4.60E-04 Extracellular region 24 7.00E-05 Extracellular region 14 5.70E-04 Extracellular space 18 1.20E-04 Extracellular matrix 5 2.38E-03 Extracellular region part 21 1.70E-04 Cell periphery 25 6.60E-03 Plasma membrane 45 1.08E-03 Protein C inhibitor-TMPRSS7 complex 1 6.88E-03 Molecular function Deaminase activity 3 4.90E-04 Icosanoid receptor activity 2 1.20E-03 Receptor activity 17 6.30E-04 Chloride channel activity 3 2.70E-03 Hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in cyclic amidines 3 6.40E-04 Drug binding 3 4.20E-03 Glycogen binding 2 6.50E-04 Chloride transmembrane transporter activity 3 4.20E-03 Receptor binding 24 6.70E-04 Anion channel activity 3 4.80E-03 In addition, TNFAIP6, CYP11A1 and CD36 were found to be differentially expressed when comparing Uni-S vs Mul- S, while PTGFR and SERPINA5 were found to be differen- tially expressed when comparing Uni-L vs Mul-L. CD36 knockdown has been shown to increase proliferation and expression of survival and angiogenic markers in GCs [70]. In this study, CD36 expression was shown to be downregulated in multiple goats and it was associated with specific hematopoietic cell lineages, suggesting that the low expression of CD36 expression in multiple individuals may promote follicular maturation by stimulating GCs prolifera- tion or angiogenesis, which then increases the kidding rate. Overall, TNFAIP6 and CYP11A1 played a positive role in the regulation of ovulation, while CD36 contributed to fol- licular atresia. Discussion DEmRNAs were screened according to FPKM > 1 of at least three samples per group. Ellipses and triangle represent DEmRNAs and DEmiRNAs, respectively. Red and blue colored nodes represent upregulation and downregulation, respectively Fig. 3 The original and selected DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S and Uni-L vs Mul-L. a The original DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S. b The original DEmRNA-DEmiRNA interaction network of Uni-L vs Mul-L. c The selected DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S. DEmRNAs were screened according to FPKM > 1 of at least three samples per group. d The selected DEmRNA-DEmiRNA interaction network of Uni-L vs Mul-L. DEmRNAs were screened according to FPKM > 1 of at least three samples per group. Ellipses and triangle represent DEmRNAs and DEmiRNAs, respectively. Red and blue colored nodes represent upregulation and downregulation, respectively in basic reproductive activities. miR-200a was frequently overexpressed and is closely related to the migratory, pro- liferative, and invasive abilities of OC cells [80–82]. miR- 141 was shown to be significantly upregulated in OC cell lines and advanced metastatic OC [83, 84], and can inhibit GC apoptosis by targeting DAPK1 through the MAPK sig- naling pathway, leading to the development of polycystic ovary syndrome (PCOS) [85]. In this study, miR-200a and miR-141 expression were upregulated in the small follicles of uniparous goats, suggesting that these miRNAs might affect normal GC development, thereby affecting follicular development. miR-206 and miR-1 were potential tumor suppressors that have been shown to be downregulated in OC tissues, to inhibit c-Met expression, and to regulate cell proliferation, migration, and invasion [86, 87]. And miR-206 has also been shown to induce apoptosis [88, 89]. Here, miR-206 and miR-1 expression was downregu- lated in the large follicles of multiple goats, which may be related to oocyte maturation and ovulation in goats. Mem- bers of the miR-133 family (miR-133a-3p and miR-133b) have been shown to be involved in the regulation of vari- ous cellular processes, such as cell proliferation, apoptosis, SERPINA5 expression was shown to be downregulated in ovarian cancer (OC) [77–79]. Here, we found that SER- PINA5 expression was upregulated in the large follicles of multiple goats, suggesting that SERPINA5 may affect fol- licular development and the kidding rate in goats. Taken together, downregulation of PTGFR and upregulation of SERPINA5 in large follicles may represent useful strategies for increasing the ovulation rate in multiple goats. Discussion The expression of CD36 was found to be follicle-type dependent with the greatest expression in atretic follicles, and the lowest expresssion in healthy follicles [68–71]. PTGFR was a regulatory factor in follicular development that affected mammalian reproductive pathways [72–74]. In the GCs of periovulatory follicles of mice, the expres- sion of PTGFR was shown to be drastically reduced [75].. Our results showed that PTGFR expression was downreg- ulated in the large follicle from multiple goats, suggesting that the greater reduction of PTGFR expression observed in multiple goats may contribute to a the higher ovulation rate. Previous studies have reported that SERPINA5, a protease inhibitor, was expressed in the reproductive tract of adult mice and in the GCs of bovine follicles and that was highly expressed in bovine healthy follicles [60, 76]. Zou et al. BMC Genomics (2020) 21:267 Page 8 of 15 Fig. 3 The original and selected DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S and Uni-L vs Mul-L. a The original DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S. b The original DEmRNA-DEmiRNA interaction network of Uni-L vs Mul-L. c The selected DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S. DEmRNAs were screened according to FPKM > 1 of at least three samples per group. d The selected DEmRNA-DEmiRNA interaction network of Uni-L vs Mul-L. DEmRNAs were screened according to FPKM > 1 of at least three samples per group. Ellipses and triangle represent DEmRNAs and DEmiRNAs, respectively. Red and blue colored nodes represent upregulation and downregulation, respectively action network of Uni-S vs Mul-S and Uni-L vs Mul-L. a The original DEmRNA-DEmiRNA mRNA-DEmiRNA interaction network of Uni-L vs Mul-L. c The selected DEmRNA-DEmiRNA eened according to FPKM > 1 of at least three samples per group. d The selected . DEmRNAs were screened according to FPKM > 1 of at least three samples per group. ectively. Red and blue colored nodes represent upregulation and downregulation, respectively Fig. 3 The original and selected DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S and Uni-L vs Mul-L. a The original DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S. b The original DEmRNA-DEmiRNA interaction network of Uni-L vs Mul-L. c The selected DEmRNA-DEmiRNA interaction network of Uni-S vs Mul-S. DEmRNAs were screened according to FPKM > 1 of at least three samples per group. d The selected DEmRNA-DEmiRNA interaction network of Uni-L vs Mul-L. Discussion a The expression level of six genes were validated by qRT-PCR and compared with the results of RNA-seq. 3BHSD, STAR and LEPR were selected from Uni-S vs Mul-S, and CCL21, RARRES1 and DPT were selected from Uni-L vs Mul-L. b The expression level of five miRNAs were validated by qRT-PCR and compared with the results of RNA-seq. These five DEmiRNAs were selected from Uni-L vs Mul-L. Data were presented as expression values of genes and miRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L. For qRT-PCR data, mRNA expression was normalized to β-actin in the same cDNA sample, and miRNA expression was normalized to U6 Comparing Uni-S with Mul-S and Uni-L with Mul-L, miR-182 and miR-122 were found to be differentially expressed in both groups. miR-182 was shown to be up- regulated in the follicular fluid of patients with PCOS [94], and its expression was significantly increased in OC cell lines [95]. miR-122 inhibited epithelial mesenchymal tran- sition by regulating P4HA1 expression in OC cells [43]. In addition, miR-122 regulated LHCGR expression, which was crucial for mediating LH action in growing follicles via modulating LRBP levels during FSH-induced follicle growth [96]. miR-122 has been shown to increase LHR mRNA levels by modulating the expression of LRBP through the regulation of SREBP activation, which was migration, and invasion [90, 91]. FOXL2 was a conserved, early-acting gene in vertebrate ovarian development, and it played an important role in GC proliferation and oocyte maturation [68, 69]. Recently, FOXL2 expression has been reported to be regulated by miR-133b. miR-133b can bind to the FOXL2–3′UTR in GCs to inhibit the expression of the downstream genes, STAR and CYP19A1, which simul- taneously promoted estrogen secretion in GCs [92]. miR- 133b has been shown to be upregulated more than 30- fold in metaphase I oocytes after IGF-1 treatment, and it may play important roles in oocyte growth and matur- ation by regulating the expression of its potential target gene TAGLN2 [93]. migration, and invasion [90, 91]. FOXL2 was a conserved, early-acting gene in vertebrate ovarian development, and it played an important role in GC proliferation and oocyte maturation [68, 69]. Recently, FOXL2 expression has been reported to be regulated by miR-133b. miR-133b can bind to the FOXL2–3′UTR in GCs to inhibit the expression of the downstream genes, STAR and CYP19A1, which simul- taneously promoted estrogen secretion in GCs [92]. Discussion g g We identified 17 and 16 DEmiRNAs in the Uni-S vs Mul-S and Uni-L vs Mul-L groups, respectively. The miR- NAs, miR-200a, miR-451-5p, miR-141, miR-182, miR-206, and miR-122 were highly expressed in the small follicles of multiple goats, while miR-1, miR-206, miR-133a-3p, miR-133b, miR-182, miR-215-5p, miR-122, and miR-451- 5p were highly expressed in the large follicles of multiple goats. However, only miR-200a has been previously re- ported to be highly expressed in goat ovaries [1], demon- strating that the the independence of the expression of miRNAs in whole ovary, small follicles, and large follicles. Of these 14 highly expressed DEmiRNAs, miR-200a, miR- 141, miR-1, miR-206, miR-133b, miR-133a-3p, miR-182, and miR-122 have been reported to play important roles Zou et al. BMC Genomics (2020) 21:267 Page 9 of 15 migration, and invasion [90, 91]. FOXL2 was a conserved, Comparing Uni-S with Mul-S and Uni-L with Mul-L, Fig. 4 Verification of differently expressed genes and miRNAs by qRT-PCR. a The expression level of six genes were validated by qRT-PCR and compared with the results of RNA-seq. 3BHSD, STAR and LEPR were selected from Uni-S vs Mul-S, and CCL21, RARRES1 and DPT were selected from Uni-L vs Mul-L. b The expression level of five miRNAs were validated by qRT-PCR and compared with the results of RNA-seq. These five DEmiRNAs were selected from Uni-L vs Mul-L. Data were presented as expression values of genes and miRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L. For qRT-PCR data, mRNA expression was normalized to β-actin in the same cDNA sample, and miRNA expression was normalized to U6 Fig. 4 Verification of differently expressed genes and miRNAs by qRT-PCR. a The expression level of six genes were validated by qRT-PCR and compared with the results of RNA-seq. 3BHSD, STAR and LEPR were selected from Uni-S vs Mul-S, and CCL21, RARRES1 and DPT were selected from Uni-L vs Mul-L. b The expression level of five miRNAs were validated by qRT-PCR and compared with the results of RNA-seq. These five DEmiRNAs were selected from Uni-L vs Mul-L. Data were presented as expression values of genes and miRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L. For qRT-PCR data, mRNA expression was normalized to β-actin in the same cDNA sample, and miRNA expression was normalized to U6 Fig. 4 Verification of differently expressed genes and miRNAs by qRT-PCR. Discussion miR- 133b has been shown to be upregulated more than 30- fold in metaphase I oocytes after IGF-1 treatment, and it may play important roles in oocyte growth and matur- ation by regulating the expression of its potential target gene TAGLN2 [93]. Page 10 of 15 Page 10 of 15 Page 10 of 15 Zou et al. BMC Genomics (2020) 21:267 crucial for regulating key reproductive processes, such as ovulation and CL function [97]. In this study, both miR- 182 and miR-122 were upregulated in uniparous goats, suggesting that they may affect the ovulation rate by af- fecting follicular growth in uniparous goats. access to food and water. Five goats were uniparous with only one kid per birth, and six goats were multiple with an average of 2.8 kids per birth. Mul: the six goats had three litters whose kidding ≥2. Uni: the five goats had three litters whose kidding = 1. Taken together, the genes CD36, TNFAIP6, CYP11A1, SERPINA5, and PTGFR were reported to be crucial for regulating the proliferation, migration, invasion and apop- tosis of follicular somatic cells. The present study showed that TNFAIP6, CYP11A1, and CD36 were found to be dif- ferentially expressed when comparing Uni-S with Mul-S, and PTGFR and SERPINA5 were found to be differentially expressed when comparing Uni-L with Mul-L, suggesting that they may affect follicular development by affecting the growth of follicular somatic cells in goats. Further- more, we predicted the target genes for DEmiRNAs and some of the targets showed high expression including CD36 (miR-122, miR-200a), TNFAIP6 (miR-200a, miR- 182), CYP11A1 (miR-122), SERPINA5 (miR-1, miR-206, miR-133a-3p, and miR-133b), and PTGFR (miR-182 and miR-122); these expression regulation mechanisms may be related to ovulation and kidding rates. In order to achieve estrus synchronization, each goat was injected intramuscularly with 0.1 mg chloroprostenol. Eighteen days later, male goats (vasectomy and ligation of vas deferens) were used to confirm whether the estrous was synchronized. At 24 h after estrus (in the middle of es- trus), all goats were weighed and slaughtered at a local slaughterhouse. The intact ovaries were rapidly collected and washed with 75% alcohol thrice. Then they were soaked into phosphate buffered saline (PBS). The follicles were achieved by using micro-blades and tweezers under surgical dissecting microscope within 30 min. Discussion Isolated folli- cles from each ovarian were washed with PBS to eliminate debris, then froze in liquid nitrogen instantly and stored at −80 °C for generating RNA libraries. The small follicles (S, d < 3 mm) or large antral follicles (L, d > 1 cm) were isolated from the ovarian stromal tissues with tweezers by immersing the ovary in PBS, and then placed in liquid nitrogen (Table 5). The small and large follicles were col- lected at the same time within 30 min. mRNA sequencing and data processing A total of 3 μg RNA per sample was used as input ma- terial for removing ribosomal RNA using the Ribo-Zero Magnetic kit (EpiCentre, Madison, WI, USA). RNA was Table 5 Sample characteristics Index Number a(n) Age (years old) Sb (d < 3 mm) /per onec Ld (d > 1 cm) /per onee Uniparous (Uni) 5 3.5–4.5 8–10 1–2 Multiple (Mul) 6 3.5–4.5 8–10 1–3 a The number of uniparous (Uni) and multiple (Mul) goats selected in this experiment. b Small follicles c Eight to ten small follicles were collected and pooled from each goat d Large follicles e One to three large follicles were collected from each goat Table 5 Sample characteristics Index Number a(n) Age (years old) Sb (d < 3 mm) /per onec Ld (d > 1 cm) /per onee Uniparous (Uni) 5 3.5–4.5 8–10 1–2 Multiple (Mul) 6 3.5–4.5 8–10 1–3 Table 5 Sample characteristics Conclusions Identifying the specific subset of genes and miRNAs in- volved in follicular development is essential for fully com- prehending the cascade of events leading to ovulation and will likely contribute to an improved ability to control fertility. This study was the first to reveal the DEmRNAs and DEmiRNAs as well as their interaction in the follicles of uniparous and multiple goats at the estrus phase using RNA-seq technology. Numerous DEmiRNAs were more highly expressed in the small follicular libraries of multiple goats (miR-200a, miR-451-5p, miR-141, miR-182, miR- 206, and miR-122), and while other DEmiRNAs were more highly expressed in the large follicular libraries of multiple goats (miR-1, miR-206, miR-133a-3p, miR-133b, miR-182, miR-215-5p, miR-122 and miR-451-5p). The higher expression of TNFAIP6, CYP11A1 and CD36 in the small follicles of multiple goats, and the higher expression of PTGFR and SERPINA5 in the large follicles of multiple goats may play a critical role in goat prolificacy. Our find- ings provide a basic foundation for elucidating the regula- tory mechanisms of mRNAs and miRNAs in CZ black goats and a unique source for exploring the corresponding targets of the miRNAs in the future. RNA extraction and qualification Total RNA was extracted from the whole ovarian follicle using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA qual- ity was evaluated with the NanoDrop ND-2000 spectro- photometer (Thermo Fisher Scientific, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technolo- gies, Palo Alto, CA, USA). RNA integrity was evaluated using 1% agarose gel. Purified RNA was stored at −80 °C until further use. RNA with amount > 6 μg, concentra- tion ≥200 ng/mL, 1.8 < OD260/280 < 2.2, and RNA in- tegrity number (RIN) > 8.5 was used for the preparation of cDNA libraries. Animals and sample preparation In the present study, CZ goats were obtained from the South China Agriculture University, Guangdong, China. A total of eleven healthy female goats of the same age (about 3.5–4.5 years old) with more than three litters were raised under natural light conditions with free Page 11 of 15 Zou et al. BMC Genomics (2020) 21:267 Zou et al. BMC Genomics (2020) 21:267 Raw reads were processed with the script developed by consisting of index trimming, read alignment, and read counting. To obtain clean reads, raw reads were further filtered according to the following rules: (1) Removing low quality reads containing more than one low quality (Q- value ≤20) base or containing unknown nucleotides (N); (2) Removing reads without 3′ adapters; (3) Removing reads containing 5′ adapters; (4) Removing reads contain- ing 3′ and 5′ adapters but no small RNA fragment between them; (5) Removing reads containing poly A in the small RNA fragment; and (6) Removing reads shorter than 18 nt (not including adapters). The resected clean reads were mapped to the goat reference genome (GCF_001704415.1_ ARS1) Ensembl V96 using miRDeep2, in which the map- per.pl program invokes Bowtie for the alignment between the de-repeat sequence and the reference genome sequence. The de-repeat sequences were aligned to the ma- ture miRNA and precursor miRNA sequences of the spe- cies in the miRBase (http://www.mirbase.org/) [98], and the detected miRNA was annotated. Using mireap to analyze unannotated sequences of information, a new miRNA pre- diction analysis was carried out. According to the number of mature miRNA sequences of this species, the read count values of miRNA were calculated. then fragmented into 200–300 bp by ion interruption. The first cDNA strand was synthesized using 6-base random hexamer primers and reverse transcriptase, and the sec- ond cDNA strand was synthesized with dUTP instead of dTTP. The library was constructed and amplified accord- ing to the size of the fragments (300–400 bp) by PCR using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The hybrid library was uniformly di- luted to 2 nM through proportionally mixing the libraries containing different index sequences and forming a single chain library using TruseqTM RNA sample prep kit (Illu- mina, San Diego, CA, USA). The libraries were sequenced by paired-end sequencing on the HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). Identification of DEmRNAs and DEmiRNAs For RNA-seq analysis, the criteria for measuring mRNA and miRNA expression levels were FPKM and CPM (CPM = C/N × 1,000,000; C is the total number of reads mapped onto the gene, and N is the total number of mapped reads) values, respectively. Differential expres- sion in each group was identified using DESeq version 1.18.0 with R package, and DEmRNAs and DEmiRNAs were identified with the cut-off criteria of \|log2Fold- Change\| > 1 and P-value < 0.05. After trimming the raw reads, the clean reads were mapped to the goat reference genome (GCF_001704415.1_ ARS1) Ensembl V96 using Tophat2. The mismatch of de- fault reads and reference genomic sequence was within 2, and the mapping ratio is generally higher than 70%. The volcano plots of differentially expressed genes were generated using the ggplots2 package in R. All genes and samples were clustered using the heatmap package in R software. The Euclidean distance was cal- culated based on the expression level of the same gene in different samples and the expression patterns of dif- ferent genes in the same sample. The complete linkage hierarchical clustering method, which uses the largest intercluster distance, was used for clustering. Shared DEmRNAs or DEmiRNAs were identified in both Uni-S vs Mul-S and Uni-L vs Mul-L groups. Small RNA library construction, sequencing, and data processing Following extraction and purification, about 2 μg of total RNA per sample was used to construct the small RNA library using the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego CA, USA). All libraries for high- throughput sequencing of miRNA were amplified using PCR by adding the sequencing connector and the index part. Next, the 18–36 nucleotide RNA was purified using 6% Novex TBE PAGE gel (1.0 mm, 10 well) and quanti- fied using the Agilent 2100 Bioanalyzer. Single-stranded cDNA template was subjected to bridge PCR followed by Illumina single-end sequencing on the HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). Prediction of the target genes of the DEmiRNAs and construction of mRNA-miRNA interaction network Systematic bioinformatic analysis was developed based on possible functional relationships between DEmiRNAs and DEmRNA. In this study, miRanDa was used to pre- dict the target genes of DEmiRNAs using the 3’UTR mRNA sequence of the species as the target sequence. Animals and sample preparation g By using base calling, raw data obtained from high- throughput RNA-seq were translated into raw FASTQ se- quence data. Raw reads of FASTQ format were then proc- essed with Perl scripts to assess the quality of data used for subsequent analysis. To obtain clean reads from RNA-seq data for mRNA analysis, low quality sequences, including adaptor sequences, sequences with quality score < 20, and sequences with N base rate of raw reads > 10% were re- moved using cutadapt (http://cufflinks.cbcb.umd.edu/). Next, low quality sequences, including adaptor sequences, sequences with quality score < 20, sequences with N base, and sequences less than 18 bp were removed using Fastx- Toolkit (http://hannonlab.cshl.edu/f astx toolkit/). After fil- tering the raw reads, clean reads were obtained. Statistical analysis was performed to evaluate its quantity and quality, including Q30 (the percentage of the number of bases with phared score > 30 in the original data to the total number of bases) statistics, data quantity statistics, base content statistics, etc. Statistical analysis One-way analysis of variance was conducted using JMP 8.0 software (SAS Institute, Cary, NC). All results were expressed as means ± standard error, and P-values below 0.05 were considered to indicate statistically significant differences. Functional enrichment analysis GO (http://geneontology.org/) and KEGG (http://www. kegg.jp/) pathway enrichment analysis were used to analyze DEmRNAs and target genes of DEmiRNAs. GO and KEGG pathway analysis of the differentially expressed and target genes was performed with the software DAVID (https://david.ncifcrf.gov). Degree of enrichment was mea- sured by Rich factor, FDR, and the number of genes that were enriched in the pathway. Both GO terms and KEGG pathways were corrected. A P-value ≤0.05 was considered to be significantly enriched. RNA preparation and qRT-PCR First, total RNA was extracted from the ovarian follicles with the Total RNA Kit II (OMEGA, USA) for qRT-PCR analysis of DEmRNAs and DEmiRNAs. Second, the levels of DEmRNAs were measured using the PrimeScript® RT Reagent Kit with gDNA Eraser (TaKaRa, China) and qRT- PCR was performed using SYBR® Green PCR Supermix (Bio-Rad, USA). Each 20 μL reaction included 10 μL of SYBR® Green PCR Supermix, 1 μL of each divergent primer, 1 μL of cDNA, and 7 μL of RNase-free water. The cycling conditions included an initial single cycle (95 °C for 1 min), followed by 34 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. Melting-curve analysis was performed to verify the product identity. The levels of DEmiRNAs were then measured by qRT-PCR using miD- ETECT A Track™miRNA qRT-PCR Starter kit (RiboBio, Guangzhou, China). Each 20 μL reaction included 10 μL of SYBR Green Mix, 0.5 μL of each divergent primer, 1 μL of cDNA, and 8 μL of RNase-free water. The cycling condi- tions included an initial single cycle (95 °C for 10 min), Prediction of the target genes of the DEmiRNAs and construction of mRNA-miRNA interaction network Systematic bioinformatic analysis was developed based on possible functional relationships between DEmiRNAs and DEmRNA. In this study, miRanDa was used to pre- dict the target genes of DEmiRNAs using the 3’UTR mRNA sequence of the species as the target sequence. Zou et al. BMC Genomics (2020) 21:267 Page 12 of 15 Table 6 Primers of DEmRNAs Gene name Forward primer (5′-3′) Reverse primer (5′-3′) Product length/bp Annealing temperature mRNA version 3BHSD agggcatctcagtggtca ggataaagactggcacgcta 144 57.4 °C NM_001285716.1 LEPR ccattgagaagtatcagttcagtc catgctggtgtttttcatcatcttg 105 58.4 °C XM_018045220.1 STAR cagaagggtgtcatcagagc tgagcagccaggtgagttt 97 58.6 °C XM_013975437.2 CCL21 ccgaaagaagattcccgcca ggcgagaacaggatagctgg 90 60.1 °C XM_005684096.3 RARRES1 gcgcgtgggttaatcagaag acattaacagctggtctgggtt 148 59.8 °C XM_018048385.1 DPT gtaccagacatgctccaacaa ctgttgtcagccagcaggaa 137 59.4 °C XM_005690613.3 β-actin tgcttctaggcggactgatt tacaatcaaagtcctcggccac 106 59.7 °C NM_001314342.1 followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. Melting-curve analysis was performed to verify the product identity. The parameters used to determine miRNA-target pre- dicted interaction were the mapping score more than 140 and the free energy less than 1.0. Due to multiple target genes, data were re-filtered and the criterion was that the gene could only be retained in FPKM > 1 of at least three samples per group. Based on the re-filtered data, DEmRNA-DEmiRNA pairs in Uni-S vs Mul-S and Uni-L vs Mul-L were constructed and visualized using Cytoscape (v3.5.1) software. According to the results of RNA-Seq, 6 DEmRNAs (FPKM > 1) and 5 DEmiRNAs (CPM > 1) were selected for validation by qRT-PCR. Primers for DEmRNAs were designed by Premier 5 and obtained from Sangon Bio- tech (Shanghai, China), and primers for DEmiRNAs were designed and obtained from RiboBio Company (Guangzhou, China). Information regarding the quanti- tative primers used in this experiment is listed in Table 6. β-actin and U6 were used as endogenous controls for mRNA and miRNA, respectively, and all reactions were performed in triplicate. Relative expression levels were calculated using the 2−ΔΔCt method. The correlation between qRT-PCR and RNA-seq results was calculated with Microsoft Excel 2019. Acknowledgements We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript. 8. Buratini J, Price CA. Follicular somatic cell factors and follicle development. Reprod Fertil Dev. 2011;23(1):32–9. 8. Buratini J, Price CA. Follicular somatic cell factors and follicle development. Reprod Fertil Dev. 2011;23(1):32–9. Funding Th This research was supported by the Guangdong Provincial Department of Education Youth Innovative Talents Project (2017KQNCX014), Modern Agricultural Industrial Technology System of Guangdong Province (2019KJ127), the Guangdong Public Welfare Research and Capacity Building Project (2017B020201014, 2017A020208050), the Guangdong Provincial Promotion Project on Preservation and Utilization of Local Breed of Livestock and Poultry, and the Guangdong Special plan young top-notch talent (2015TQ01N843). 12. Wang X, Zou P, He Y, Meng K, Quan F, Zhang Y. Effect of luteinizing hormone on goat theca cell apoptosis and steroidogenesis through activation of the PI3K/AKT pathway. Anim Reprod Sci. 2018;190:108–18. 13. Zhang GM, Deng MT, Lei ZH, Wan YJ, Nie HT, Wang ZY, Fan YX, Wang F, Zhang YL. Effects of NRF1 on steroidogenesis and apoptosis in goat luteinized granulosa cells. Reproduction. 2017;154(2):111–22. 14. Han P, Xin H, Peng J, Hou J, Zhang L, Song Y, Li G, Cao B, An X. 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(D) Unsupervised clustering analysis showing expres- sion profiles of miRNAs between Uni-L and Mul-L groups. Additional file 2. RNA-seq data of DEmRNA expression in large and small follicles from uniparous and multiple goats. Additional file 3. RNA-seq data of DEmiRNA expression in large and small follicles from uniparous and multiple goats. Additional file 4. The significant Go term of DEmRNAs in large and small follicles from uniparous and multiple goats. Page 13 of 15 Page 13 of 15 Zou et al. BMC Genomics (2020) 21:267 genes between prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus). Gen Comp Endocrinol. 2013;187:1–5. genes between prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus). Gen Comp Endocrinol. 2013;187:1–5. Additional file 5. The significant KEGG pathways of DEmRNAs in large and small follicles from uniparous and multiple goats. 3. 3. Cui HX, Zhao SM, Cheng ML, Guo L, Ye RQ, Liu WQ, Gao SZ. 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https://openalex.org/W2605400505	https://respiratory-research.biomedcentral.com/track/pdf/10.1186/s12931-017-0538-5	English	null	Determining the presence of asthma-related molecules and salivary contamination in exhaled breath condensate	Respiratory research	2,017	cc-by	18,411	* Correspondence: Nichole.Reisdorph@UCDenver.edu Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 East Montview Boulevard, Aurora, CO 80045-2605, USA © 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. Determining the presence of asthma- related molecules and salivary contamination in exhaled breath condensate Charmion Cruickshank-Quinn, Michael Armstrong, Roger Powell, Joe Gomez, Marc Elie and Nichole Reisdorph* Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 DOI 10.1186/s12931-017-0538-5 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 DOI 10.1186/s12931-017-0538-5 Background pre-concentration, stable isotope dilution, and immu- noaffinity. Since the α-amylase activity in samples did not exceed 0.1% of the saliva activity, the investigators excluded significant salivary contamination of EBC. However, small amounts of saliva molecules can be de- tected by liquid chromatography tandem mass spectrom- etry (LC-MS/MS); Gaber et al reported that the main source of LTB4 detected in EBC was from saliva [25]. LTB4 and α-amylase activity were measured in saliva and α-amylase activity was measured in undiluted EBC. The authors observed that spiking EBC with saliva consistently increased LTB4 levels in EBC and concluded that α- amylase assay may not be sufficiently sensitive to show the presence of saliva in small quantities. Exhaled breath condensate (EBC) is comprised of volatile gases (e.g. nitric oxide) [1] and non-volatile compounds such as eicosanoids and cytokines [2]. EBC is increasingly used as a tool for biomarker discovery; it can be obtained non-invasively and reflects the physiology of the airway lining, thereby providing vital information about lung health. Publications have reported on the benefits of EBC as a quick screening tool for lung diseases such as asthma [3–6], pneumonia [5], chronic obstructive pulmonary dis- ease (COPD) [5–8], cystic fibrosis [4, 9, 10], and pneumo- coniosis [11]; these suggest the potential for EBC to be used in point-of-care diagnostics. Several EBC studies have focused on eicosanoids due to their known relationship to lung disease and the fact that these compounds are released from mast cells and eosinophils during inflammatory responses [12]. Leuko- triene B4 (LTB4), for example, has been shown to be re- leased by activated alveolar macrophages in sarcoidosis patients [13]. Moreover, levels of cysteinyl-leukotrienes (CysLT) and 8-isoprostane have been reported to be in- creased in EBC of moderate and severe asthma patients compared to healthy controls [14]. Antczek et al. [15] investigated CysLT, LTB4, prostaglandin E4 (PGE4), and 8-isoprostane in longitudinal EBC samples of 16 COPD patients at 4 time points: day 1, during treatment, after therapy, and when stable. Their results showed (1) a decrease in CysLT, LTB4, and 8-isoprostane after anti- biotic therapy and (2) that eicosanoids were elevated in the airways of stable COPD patients compared to healthy sub- jects. Although numerous studies demonstrate the potential for EBC in point-of-care diagnostics, eicosanoid detection in EBC can be marked by loss of analyte due to adsorption in plastic collection tubes. Abstract Background: Researchers investigating lung diseases, such as asthma, have questioned whether certain compounds previously reported in exhaled breath condensate (EBC) originate from saliva contamination. Moreover, despite its increasing use in ‘omics profiling studies, the constituents of EBC remain largely uncharacterized. The present study aims to define the usefulness of EBC in investigating lung disease by comparing EBC, saliva, and saliva-contaminated EBC using targeted and untargeted mass spectrometry and the potential of metabolite loss from adsorption to EBC sample collection tubes. Methods: Liquid chromatography mass spectrometry (LC-MS) was used to analyze samples from 133 individuals from three different cohorts. Levels of amino acids and eicosanoids, two classes of molecules previously reported in EBC and saliva, were measured using targeted LC-MS. Cohort 1 was used to examine contamination of EBC by saliva. Samples from Cohort 1 consisted of clean EBC, saliva-contaminated EBC, and clean saliva from 13 healthy volunteers; samples were analyzed using untargeted LC-MS. Cohort 2 was used to compare eicosanoid levels from matched EBC and saliva collected from 107 asthmatic subjects. Samples were analyzed using both targeted and untargeted LC-MS. Cohort 3 samples consisted of clean-EBC collected from 13 subjects, including smokers and non-smokers, and were used to independently confirm findings; samples were analyzed using targeted LC-MS, untargeted LC-MS, and proteomics. In addition to human samples, an in-house developed nebulizing system was used to determine the potential for EBC samples to be contaminated by saliva. Results: Out of the 400 metabolites detected in both EBC and saliva, 77 were specific to EBC; however, EBC samples were concentrated 20-fold to achieve this level of sensitivity. Amino acid concentrations ranged from 196 pg/mL – 4 μg/mL (clean EBC), 1.98 ng/mL – 6 μg/mL (saliva-contaminated EBC), and 13.84 ng/mL – 1256 mg/mL (saliva). Eicosanoid concentration ranges were an order of magnitude lower; 10 pg/mL – 76.5 ng/mL (clean EBC), 10 pg/mL – 898 ng/mL (saliva-contaminated EBC), and 2.54 ng/mL – 272.9 mg/mL (saliva). Although the sample size of the replication cohort (Cohort 3) did not allow for statistical comparisons, two proteins and 19 eicosanoids were detected in smoker vs. non-smoker clean-EBC. Conclusions: We conclude that metabolites are present and detectable in EBC using LC-MS; however, a large starting volume of sample is required. Keywords: EBC, Saliva, Lung, Metabolomics, Proteomics, LC-MS, Amino acids, Eicosanoids, Leukotriene, Asthma Page 2 of 22 Page 2 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Background Therefore, recent investigators have examined the use of glass tubes coated with surfac- tants, such as Tween 20, as an alternative to improve eicos- anoid analysis in EBC [16]. We sought to define the usefulness of EBC in investi- gating lung disease by characterizing its constituents and delineating if the molecules reportedly detected in EBC are a result of saliva contamination during sample col- lection. This was achieved using three strategies. First, we used an in-house developed nebulizing system to val- idate methods and to determine loss of molecules during collection. Second, since they have been reported as in- creased in lung disease, we specifically measured amino acids and eicosanoids in saliva and EBC in both healthy and asthmatic individuals. Finally, we used untargeted metabolomics and proteomics to determine the compo- nents of a highly concentrated EBC sample. RTube EBC simulation setup The RTube was constructed with an alternative material (Borosilicate glass) and the performance compared with the plastic RTube. The EBC simulation apparatus uti- lized the following components: syringe pump, a plastic 10 mL luer lock BD syringe, a 2 foot section of 0.17 mm PEEK tubing with an inline 2 μm frit and luer lock adapter, an Agilent electrospray nebulizer and column stand, nitrogen (98% pure or better) supplied at 10psi, an aluminum condenser (for plastic RTubes) stored at -80 °C, and water ice condenser fabricated with a zip-lock bag (for glass RTubes) stored at -80 °C. Before each simulation, the syringe, tubing and nebulizer assembly was rinsed with 200 μL of LC-MS methanol. 8-9 mL of low or high level standard was poured into the 10 mL syringe and placed into the syringe pump. The syringe was attached to the tubing and nebulizer. The gas port of the nebulizer was blocked with a blank nut. The syringe pump was set to pump 1.5 mL of standard solution for 5 min (0.3 mL/min). A first 5 min infusion was run and discarded to purge the line; a second 5 min infusion was run and collected in a 2 mL glass auto- sampler vial as a control sample. Nitrogen at 10 psi was then attached to the nebulizer. The syringe pump was op- erated for 20 s to observe and verify the nebulizer spray quality. A glass or plastic RTube was attached to the column stand. The nebulizer was then attached to the column stand with the nebulizer tip protruding 1-5 mm above the RTube duckbill. All standards and deuterated internal standards used for LC-MS/MS analysis of arachidonic acid, docosahex- aenoic acid derived lipid mediators were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All HPLC solvents and extraction solvents were HPLC grade or better. Study population Adults were recruited from asthmatic patients at National Jewish Health through flyers and through the asthma clinic. Studies were approved by the Western Institutional Review Board or National Jewish Health IRB. Informed consent was obtained from all participants. The 133 volunteers were adult males and females ranging in age 27-64 years old; subjects filled out questionnaires detailing the last time they ate, drank, brushed their teeth, flossed, or used mouthwash. In addition to eicosanoids, other EBC studies have fo- cused on the measurement of nitrogen oxide [17], glucose [10], proteins [18], and amino acids [19]. Amino acids have been shown to be markers of lung function [20], are dysregulated in COPD [21, 22], and are perturbed in smokers [23]. Although amino acids and eicosanoids have been reported in EBC [19, 24, 25], the contribution from saliva remains in question. Saliva contains more than 200 metabolites [26, 27] and has been suggested as a source of contamination in EBC during sample collection. Of the EBC collection devices commercially available, only two (ECoScreen, and TURBO-DECCS) contain saliva traps [28]. Cohort 1: This cohort included 13 healthy subjects with no pre-existing conditions who provided clean-EBC, saliva-EBC, and/or clean saliva. Not all subjects were able to provide both EBC and saliva: 80% of subjects who pro- vided clean-EBC provided saliva; 100% of subjects who provided saliva-EBC also provided saliva; 5 subjects who provided saliva did not provide EBC. Cohort 2: This cohort consists of 107 asthmatic sub- jects who had matched saliva and EBC collected. One subject who provided EBC did not provide saliva. Cohort 3: This cohort refers to a separate group of 13 subjects of various health statuses – healthy smoker, healthy non-smoker, common cold, nasal congestion – who all provided clean-EBC. The smoker group is desig- nated as smoking at least one cigarette per day, while healthy indicated no other pre-existing conditions. Because there is potential for saliva contamination in EBC, many investigators test for saliva contamination by measuring hydrolytic α-amylase activity. Syslova et al [29] investigated CysLTs using a rapid method comprising Page 3 of 22 Page 3 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Cruickshank-Quinn et al. Chemicals and reagents All solvents for untargeted mass spectrometry were LC- MS grade. Acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (Fairlawn, New Jersey); Water was purchased from Honeywell (Muskegon Michigan); 1-methylhistidine, 3-methylhistidine, α-amino- n-butyric acid, alanine, anserine, arginine, asparagine, aspartic acid, β-aminoisobutryic acid, β-alanine, carnosine, citrulline, creatinine, cystathionine, cysteine, ethanol- amine, γ-aminobutyric acid, glutamic acid, glutamine, glycine, histidine, homocystine, hydroxylysine, hydroxy- proline, isoleucine, L-aminoadipic acid, L-cystine, leucine, lysine, proline, methionine, ornithine, phenylalanine, phos- phoserine, phosphoethanolamine, sarcosine, serine, tau- rine, threonine, tryptophan, tyrosine, urea, and valine were purchased from Sigma Aldrich (St. Louis, Missouri); 10(S),17(S)-DiHDoHE (Protectin DX), 11β-PGF2α, 14(S)- HDHA, 15R-PGF2α, 17(S)-HDHA, 8-iso-15R-PGF2α, 8-iso-PGF2α, Lipoxin A4 (LXA4), LTB4, LTC4, LTD4, LTE4, PGE2, PGF2α, Resolvin D1 (RVD1), and Resolvin D2 (RVD2) were purchased from Cayman Chemical (Ann Arbor, Michigan). Study population Respiratory Research (2017) 18:57 RTube EBC simulation standards RTube EBC simulation standards The deuterated internal standard solution contained LTB4-d4, LTE4-d3 and PGE2-d4 at concentrations of 500 pg/ml in ethanol and stored at -20 °C until use. The calibration standards (LTB4, LTC4, LTD4, and LTE4) were prepared in LC-MS water to final concentrations of 5, 10, 25, 50, 100, and 250 pg/mL and they were kept on ice until use. Standards were prepared for analysis by adding 200 μL of deuterated internal standard solution and 800 μL of calibration standard into a 2 mL autosam- pler vial and vortexing for 5 s. The 10 pg/mL (low level) and 100 pg/mL (high level) calibration standards were used for infusion in the EBC simulation experiment. Additional demographic data is unavailable for the cohorts as patient samples were de-identified and add- itional information was no longer available following completion of the study. Because only minimal informa- tion was available, no statistical claims are being made based on health status, gender, or age in these cohorts. TURBO DECCS EBC simulation collection EBC simulation apparatus was the same as the RTube setup with the following changes: the 0.12 mm peek tub- ing was replaced with 0.17 mm stainless steel tubing to reduce back pressure on the syringe pump. Nitrogen was supplied at 60 psi which produced a finer spray to help prevent condensation in the PET tube. Before each simulation, the syringe, tubing and nebulizer assembly was rinsed with 200 μl of LC-MS methanol. The dispos- able DECCS sampling assembly was placed into the Turbo cooler and allowed to reach -5 °C +/-0.5 °C. 8-9 mL of low or high level standard was poured into the 10 mL syringe and placed into the syringe pump. The syringe was at- tached to the tubing and nebulizer. The gas port of the nebulizer was blocked with a blank nut. The syringe pump was set to pump 2 mL of standard for 7.5 min (0.3 mL/ min). The first 2 min of the infusion sample were dis- carded. The next three 2.0 mL infusions were collected in a 2 mL autosampler vial and set aside in 4 °C fridge until ready to aliquot for analysis. Nitrogen at 60 psi was then attached to the nebulizer. The syringe pump was operated for 20 s to observe and verify the nebulizer spray quality. The nebulizer was then placed into the assembly and the syringe pump was programmed to run for 10 min (infuse 3 mL of condensate). At the completion of the condensate collection, the 50 mL collection tube was centrifuged for 2 min and the condensate was aliquoted into a 2 mL auto- sampler vial for analysis. Immediately after collection, the saliva in the falcon tube was then centrifuged for 10 min at 3000 rpm at 4 °C to separate the clear liquid component of the saliva from the stringy mucus portion of the sample. The supernatant was pipetted into an amber autosampler vial for untar- geted LC-MS analysis. For targeted analysis, 1 mL of the centrifuged saliva was placed into a new falcon tube con- taining 50 μL of internal standard (LTB4-d4, LTC4-d5, LTD4-d5, LTE4-d5 and PGE2-d4 at 2.5 pg/μL in ethanol) and 4 mL of acetonitrile. Samples were then vortexed for 10 s. After addition of the acetonitrile to the clarified sal- iva, some additional stringy mucus may appear, so the samples were then allowed to settle. TURBO DECCS EBC simulation collection Taking care not to disturb the stringy mucus, 4 separate aliquots of the sample were pipetted into 1.5 mL centrifuge tubes and centrifuged at 14,000 rpm for 10 min at 4 °C. 950 μL of supernatant was removed and placed in a new centri- fuge tube. The sample was then frozen at -80 °C until analysis was performed. Prior to MS analysis, samples were dried in the centrifugal evaporator until ~200 μL of sample remained. The remaining supernatant was transferred to a 1.8 mL glass autosampler vial. 200 μL of ethanol was added to the centrifuge tube and vor- texed for ~5 s. The ethanol was transferred to the vial with the rest of the supernatant. The sample was then diluted to 1 ml with LC-MS water and analyzed. An initial experiment with the standard DECCS sam- pling device showed excessive adsorption of cysteinyl leukotrienes. The source of the adsorption was deter- mined using 3 separate experimental setups (n = 3 for each parameter). Setup #1: DECCS assembly minus the mouthpiece with a solvent-rinsed 30 mL glass Corex centrifuge tube placed inside the 50 mL plastic collection tube; Setup #2: Same as setup #1 but minus the PET tube. The nebulizer was placed directly on top of the diffusing tube and the collecting tube; Setup #3: Same as setup #2 minus the 30 mL glass Corex centrifuge tube. These dif- ferent parameters allowed the elimination of the PET tube EBC spike recovery experiments: evaluation of adsorption to glass versus plastic tubes To evaluate adsorption of eicosanoids during collection and analysis, simulated experiments were conducted with different types of collection tubes and analysis was performed with two EBC devices to determine com- pound adsorption to these different tubes. Glass and plastic collection tubes were tested with the RTube de- vice (a disposable collection system which separates sal- iva from the exhaled breath) (Respiratory Research, Inc., Charlottesville, VA). Glass with polyethylene terephthal- ate (PET), glass without PET and plastic without PET were tested with the TURBO DECCS device (a trans- portable unit for use in research on biomarkers obtained from disposable exhaled condensate collection systems) (Medivac, Italy). EBC production and deposition was simulated using the EBC sampling devices without hu- man subjects. The appropriate condenser was slid over the RTube and careful observation was made to ensure that the nebulizer needle was centered in the duckbill. The syringe pump was allowed to flow for 5 min. The glass RTube required a small amount of isopropanol (20-50 μL) to be injected with a spinal needle between the bottom of the duckbill and the walls of the condenser to prevent the duckbill from sticking to the walls during the EBC recovery. Con- densate was aliquoted into a 2 mL glass autosampler vial and placed on ice. Samples were prepared for analysis by adding 800 μL of condensate and 200 μL of deuterated Page 4 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 and the 50 mL collection tube as possible sites of adsorp- tion for the Cys-LTs. internal standard to a 2 mL glass autosampler vial and vortexing for 5 s. TURBO DECCS EBC simulation standards Each volunteer from Cohort 1 was provided with a 50 mL conical tube and saliva was allowed to flow natur- ally into the tube for 15-30 min. Approximately 3-5 mL of saliva was collected per volunteer. Samples were col- lected between 9:45 am and 10:30 am, approximately 2-3 h after eating breakfast. For Cohort 2, a “dirty” saliva sample was collected by having the subject spit into a 15 mL con- ical tube without rinsing their mouth. The subject then rinsed their mouth out 3 times with drinking water. A “clean” saliva sample was produced by having the subject chew a piece of chewing gum for 15-30 s, removing gum, and then spitting into 15 mL conical falcon tube; subjects were cautioned not to swish the sample in their mouth prior to spitting. Standards were prepared using the same protocol as the RTube experiments with the following changes: calibra- tion standards were prepared in LC-MS water to final concentrations of 1, 5, 10, 25, 50 pg/mL and kept on ice until use. All calibration standards were infused and collected in autosampler vials using the EBC simulation apparatus without nebulizer gas. Three additional aliquots of the 10 pg/mL standard were prepared for the EBC simulation experiments. EBC sample collection and preparation EBC control experiment A TURBO-DECCS (Medivac, Italy) apparatus was set up to mimic a human subject breathing into an EBC collec- tion tube (Fig. 2a) and included a saliva trap. A syringe was rinsed thoroughly with LC-MS grade water followed by LC-MS grade methanol and then attached to a syr- inge pump at one end. At the other end, the syringe was connected to the mouthpiece and secured with parafilm. Page 5 of 22 Page 5 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 The syringe pump flow rate was adjusted to allow 3 mL of sample to be collected in 15 min (average amount of EBC per person). A nitrogen dryer was attached to the side of the mouthpiece using tubing to mimic exhal- ation. Two blank samples were run through the device using 100% LC-MS grade water; sample was collected for analysis and referred to as the ‘blank water control’. This was followed by running spiked LC-MS grade water containing 42 amino acids (10 μM) and 16 eicosanoids (10 ng/mL) as a spiked control; this sample was desig- nated ‘spiked water control’. A new TURBO-DECCS nozzle and collection tube was used for each sample. Each nozzle and tube was discarded after use. trap was used to prevent saliva contamination in EBC sam- ples in Cohort 3 as well as visual observation of each sam- ple following collection. Water spiked with amino acids and eicosanoids (referred to as standards solution) was used as reference. Overall, samples were concentrated 50-fold and divided into aliquots for untargeted LC-MS, targeted LC-MS, and proteomics analysis. Quality control To reduce false positives due to cross contamination or carryover, a new column was used. Instrument blanks (100% methanol) were injected onto a new column and followed by ‘water blanks’ prior to sample analysis. Sam- ples were run in the following order: control “blank” water, clean-EBC, saliva-EBC, spiked water control sam- ple. Solvent blanks were run between each sample to eliminate carryover. Saliva samples were run last, followed by a series of additional blanks and laboratory instrument QCs to ensure that the instrument was oper- ating at optimal conditions. This process greatly reduced the chances of any potential carryover from any of the samples. g The lyophilized samples were each reconstituted in 1 mL of 80:20 methanol: water, briefly vortexed and centri- fuged. EBC samples were then dried down in a vacuum centrifugal concentrator at 55 °C for 1.5 h, reconstituted in 20 μL of LC-MS starting buffer (2% of 90:10 acetonitrile:- water with 0.1% formic acid), spun for 5 s to remove resi- due from the sides of the centrifuge tube, and transferred to an amber autosampler vial. The entire amount of each human-derived and control EBC sample was used for untargeted LC-MS analysis. For targeted analysis, immedi- ately after collection, 400 μL of internal standard (LTB4-d4, LTC4-d5, LTD4-d5, LTE4-d5 and PGE2-d4 at 0.125 pg/μL in ethanol) was added to the EBC, vortexed and then cen- trifuged for 10 minutes at 3000 rpm at 4 °C. The total con- densate volume was measured and recorded and split evenly between 2 centrifuge tubes. LC-MS water was added to each tube to reach a final volume of 1 mL. The samples were then frozen at -70 °C until analysis. Note that a saliva EBC sample collection and preparation p p p The TURBO-DECCS collection device (Medivac, Italy) was set up at room temperature and the condenser temperature was allowed to decrease until stable at -5.5 °C. This EBC collection device contains a saliva trap. A new, unused mouth piece and nozzle was sup- plied for each individual. Nose clips were optional. EBC was collected per ATS/ERS recommendations [30]. Subjects breathed normally into the EBC breathing ap- paratus for 15-20 min each. Subjects did not eat, drink, or exercise for at least two hours prior to sample col- lection. Approximately 2-5 mL of EBC was collected per person. For Cohort 1, samples were examined for possible salivary contamination and marked as “saliva” or “clean”. Human-derived EBC samples were pooled into two groups (‘clean’ EBC, 12.5 mL, (n = 5 subjects), and ‘saliva’ EBC, 8.5 mL (n = 3 subjects)) as shown in Fig. 2e, and aliquoted into 15 mL falcon tubes. Human- derived EBC samples and control EBC samples (blank water control and spiked water control) were immediately frozen at -80 °C overnight, and lyophilized the following morning. Untargeted metabolomics analysis Untargeted LC-MS Liquid chromatography was performed on an Agilent Series G2226A pump by injecting 5 μL sample onto an Agilent Zorbax SB-Aq Rapid Resolution HT 2.1 x 100 mm, 1.8 μm, 600 bar analytical column, coupled to an Agilent Zorbax SB-Aq Narrow-Bore 2.1 x 12.5 mm, 5 μm guard column. The autosampler tray temperature was set at 4 °C and column compartment was set at 30 °C. Samples were run at a flow rate of 0.3 mL/min, using mobile phase A (water with 0.1% formic acid), and mobile phase B (90:10 acetonitrile:water with 0.1% formic acid). Gradient elution was as follows: 0-3 min 2% B, 3-5 min 2-40% B, 5-20 min 40-100% B, 20-30 min 100% B, followed by column re- equilibration. An Agilent 6520 quadrupole time-of-flight mass spectrometer (Q-TOF-MS) was used to analyze sam- ples in positive and negative ionization mode at mass range 50-1700 m/z, scan rate 2.22 spectra/s, gas temperature 300oC, and gas flow 10 L/min. The nebulizer was 30 psi, skimmer 60 V, capillary voltage 4000 V, and fragmentor 120 V in positive mode and 140 V in negative mode, with reference masses 121.050873 and 922.009798 for positive mode and 112.985628 and 966.000725 for negative mode (Agilent reference mix). MS/MS analysis Tandem MS was performed on an Agilent 6560 IMMS Q-TOF for selected metabolites. MS/MS data was col- lected with a 500 ms/spectra acquisition time. Precursor ions were isolated with a 4 m/z isolation width and 1 min delta retention time. Collision energies of 10, 20, and 40 eV were applied. Fragmentation data was exported to the freely available NIST MS Search v.2.2 g GUI program [31] (NIST, Gaithersburg, MD, USA) and were matched to spectra in the NIST 14 Mass Spectral Library. This library contains 193,119 spectra representing 43,912 precursor ions and 8,351 compounds; a detailed description of the library is available [32]. Automated li- brary searching was performed using spectrum search type ‘Identity’, search with “MS/MS”, and default program settings. The search m/z tolerance was ± 0.4 for precursor ions and ± 0.4 for product ions without ignoring the pre- cursor ion. The MS search program outputted a list of matched chemical compounds including several measures of spectral similarity [33]. The Match Factor (MF) is the normalized dot product with square-root scaling of the ex- perimental mass spectrum and a library mass spectrum, using all the elements in the experimental mass spectrum. The Reverse Match Factor (RMF) is the normalized dot product with square-root scaling of the experimental mass spectrum and the library mass spectrum, but the elements that are not present in the library mass spectrum are not included. 100 μl of extracted saliva or EBC was injected onto an Agilent Poroshell EC-C18 2.1x5 mm 2.7 μm trapping column using pump 1 at 0.5 mL/min for 0.5 min with a solvent composition of 95% solvent A: 5% solvent B. At 0.51 min the switching valve changed the flow to the trapping column from pump 1 to pump 2. The flow was reversed and the trapped lipid mediators were eluted onto an Agilent Poroshell EC-C18 2.1x150 mm 2.7 μm analytical column using the following gradient at a flow rate of 0.4 mL/min: hold at 75% solvent A:25% solvent D from 0-0.5 min, then a linear gradient from 25-45% D over 8.5 min followed by an increase from 45-48% D from 8.5-11 min, then from 48% D to 65% D over 4 min, and then from 65-100% D in 0.01 min, finally holding at 100% D for 1 min. During the analytical gradient pump 1 washed the injection loop with 100% B for 4 min at 0.5 mL/min. Data extraction and analysis MassHunter Profinder software (Agilent) was used to analyze the spectral data. An initial naïve feature finding algorithm was used to detect and extract peaks present in the spectrum of the samples using the following parame- ters: peak heights ≥300 counts, ion species + H, +Na, +K in Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 6 of 22 Page 6 of 22 positive mode, -H, +Br, +HCOO, +CH3COO in negative mode, charge state maximum of 2, ion threshold of two or more ions, alignment using 0.3 min retention time and 20 ppm mass window, absolute height ≥1100 counts, MFE score ≥90, and a compound must be present in at least two sample files. A formula generation algorithm was used to re-mine the spectral data and reduce missing values using the following parameters: symmetric ppm of 20, matching score > 75, absolute peak height ≥1000 counts, absolute ion filter height ≥1000 counts, and a compound must be present in at least two sample files. Compounds were imported into Mass Profiler Professional (Agilent) using a minimum abundance threshold of 3000 counts. Solvent blanks and the unspiked water controls were background subtracted to eliminate any contamination from the sam- ples during sample collection, preparation or instrument analysis; this removed 13 compounds in control water and 2 compounds in the instrument blank from the EBC samples. positive mode, -H, +Br, +HCOO, +CH3COO in negative mode, charge state maximum of 2, ion threshold of two or more ions, alignment using 0.3 min retention time and 20 ppm mass window, absolute height ≥1100 counts, MFE score ≥90, and a compound must be present in at least two sample files. A formula generation algorithm was used to re-mine the spectral data and reduce missing values using the following parameters: symmetric ppm of 20, matching score > 75, absolute peak height ≥1000 counts, absolute ion filter height ≥1000 counts, and a compound must be present in at least two sample files. Compounds were imported into Mass Profiler Professional (Agilent) using a minimum abundance threshold of 3000 counts. Solvent blanks and the unspiked water controls were background subtracted to eliminate any contamination from the sam- ples during sample collection, preparation or instrument analysis; this removed 13 compounds in control water and 2 compounds in the instrument blank from the EBC samples. of ±0.5 min for retention time window and ±10 ppm for m/z. Compound identification Amino acids and eicosanoids in EBC and saliva were identified by matching their exact mass, isotope ratios, and retention times to purchased standards. An in-house database comprising METLIN, LIPID MAPS, Kyoto Encyclopedia of Genes and Genomes (KEGG), and human metabolome database (HMDB) was used to annotate add- itional detected metabolites in the untargeted data using exact mass, isotope ratio, and isotopic distribution with a mass error of ≤10 ppm and a minimum database score of 70/100. Spectra were manually inspected for quality. Targeted analysis of lipid mediators by LC-MS Quantitation of lipid mediators was performed using 2 dimensional reverse phase HPLC tandem mass spectrom- etry (LC-MS/MS). The HPLC system consisted of an Agi- lent 1260 autosampler (Agilent Technologies, Santa Clara, CA), an Agilent 1260 binary loading pump (pump 1), an Agilent 1260 binary analytical pump (pump 2) and a 6 port switching valve. Pump 1 buffers consisted of 0.1% for- mic acid in water (solvent A) and 9:1 v:v acetonitrile:water with 0.1% formic acid (solvent B). Pump 2 buffers con- sisted of 0.01% formic acid in water (solvent C) and 1:1 acetonitrile:isopropanol (solvent D). Data extraction and analysis Quantitation of amino acids and eicosanoids was based on peak areas of amino acids and eicosanoids in the samples against known spiked standards [34, 35]. MS/MS analysis Both the trapping column and the analyt- ical column were re-equilibrated at starting conditions for 4 min before the next injection. Mass spectrometric analysis was performed on an Agi- lent 6490 triple quadrupole mass spectrometer in nega- tive or positive ionization mode, based on compound chemistry (Additional file 1). The drying gas was 250 °C at a flow rate of 15 mL/min. The sheath gas was 350 °C at 12 mL/min. The nebulizer pressure was 35 psi. The Results Experimental set up for control experiments In order to test recoveries and determine background contamination, a system was developed that mimicked EBC collection. As shown in Fig. 1a, a syringe and syr- inge pump were attached to a nebulizer; this was used to administer sample at a controlled rate into a TURBO- DECCS EBC collection apparatus. An unspiked, “blank” water sample was used as a control to monitor contami- nants that may be present in the collection tubing. Water spiked with amino acids and eicosanoids was used to measure recoveries; this “spiked control” was also used as a reference to verify metabolite identities in EBC using mass and retention time. As expected, no compounds Proteomics analysis An aliquot of the EBC samples collected from the 13 volunteers in the replication study (Cohort 3) and grouped into 4 categories based on health status was used for proteomics analysis; 120 μL healthy non- smoker, 8 μL healthy smoker, 28 μL non-smoker com- mon cold, and 6.6 μL non-smoker nasal congestion. Samples were dried in a centrifugal evaporator at 45 °C. The 4 samples then underwent a trifluoroethanol (TFE) in-solution digestion overnight with trypsin. Digests were dried at 45 °C and resuspended in 10 μL of 3% acetonitrile with 0.1% formic acid. Adsorption of compounds on EBC collection tubes: leukotriene recovery experiment A second control experiment (Fig. 2) was performed to determine recoveries of commonly reported EBC mole- cules. Because plastic tubes may result in adsorption of certain molecules, glass and plastic tubes and the addition of a coating were compared. For this experi- ment, known amounts of leukotrienes (10 pg/mL and 100 pg/mL) were injected into TURBO DECCS tubes using the apparatus in Fig. 1; RTubes were attached to the syringe pump as shown in Fig. 2. Overall, recoveries of the leukotrienes ranged between 96.9 – 112% depending on the collection tube used (Fig. 2c). Recoveries were higher with the glass RTube device (58.4 – 98.9%) com- pared to the plastic RTube device (38.6 – 68.5%). In experi- ments using the TURBO DECCS, recoveries were higher using the glass tube without PET (48.01 – 83.58%), com- pared to the glass tube with PET (20.24 – 87.57%), and the plastic tube without PET (49.11 – 73.78%). These recovery experiments showed significant adsorption of cysteinyl leu- kotrienes to the plastic RTube by ~60% compared to the glass RTube (Fig. 2c). There was also 10-22% less adsorp- tion of the leukotrienes to the TURBO-DECCS plastic tube compared to the plastic RTube at the 10 pg/mL spike levels (Fig. 2d). In addition, polyethylene terephthalate (PET) caused significant adsorption of LTC4, LTD4, and LTE4 to the coated collection tubes compared to the uncoated tubes (Fig. 2d). 5 μL of each sample was injected onto a ProntoSil C18AQ (0.1X150mm) column from NanoLCMS Solu- tions. Samples were analyzed using a nanoAdvance nano flow LC (Bruker) on the front of an Impact HD Q-TOF (Bruker) with a gradient elution from 5-50% over 30 min at 40 °C and 800 nL/min flow rate. Buffer A was water with 0.1% formic acid, and buffer B was acetonitrile with 0.1% formic acid. Data was acquired at 2Hz over a range of 150-2200 m/z. Data was processed using DataAnalysis 4.2 (Bruker), database searches were performed with Mascot v2.4 (Matrix Science), and protein assessment/ scoring was performed with ProteinScape 3.1 (Bruker). Amino acid and eicosanoid data analysis Amino acid and eicosanoid data analysis The raw LC-MS .d files from the standards and samples were imported into MassHunter Quantitative Analysis Software (v.B.07.00) (Agilent Technologies, Santa Clara, CA) to extract m/z and retention times using tolerances Page 7 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 7 of 22 capillary voltage was 3500 V in negative mode and 4000 V in positive mode. were detected in the “blank” controls. In total, 30 out of the 42 spiked amino acids and 16 spiked eicosanoids were detected in the “spiked control” samples. Following method validation, EBC (n = 8) was collected from healthy volunteers, and underwent minimal sample preparation to reduce potential for contamination. EBC was divided into clean (n = 5) and saliva-contaminated (n = 3) based on vis- ual inspection of the samples. Note that spikes were not added to the EBC samples; this was in an effort to reduce false positives that may be present as a result of degrad- ation of spiked standards. Figure 1b, c and d show repre- sentative total ion chromatograms (TIC) of the blank, spiked water and “clean” EBC samples respectively. Figure 1e shows EBC in tubes following collection; saliva-contaminated EBC was clearly distinguishable from non-contaminated EBC. Data for lipid mediators was acquired in dynamic MRM mode using experimentally optimized collision energies obtained by flow injection analysis of authentic standards (Additional file 1). Calibration standards for each lipid mediator were analyzed over a range of concentrations from 0.04 pg/mL-8 pg/mL. Calibration curves for each lipid mediator were constructed using Agilent MassHunter Quantitative Analysis software. The results were calculated by obtaining the ratio of the target compound/internal standard and then using the linear equation obtained from the calibration curve (y = mX + b) to get the final concen- tration in pg/mL. Comparison of eicosanoids and amino acids in healthy saliva, healthy EBC, and healthy saliva-EBC Saliva and EBC were collected from 13 healthy subjects (Cohort 1) and analyzed using LC-MS. Table 1 shows concentrations of the amino acids and eicosanoids de- tected in EBC and saliva of these subjects. Additional file 2 shows the extracted peak areas of selected compounds and Additional file 3 shows the separation of PGF2αisomers. Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 8 of 22 Fig. 1 Experimental setup and total ion chromatograms (TIC) of exhaled breath condensate (EBC) controls. a Setup of the control experiment using a syringe and syringe pump to administer control unspiked water and a control spiked water sample through the TURBO-DECCS EBC collection apparatus; b TIC of unspiked water. * indicates a contaminant peak at 0.933 min which was putatively identified as propiolic acid (8.69 ppm error, 82.8 score), c TIC of spiked water; d TIC of a ‘clean’ EBC sample; e Visualization of EBC samples collected from volunteers showing the clean EBC from non-droolers, compared to EBC collected from droolers which show the presence of saliva in the samples Fig. 1 Experimental setup and total ion chromatograms (TIC) of exhaled breath condensate (EBC) controls. a Setup of the control experiment using a syringe and syringe pump to administer control unspiked water and a control spiked water sample through the TURBO-DECCS EBC collection apparatus; b TIC of unspiked water. * indicates a contaminant peak at 0.933 min which was putatively identified as propiolic acid (8.69 ppm error, 82.8 score), c TIC of spiked water; d TIC of a ‘clean’ EBC sample; e Visualization of EBC samples collected from volunteers showing the clean EBC from non-droolers, compared to EBC collected from droolers which show the presence of saliva in the samples Fig. 1 Experimental setup and total ion chromatograms (TIC) of exhaled breath condensate (EBC) controls. a Setup of the control experiment using a syringe and syringe pump to administer control unspiked water and a control spiked water sample through the TURBO-DECCS EBC collection apparatus; b TIC of unspiked water. Comparison of eicosanoids and amino acids in healthy saliva, healthy EBC, and healthy saliva-EBC * indicates a contaminant peak at 0.933 min which was putatively identified as propiolic acid (8.69 ppm error, 82.8 score), c TIC of spiked water; d TIC of a ‘clean’ EBC sample; e Visualization of EBC samples collected from volunteers showing the clean EBC from non-droolers, compared to EBC collected from droolers which show the presence of saliva in the samples Three amino acids (anserine, hydroxyproline, and cysteine) were undetected in all samples including the spiked water; these molecules may be below our limit of detection or are not detectable in our system (for example due to inability to ionize). Carnosine, homocystine, lysine, methionine, and phosphoethanolamine were only detected in the spiked water; this indicates that their concentration in EBC and saliva were below detection limits or that they may not be present in these biological fluids. Seven eicosanoids (pro- tectin DX, 17(S)-HDHA, LTC4, LTD4, LTE4, RvD1, and RvD2) were less than half the concentration in clean-EBC compared to saliva-EBC; they were an order of magnitude lower in concentration in clean-EBC compared to saliva. Overall, in the clean-EBC, the eicosanoids were present at concentrations ranging 10 pg/mL – 76.5 ng/mL; amino acids ranged from 196 pg/mL – 4 μg/mL. acids: 1-methylhistidine/3-methylhistidine, arginine, cys- tathionine, ethanolamine, glutamine, L-aminoadipic acid, L-cystine, leucine/isoleucine, ornithine, phenylalanine, phosphoserine, proline, sarcosine, taurine, urea, and val- ine. These also include eight eicosanoids: protectin DX, 17(S)-HDHA, LTC4, LTD4, LTE4, PGE2, RvD1, and RvD2. These compounds are most likely elevated due to salivary contamination of the EBC samples. Ten compounds were detected at similar concentra- tions in both the clean-EBC and the saliva-EBC samples. These were alanine, aspartic acid, citrulline, creatinine, γ-aminobutyric acid, glycine, hydroxylysine, LXA4, LTB4, and PGF2α. Four amino acids (α-aminobutyric acid, glu- tamic acid, threonine, tryptophan) and five eicosanoids (11β-PGF2α, 14(S)-HDHA, 15R-PGF2α, 8-iso-15R-PGF2α, 8-iso-PGF2α) had higher concentrations in the clean-EBC compared to the saliva-EBC samples. Four compounds (asparagine, taurine, tyrosine, RvD2) were undetected in EBC compared to saliva. Alanine was lower in concentra- tion in saliva compared to clean-EBC and saliva-EBC while an additional six compounds (cystathionine, 11β- Forty compounds were of higher concentration in sal- iva compared to clean-EBC or saliva-EBC. In addition, twenty-five compounds were present at higher concen- trations in the saliva-EBC samples compared to the clean-EBC. These included the following fifteen amino Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 9 of 22 Fig. Comparison of eicosanoids and amino acids in healthy saliva, healthy EBC, and healthy saliva-EBC 2 Experimental setup and recoveries of leukotrienes using various exhaled breath condensate (EBC) collection devices. a EBC simulation device connected to a syringe pump, showing the plastic tube and condenser; b Glass (left) versus plastic (right) EBC tube; c RTube spike recovery comparison for cysteinyl leukotrienes; d TURBO-DECCS spike recovery comparison for leukotrienes. PET: polyethylene terephthalate Fig. 2 Experimental setup and recoveries of leukotrienes using various exhaled breath condensate (EBC) collection devices. a EBC simulation device connected to a syringe pump, showing the plastic tube and condenser; b Glass (left) versus plastic (right) EBC tube; c RTube spike recovery comparison for cysteinyl leukotrienes; d TURBO-DECCS spike recovery comparison for leukotrienes. PET: polyethylene terephthalate PGF2α, 15R-PGF2α, 8-iso-15R-PGF2α, 8-iso-PGF2α, and PGE2) were undetected in the saliva samples but were de- tected in the clean-EBC and saliva-EBC. or annotated using molecular formula (Additional file 4). Tandem MS provided additional confidence in the identi- fication of acetylsalicylic acid, 4-chloro-L-phenylalanine, 3,4-furandicarboxylic acid, shikimic acid, succinic acid, and citric acid (Additional file 5). Analysis of eicosanoids in saliva and EBC of asthmatics LCMS-based metabolomics was used to determine what metabolites could be detected and identified in Cohort 1 (clean EBC, saliva-EBC, and saliva) using an untargeted approach. The overlap in metabolites from the LC-MS analysis of these samples is shown in Fig. 3. Overall, there were 400 metabolites detected in all samples; 306 were present in both saliva and saliva-EBC; 77 metabo- lites were detected in the clean-EBC and the saliva-EBC but were undetected in the saliva samples. From the 77 metabolites detected in the EBC and saliva-EBC samples, 40 metabolites were annotated using freely available small molecule databases (Table 2); 37 were either unannotated Analysis of eicosanoids in saliva and EBC of asthmatics Since EBC has potential as a clinical diagnostic in lung diseases, we sought to determine if previously reported eicosanoid molecules could be detected in clean EBC or if their detection was the result of saliva contamination. Matched saliva and EBC was collected from 107 asth- matic subjects (Cohort 2) for this purpose (Fig. 4). Eicos- anoid analysis was performed using multiple reaction monitoring (MRM) on a triple quadrupole mass spec- trometer (QQQ-MS). Sixteen eicosanoids comprised the panel as detailed in the methods. Only one molecule, 8- iso-15R-PGF2α, was detected in EBC and in only one Cruickshank-Quinn et al. Analysis of eicosanoids in saliva and EBC of asthmatics Compound identifiers are ChEBI except for 5 eicosanoids with CAS identifiers. aindicates undetected in all three sample groups In order to determine if eicosanoids were present in EBC but below the limit of detection, remaining EBC from the asthmatics subjects (n = 107) was pooled into a single 13 mL aliquot and spiked with five deuterated ei- cosanoid standards (PGE2-d4, LTB4-d4, LTC4-d5, LTD4- d5, LTE4-d5). The sample was lyophilized, reconstituted in 20 μL of HPLC buffer, and the entire amount ana- lyzed via LC-MS in full scan positive ionization mode. After subtracting solvent blanks, 97 metabolites were de- tected in EBC, including tentative identifications for LTE3, thromboxane, 11-trans-LTE4, 11-trans-LTC4, and 12-oxo-LTB4 (Additional file 6). While it is possible that these molecules were the result of degradation of the standards, this result also suggests that eicosanoids may be present in EBC, albeit at very low concentrations. subject. Conversely, ten out of the sixteen eicosanoids were detected in a significant number of saliva samples including 8-iso-15R-PGF2α (n = 47), 8-iso-PGF2α (n = 102), PGF2α (n = 56), PGE2 (n = 77), LTB4 (n = 105), LTC4 (n = 16), LTD4 (n = 13), LTE4 (n = 13), 17(S)-HDHA (n = 79), and 14(S)-HDHA (n = 106). One eicosanoid, 14(S)-HDHA, was detected in all 106 saliva samples; this may be due to its higher concentration levels (12 pg/mL – 4014 pg/mL) compared to the other eicosa- noids (0.15 pg/mL – 535 pg/mL). subject. Conversely, ten out of the sixteen eicosanoids were detected in a significant number of saliva samples including 8-iso-15R-PGF2α (n = 47), 8-iso-PGF2α (n = 102), PGF2α (n = 56), PGE2 (n = 77), LTB4 (n = 105), LTC4 (n = 16), LTD4 (n = 13), LTE4 (n = 13), 17(S)-HDHA (n = 79), and 14(S)-HDHA (n = 106). One eicosanoid, 14(S)-HDHA, was detected in all 106 saliva samples; this may be due to its higher concentration levels (12 pg/mL – 4014 pg/mL) compared to the other eicosa- noids (0.15 pg/mL – 535 pg/mL). Fig. 3 Overlap of metabolites detected in clean EBC, saliva-contaminated EBC, and saliva samples of healthy volunteers in Cohort 1. Untargeted metabolomics was performed on EBC and saliva from healthy volunteers. Metabolite peaks were extracted using MassHunter Profinder software (Agilent). Samples were filtered using a 3000 abundance cutoff and a presence in at least two of the three sample groups. Analysis of eicosanoids in saliva and EBC of asthmatics A total of 77 metabolites were determined to be unique to EBC Analysis of eicosanoids in saliva and EBC of asthmatics Respiratory Research (2017) 18:57 Page 11 of 22 Table 1 Amino acid and eicosanoid concentrations in healthy human saliva and healthy EBC (Continued) 17(S)-hydroxy Docosahexaenoic Acid C22H32O3 155976-53-7 1.07 4.28 798.09 8-iso-15R-PGF2α C20H34O5 214748-65-9 69.0 9.60 ND 8-iso-PGF2α C20H34O5 34505 72.3 9.60 ND Lipoxin A4 (LXA4) C20H32O5 6498 10.4 14.9 906 Leukotriene B4 (LTB4) C20H32O4 15647 1.56 1.78 105 Leukotriene C4 (LTC4) C30H47N3O9S 16978 3.68 59.4 10 898 Leukotriene D4 (LTD4) C25H40N2O6S 28666 0.26 2.85 175.71 Leukotriene E4 (LTE4) C23H37NO5S 15650 1.05 29.5 1842 Prostaglandin E2 (PGE2) C20H32O5 15551 3.87 31.0 ND Prostaglandin F2α (PGF2α) C20H34O5 15553 0.010 0.010 2.54 Resolvin D1 (RVD1) C22H32O5 81564 3.08 6.91 2193 Resolvin D2 (RVD2) C22H32O5 81565 ND 898.5 272 983 Saliva, clean-EBC, and saliva-EBC were collected from 13healthy volunteers (Cohort 1) as described in methods. Samples were pooled and 5 μL of each sample was injected onto an analytical column. Amino acids and eicosanoids were spiked into a control water sample and underwent the EBC sample collection procedure as shown in Fig. 2a. These authentic amino acid and eicosanoid standards were used to confirm compound identities in the EBC and saliva samples using exact mass, isotope ratios and retention time matching. Isomers could not be separated or differentiated using the LC-MS method described, and are listed together.NDindicates not detected. ng/mL indicates the calculated concentration of amino acids and eicosanoids in each of the pooled samples. Compound identifiers are ChEBI except for 5 eicosanoids with CAS identifiers. aindicates undetected in all three sample groups Table 1 Amino acid and eicosanoid concentrations in healthy human saliva and healthy EBC (Continued) eicosanoid concentrations in healthy human saliva and healthy EBC (Continued) Saliva, clean-EBC, and saliva-EBC were collected from 13healthy volunteers (Cohort 1) as described in methods. Samples were pooled and 5 μL of each sample was injected onto an analytical column. Amino acids and eicosanoids were spiked into a control water sample and underwent the EBC sample collection procedure as shown in Fig. 2a. These authentic amino acid and eicosanoid standards were used to confirm compound identities in the EBC and saliva samples using exact mass, isotope ratios and retention time matching. Isomers could not be separated or differentiated using the LC-MS method described, and are listed together.NDindicates not detected. ng/mL indicates the calculated concentration of amino acids and eicosanoids in each of the pooled samples. Analysis of eicosanoids in saliva and EBC of asthmatics Respiratory Research (2017) 18:57 Page 10 of 22 n healthy human saliva and healthy EBC Formula Identifier Clean-EBC(ng/mL) Saliva-EBC (ng/mL) Saliva (ng/ C7H11N3O2 70958/70959 518.0 1276.1 1 256 059 d C4H9NO2 35621 316.3 41.3 90 579 C3H7NO2 16449 58.1 51.9 13.84 C10H16N4O3 18323 ND ND ND C6H14N4O2 29016 1211.6 6016.8 101 238 C4H8N2O3 22653 ND 8.6 7575 C4H7NO4 22660 10.7 19.4 11 387 C9H14N4O3 15727 ND ND ND C6H13N3O3 18211 302.2 304.8 16 922 C4H7N3O 16737 237.5 396.9 88 368 C7H14N2O4S 17755 930.7 1756.2 ND C3H7NO2S 15356 ND ND ND C2H7NO 16000 52.5 230.9 20 887 C4H9NO2 16865 41.3 56.1 13 705 C5H9NO4 18237 77.0 29.6 49 725 C5H10N2O3 28300 24.6 162.4 18 423 C2H5NO2 15428 5.99 11.4 1739 C6H9N3O2 27570 401.2 316.5 14 751 C4H9NO2S 17485 ND ND ND C6H14N2O3 60175 205.6 283.3 133 890 C5H9NO3 24741 ND ND ND C6H11NO4 37024 373.3 696.5 27 008 C6H12N2O4S2 17376 55.6 295.2 60 301 C6H13NO2 25017/24898 100.2 507.1 99 237 C6H14N2O2 18019 ND ND ND C5H11NO2S 16643 ND ND ND C5H12N2O2 18257 20.3 167.8 71 403 C9H11NO2 28044 0.196 78.6 12 685 C2H8NO4P 36711 ND ND ND C3H8NO6P 37712 685.1 3926.3 329 903 C5H9NO2 26271 111.4 1088.9 25 577 C3H7NO2 16511 78.0 143.8 56 894 C3H7NO3 17822 2.17 1.98 26 140 C2H7NO3S 15891 ND 405.7 53 156 C4H9NO3 26986 4055.0 6.67 1223 C11H12N2O2 27897 431.3 245.9 30 868 C9H11NO3 18186 ND 27.1 12 607 C5H11NO2 27266 57.3 109.8 25 238 CH4N2O 16199 1.56 51.4 3651 CH4N2O 16199 2.22 65.1 5802 C22H32O4 871826-47-0 0.084 0.176 166.83 C20H34O5 27595 76.5 9.60 ND C22H32O3 119433-37-3 24.9 6.65 30.76 C20H34O5 37658-84-7 74.8 9.60 ND Table 1 Amino acid and eicosanoid concentrations in healthy human saliva and healthy EBC Compound Formula Identifier Clean-EBC(ng/mL) Saliva-EBC (ng/mL) Saliva (ng/mL Amino acids 1-methylhistidine/3-methylhistidine C7H11N3O2 70958/70959 518.0 1276.1 1 256 059 α-amino-n-butyric acid/β-aminoisobutryic acid C4H9NO2 35621 316.3 41.3 90 579 Alanine/β-Alanine C3H7NO2 16449 58.1 51.9 13.84 Anserinea C10H16N4O3 18323 ND ND ND Arginine C6H14N4O2 29016 1211.6 6016.8 101 238 Asparagine C4H8N2O3 22653 ND 8.6 7575 Aspartic acid C4H7NO4 22660 10.7 19.4 11 387 Carnosinea C9H14N4O3 15727 ND ND ND Citrulline C6H13N3O3 18211 302.2 304.8 16 922 Creatinine C4H7N3O 16737 237.5 396.9 88 368 Cystathionine C7H14N2O4S 17755 930.7 1756.2 ND Cysteinea C3H7NO2S 15356 ND ND ND Ethanolamine C2H7NO 16000 52.5 230.9 20 887 ϒ-aminobutyric acid C4H9NO2 16865 41.3 56.1 13 705 Glutamic acid C5H9NO4 18237 77.0 29.6 49 725 Glutamine C5H10N2O3 28300 24.6 162.4 18 423 Glycine C2H5NO2 15428 5.99 11.4 1739 Histidine C6H9N3O2 27570 401.2 316.5 14 751 Homocystinea C4H9NO2S 17485 ND ND ND Hydroxylysine C6H14N2O3 60175 205.6 283.3 133 890 Hydroxyprolinea C5H9NO3 24741 ND ND ND L-Aminoadipic acid C6H11NO4 37024 373.3 696.5 27 008 L-Cystine C6H12N2O4S2 17376 55.6 295.2 60 301 Leucine/Isoleucine C6H13NO2 25017/24898 100.2 507.1 99 237 Lysinea C6H14N2O2 18019 ND ND ND Methioninea C5H11NO2S 16643 ND ND ND Ornithine C5H12N2O2 18257 20.3 167.8 71 403 Phenylalanine C9H11NO2 28044 0.196 78.6 12 685 Phosphoethanolaminea C2H8NO4P 36711 ND ND ND Phosphoserine C3H8NO6P 37712 685.1 3926.3 329 903 Proline C5H9NO2 26271 111.4 1088.9 25 577 Sarcosine C3H7NO2 16511 78.0 143.8 56 894 Serine C3H7NO3 17822 2.17 1.98 26 140 Taurine C2H7NO3S 15891 ND 405.7 53 156 Threonine C4H9NO3 26986 4055.0 6.67 1223 Tryptophan C11H12N2O2 27897 431.3 245.9 30 868 Tyrosine C9H11NO3 18186 ND 27.1 12 607 Valine C5H11NO2 27266 57.3 109.8 25 238 Urea (negative mode) CH4N2O 16199 1.56 51.4 3651 Urea (positive mode) CH4N2O 16199 2.22 65.1 5802 Eicosanoids 10(S),17(S)-DiHDoHE (Protectin DX) C22H32O4 871826-47-0 0.084 0.176 166.83 11β-PGF2α C20H34O5 27595 76.5 9.60 ND 14(S)-hydroxy Docosahexaenoic Acid C22H32O3 119433-37-3 24.9 6.65 30.76 Cruickshank-Quinn et al. Replication experiment using targeted LC-MS, untargeted LC-MS, and proteomics to examine healthy, sick, and smoker EBC To confirm that small molecules are detectable in EBC and to further rule out the possibility of saliva contamin- ation, a replication experiment was performed with EBC collected from 13 subjects (Cohort 3). Samples were classified into four groups based on health status: healthy non-smoker, healthy smoker, non-smoker with the com- mon cold, non-smoker with nasal congestion. A saliva trap was used and subjects were observed to ensure that each participant did not contaminate the samples with saliva during the EBC collection procedure. Overall, a total of 172 metabolites were detected when untargeted LC-MS metabolomics was used (Fig. 5a); of these 118 were Fig. 3 Overlap of metabolites detected in clean EBC, saliva-contaminated EBC, and saliva samples of healthy volunteers in Cohort 1. Untargeted metabolomics was performed on EBC and saliva from healthy volunteers. Metabolite peaks were extracted using MassHunter Profinder software (Agilent). Samples were filtered using a 3000 abundance cutoff and a presence in at least two of the three sample groups. A total of 77 metabolites were determined to be unique to EBC Cruickshank-Quinn et al. Discussion The purpose of this study was to determine the utility of EBC in studying lung diseases, especially asthma. Therefore, we aimed to (1) evaluate whether compound adsorption to collection tubes is the reason for low-to- no detection of metabolites in some EBC studies, (2) characterize the constituents of EBC using untargeted and targeted mass spectrometry, and (3) determine if the detection of molecules in EBC is due to saliva contam- ination during sample collection. Initial EBC experiments using the RTube for sample collection yielded no detect- able levels of leukotrienes in our previous studies (data not shown). Other studies have also showed the RTube to be less sensitive than other commercially available EBC collection devices [36]. Since there is the possibility of binding of compounds to the sides of the collection tube, a control experiment was performed (Fig. 1). Results showed 10-22% less adsorption of the leukotrienes to the TURBO-DECCS plastic tube compared to the plastic RTube at the 10 pg/mL spike levels (Fig. 1d). In addition, polyethylene terephthalate (PET) caused significant ad- sorption of LTC4, LTD4, and LTE4 to the coated collection tubes (Fig. 1d). Therefore, our clinical experiments used the TURBO-DECCS plastic collection tube without PET to minimize adsorption of compounds to the plastic. Targeted analysis of 32 eicosanoids resulted in the de- tection of 19 compounds in the smoker group; these were undetected or below our limit of quantitation in the other three EBC groups (Additional file 8). Seven of those eicosanoids were part of the cyclooxygenase pathway, 9 were part of the 5-, 12-, or 15-lipoxygenase pathways, and 2 were in the lipid peroxidation/oxidative stress pathway. Four eicosanoids were detected and quantified in all four groups (Fig. 5c). The smoker and the nasal congestion group showed elevated levels of 13-HODE, 13-OxoODE, 9-HODE, and 9-OxoODE compared to the healthy and common cold groups. A comprehensive pathway analysis was performed across all sample groups (Additional File 9) which showed that more pathways were detected in the smoker EBC compared to the other groups in that cohort. Pathway analysis also included Cohorts 1 and 2, of which the saliva-based samples showed a greater number of path- ways compared to the EBC-only samples. These results were not unexpected as the saliva samples contained a larger number of compounds than the EBC samples. Replication experiment using targeted LC-MS, untargeted LC-MS, and proteomics to examine healthy, sick, and smoker EBC Respiratory Research (2017) 18:57 Page 13 of 22 Table 2 Database annotated metabolites detected in healthy EBC (Continued) S-Farnesyl Thioacetic Acid C17 H28 O2 S + 7.44 296.1797 297.1891 [M + H]+ 0.87 CAS: 135784-48-4 Terbucarb C17 H27 N O2 + 13.58 277.2047 278.2088 [M + H]+ 9.9 KEGG: C19129 CAS: 1918-11-2 8-Hydroxypinoresinol 4-glucoside C26 H32 O12 + 9.95 536.1894 537.1986 [M + H]+ 6.55 KEGG: C07149 HMDB14643 CAS: 26171-23-3 Ganglioside GM3 (d18:0/20:0) C61 H114 N2 O21 + 10.09 1210.7914 597.3970 (M + 2H) + 2[-H2O]+ KEGG: C04730 HMDB11919 Beta-Santalic acid C15 H22 O2 - 15.63 234.1620 233.1548 [M-H]- 2.93 HMDB39621 Phenylalanyl-Histidine C15 H18 N4 O3 + 7.77 302.1379 285.1354 [M + H-H2O]+ 2.81 HMDB28997 4-Hydroxyphenylacetaldehyde C8 H8 O2 - 9.28 136.0524 135.0450 [M-H]- 3.11 KEGG: C03765 HMDB03767 Pimelylcarnitine C14 H25 N O6 - 8.98 303.1682 284.1487 [M-H2O-H]- 3.87 CAS: 7339-87-9 Database annotations were obtained for 40 of the 77 compounds that were specific to the EBC samples (Fig. 3). Samples were analyzed in positive and negative ionization mode using LC-MS untargeted metabolomics on an SB-AQ analytical column. Annotations were based on an in-house database comprising KEGG, HMDB, Lipid Maps, and Metlin. + indicates detected in positive ionization mode, - indicates detected in negative ionization mode Table 2 Database annotated metabolites detected in healthy EBC (Continued) Database annotations were obtained for 40 of the 77 compounds that were specific to the EBC samples (Fig. 3). Samples were analyzed in positive and negative ionization mode using LC-MS untargeted metabolomics on an SB-AQ analytical column. Annotations were based on an in-house database comprising KEGG, HMDB, Lipid Maps, and Metlin. + indicates detected in positive ionization mode, - indicates detected in negative ionization mode healthy EBC. No proteins were detected in the nasal con- gestion or common cold EBC. healthy EBC. No proteins were detected in the nasal con- gestion or common cold EBC. database annotated and 81 metabolites were common to all four groups. While the sample numbers are too low to enable the use of statistics, it is useful to discuss the results in the context of the individual groups. The healthy group contained the least number of metabolites (112) compared to the smoker (141), common cold (150), and nasal con- gestion (164) groups (Fig. 5a, b). Amino acids and their derivatives were unique to the smoker EBC (Table 3). Replication experiment using targeted LC-MS, untargeted LC-MS, and proteomics to examine healthy, sick, and smoker EBC A complete list of annotated and MS/MS hits is available in Additional file 7. Replication experiment using targeted LC-MS, untargeted LC-MS, and proteomics to examine healthy, sick, and smoker EBC Respiratory Research (2017) 18:57 Page 12 of 22 Table 2 Database annotated metabolites detected in healthy EBC Compound Database Annotation Formula Mode RT (min) Mass m/z Adduct ppm error Identifier C25-Allenic-apo-aldehyde C25 H34 O3 + 8.24 382.2508 383.2579 [M + H]+ 0.52 KEGG: C14044 LMPR01070293 19α-19-Hydroxy-3,11-dioxo-12-ursen-28-oic C29 H42 O5 + 8.40 470.3055 471.3124 [M + H]+ 3.35 HMDB38683 2,2,4,4,-Tetramethyl-6-(1-oxopropyl)-1,3, 5-cyclohexanetrione C13 H18 O4 + 10.55 238.1205 261.1105 [M + Na]+ 5.21 HMDB33191 Planinin C21 H22 O6 - 13.10 370.1416 369.1348 [M-H]- 1.88 HMDB38236 25-Hydroxyvitamin D2-25-glucuronide C34 H52 O8 + 8.34 588.3685 606.4037 [M + NH4]+ 3.81 KEGG: C03033 HMDB10342 3-Deoxy-3-azido-25-hydroxyvitamin D3 C27 H43 N3 O + 7.69 425.3406 446.2932 [M + K-H2O]+ 2.64 LMST03020677 3-keto Fusidic acid C31 H46 O7 + 8.03 530.3267 548.3616 [M + NH4]+ 5.04 HMDB60745 Mumefural C12 H12 O9 - 8.36 300.0481 299.0388 [M-H]- 6.58 HMDB35179 3-Oxooctanoic acid C8 H14 O3 + 8.09 158.0940 159.1007 [M + H]+ HMDB10721 Fluometuron C10 H11 F3 N2 O - 9.40 232.0823 463.1564 [2 M-H]- 1.49 KEGG: C18853 Dibenzyl ether C14 H14 O - 13.73 198.1039 197.0967 [M-H]- 2.85 HMDB32078 Marmesin rutinoside C26 H34 O13 + 10.16 554.1999 537.1973 [M + H-H2O]+ 1.12 HMDB41413 Amitraz C19 H23 N3 + 7.79 293.1892 316.1790 [M + Na]+ 4.13 KEGG: C10995 CAS: 33089-61-1 Glutamyl-Glycine C10 H7 N3 O + 11.72 223.0123 224.0203 [M + K-H2O]+ 3.63 HMDB28819 de-Hypoxanthine futalosine C14 H16 O7 + 1.77 296.0896 149.0523 [M + 2H]2+ 0.78 KEGG: C17010 Diethyltoluamide (DEET) C12 H17 N O - 16.74 251.1519 250.1451 [M + CH3COO]- 0.05 KEGG: C10935 CAS: 134-62-3 3,4-Dihydroxyfluorene C13 H10 O2 + 11.38 198.0691 181.0661 [M + H-H2O]+ 6.66 KEGG: C07717 CAS: 42523-20-6 2,3-Dihydro-2,3-dihydroxy-4-(4-methoxyphenyl)- 1H-phenalen-1-one C20 H16 O4 - 7.63 320.1049 319.0981 [M-H]- 1.49 HMDB41463 3-Oxopregn-4-ene-20beta-carboxaldehyde dioxime C22 H34 N2 O2 + 8.66 358.2620 422.2817 [M + ACN + Na]+ 9.24 KEGG: C15106 Hyperin 2''-[glucosyl-(1- > 3)-rhamnoside] 6''-rhamnoside C39 H50 O25 - 12.42 918.2638 917.2567 [M-H]- 0.20 HMDB39911 Mangostanol C24 H26 O7 - 13.02 426.1679 485.1805 [M + CH3COO]- 3.73 HMDB29868 Oleoside dimethyl ester C18 H26 O11 - 9.79 418.1475 453.1160 [M + Cl]- 1.23 HMDB31350 N-Acetyl-6-O-L-fucosyl-D-glucosamine C14 H25 N O10 - 8.49 349.1370 348.1292 [M-H2O-H]- 3.03 HMDB02220 CAS: 109582-58- 3 Oleanolic acid 3-O-beta-D-glucosiduronic acid C36 H56 O9 + 8.40 632.3924 650.4312 [M + NH4]+ 6.2 KEGG: C08964 Methionyl-Arginine C11 H23 N5 O3 S + 11.97 305.1522 288.1497 [M + H-H2O]+ 2.01 HMDB28967 N-Cyclopropylammelide C6 H8 N4 O2 - 9.27 204.0403 203.0329 [M-Cl]- 8.76 KEGG: C14149 PE(34:1)-15-isoLG hydroxylactam C59 H104 N O13 P + 10.01 1065.7160 1088.7032 [M + Na]+ 7.06 KEGG: C06254 LMGP00000061 PE(44:7) C49 H84 N O8 P + 9.75 845.5867 868.5782 [M + Na]+ 5.18 KEGG: C00350 HMDB09700 Prostaglandin F2α-biotin C35 H60 N4 O6 S + 8.71 664.4234 669.3982 [M + Na-H2O]+ 7.77 Prostaglandin D2-biotin C36 H60 N4 O6 S + 8.47 676.4234 694.4563 [M + NH4]+ 1.30 Prostaglandin E2-biotin C35 H58 N4 O6 S + 8.24 662.4045 663.4127 [M + NH4]+ 1.63 Tyrosol-histidine C15 H18 N4 O4 + 7.90 300.1224 301.1300 [M + H-H2O]+ 2.79 HMDB29107 able 2 Database annotated metabolites detected in healthy EBC Cruickshank-Quinn et al. Discussion Metabolites were filtered for presence in at least two out of the four groups; b Hierarchical clustering of 172 metabolites present in at least two EBC groups. Blue sections indicate low metabolite abundances and red sections indicate high abundance levels. The healthy EBC subjects appear to have a majority of lower abundance metabolites compared to the other three groups; c Concentration levels of four eicosanoids detected in all four sample groups using targeted LC-MS. Samples were analyzed on a triple quadruple mass spectrometer Fig. 5 Distribution of compounds across EBC groups in Cohort 3. a Venn diagram depicting the overlap of metabolites in four categories based on subject from the untargeted metabolomics analysis. Metabolites were filtered for presence in at least two out of the four groups; b Hierarchical clustering of 172 metabolites present in at least two EBC groups. Blue sections indicate low metabolite abundances and red sections indicate high abundance levels. The healthy EBC subjects appear to have a majority of lower abundance metabolites compared to the other three groups; c Concentration levels of four eicosanoids detected in all four sample groups using targeted LC-MS. Samples were analyzed on a triple quadruple mass spectrometer Although a saliva trap was used, it may be possible that saliva contributed to the ng/mL concentration values ob- tained in both the clean-EBC and saliva-EBC sample group. As suggested by Gaber et al [25], even the slight- est amount of saliva can generate false positives in EBC samples when sensitive detection methods are used. Since LTB4 is present in the nasal mucosa, and the oro- pharyngeal tract contributes to the contents of EBC, a more sensitive or alternate method to alpha-amylase de- tection may be required to confirm saliva contamination. In our study, we detected small molecules in EBC and saliva-EBC which were not detected in the saliva-only samples. This suggests that these molecules may be spe- cific to EBC or have much lower concentrations in saliva; therefore, the low saliva content would not contribute to their detection in EBC. These EBC constituents may po- tentially be used as biomarkers; however, further study is required to confirm their identities and usefulness in studying lung disease. Previous investigations have reported the presence of eicosanoids in EBC [11, 15, 37, 38]. Discussion Our first goal was to identify the contribution of saliva to EBC measurements by determining concentrations of amino acids and eicosanoids in both EBC and saliva. We observed that the concentration of several eicosanoids and amino acids (Table 1) were orders of magnitude higher in saliva compared to clean-EBC and saliva-EBC. Three proteins were detected in EBC samples of Cohort 3 subjects following proteomics analysis (Table 4). Zinc finger protein 800 and myoneurin were only detected in the smoker EBC. Cytokeratin 9 was only detected in the Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 14 of 22 Fig. 4 Concentrations of ten eicosanoids in saliva and EBC samples of matched asthmatics subjects in Cohort 2. Quantitative analysis was performed on an Agilent triple quadrupole (QQQ) 6410 mass spectrometer using targeted multiple reaction monitoring (MRM); concentration units in pg/mL; blue circles are saliva samples (n = 106), red triangles are EBC samples (n = 107); black line is sample mean Fig. 4 Concentrations of ten eicosanoids in saliva and EBC samples of matched asthmatics subjects in Cohort 2. Quantitative analysis was performed on an Agilent triple quadrupole (QQQ) 6410 mass spectrometer using targeted multiple reaction monitoring (MRM); concentration units in pg/mL; blue circles are saliva samples (n = 106), red triangles are EBC samples (n = 107); black line is sample mean Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 15 of 22 Fig. 5 Distribution of compounds across EBC groups in Cohort 3. a Venn diagram depicting the overlap of metabolites in four categories based on subject from the untargeted metabolomics analysis. Metabolites were filtered for presence in at least two out of the four groups; b Hierarchical clustering of 172 metabolites present in at least two EBC groups. Blue sections indicate low metabolite abundances and red sections indicate high abundance levels. The healthy EBC subjects appear to have a majority of lower abundance metabolites compared to the other three groups; c Concentration levels of four eicosanoids detected in all four sample groups using targeted LC-MS. Samples were analyzed on a triple quadruple mass spectrometer Fig. 5 Distribution of compounds across EBC groups in Cohort 3. a Venn diagram depicting the overlap of metabolites in four categories based on subject from the untargeted metabolomics analysis. Discussion Many of these groups have reported higher concentrations of eicosa- noids than those shown in Table 1 of the current study; the majority of these studies were conducted in the con- text of lung disease such as asthma where elevated levels may due to inflammation. The healthy volunteers in our study had no history of asthma or lung inflammation; this could explain the lower levels which we report. We compared our concentration values of amino acids and eicosanoids in Table 1 to those available in the literature for EBC and saliva. LTB4 has been reported to have an average concentration in saliva of 0.467 ng/mL [25] which is similar to our estimated value of 1.56 ng/mL. Tyrosine has been reported at an average of 33.3 ng/mL [24] in EBC while Conventz et al [19] reported an average value of 15.5 ng/mL. We detected tyrosine at 27.1 ng/mL in ‘saliva-EBC’ but it was undetected in ‘clean-EBC’. Our proline estimate was 111.4 ng/mL in EBC. This was twice Cruickshank-Quinn et al. Discussion Respiratory Research (2017) 18:57 Page 16 of 22 Table 3 Compounds detected in healthy, sick, and smoker EBC Compound Smoker Nasal Cold Healthy RT (mins) Mass Formula Mode Identifier 1,3-Dicyclohexylureaa ✓ ✓ ✓ ✓ 8.876 224.1889 C13 H24 N2 O + CAS: 2387-23-7 13,14-dihydro Prostaglandin F1a ✓ ✓ ✓ ✓ 6.814 358.2736 C20 H38 O5 + CAS: 20592-20-5 1-Hydroxy-2-naphthoic acid ✓ ✓ ✓ ✓ 6.258 188.0472 C11 H8 O3 + KEGG: C03203 2-Amino-3,7-dideoxy-D-threo-hept-6-ulosonic acid ✓ ✓ ✓ ✓ 6.380 213.0620 C7 H13 N O5 + KEGG: C16850 C14 sphingosine ✓ ✓ ✓ ✓ 9.628 271.2141 C14 H29 N O2 - LMSP01040006 N-Acetyl-D-fucosamine ✓ ✓ ✓ ✓ 8.079 205.0950 C8 H15 N O5 + KEGG: C15480 p-Cymene ✓ ✓ ✓ ✓ 9.382 152.1205 C10 H14 + KEGG: C06575 Ureidoglycine ✓ ✓ ✓ ✓ 0.980 380.0903 C3 H7 N3 O3 - KEGG: C02091 15(S)-HPETE ✓ ✓ ✓ 10.185 264.2089 C20 H32 O4 + KEGG: C05966 6-hydroxy caproic acid ✓ ✓ ✓ 6.131 174.0890 C6 H12 O3 - KEGG: C06103 Homoserine ✓ ✓ ✓ 6.707 215.0406 C4 H9 N O3 - KEGG: C00263 PA(22:2/0:0) ✓ ✓ ✓ 6.118 490.3105 C25 H47 O7 P - KEGG: C00416 PI(12:0/12:0) ✓ ✓ ✓ 5.703 698.3983 C33 H63 O13 P - KEGG: C01194 Tetrahydrodipicolinate ✓ ✓ ✓ 7.780 153.0427 C7 H9 N O4 - KEGG: C03972 Threonine ✓ ✓ ✓ 8.939 233.0513 C4 H9 N O3 - KEGG: C00188 2-Oxo-4-hydroxy-5-aminovalerate ✓ ✓ 1.604 129.0426 C5 H9 N O4 - KEGG: C05941 N-Acetyl leucine ✓ ✓ 5.710 173.1055 C8 H15 N O3 - KEGG: C02710 2-Amino-m-cresola ✓ 0.811 123.0684 C7 H9 N O + CAS: 2835-97-4 3-Cyano-6-methoxycoumarina ✓ 5.936 201.0426 C11 H7 N O3 + - 3-Methylhistidinea ✓ 0.809 169.0851 C7 H11 N3 O2 + KEGG: C01152 4-Imidazoleacrylic acida ✓ 5.023 138.0429 C6 H6 N2 O2 + HMDB00301 5-Aminosalicylic acida ✓ 1.612 153.0426 C7 H7 N O3 + KEGG: C07138 Acetyl argininea ✓ 4.910 216.1222 C8 H16 N4 O3 + HMDB04620 Argininea ✓ 0.810 174.1117 C6 H14 N4 O2 + KEGG: C02385 Carnosinea ✓ 0.809 226.1066 C9 H14 N4 O3 + KEGG: C00386 D-erythro-Sphinganinea ✓ 5.186 301.2981 C18 H39 N O2 + LMSP01020001 Triethyl citratea ✓ 7.519 276.1209 C12 H20 O7 + CAS: 77-93-0 Tryptophana ✓ 4.079 204.0899 C11 H12 N2 O2 + KEGG: C00078 1alpha,24,25,28-tetrahydroxyvitamin D2 ✓ ✓ 6.213 460.3189 C28 H44 O5 - LMST03010055 MG(18:1) ✓ ✓ ✓ 8.908 373.3185 C21 H40 O4 + KEGG: C01885 PC(18:1/22:6) ✓ ✓ ✓ 6.813 831.5785 C48 H82 N O8 P + KEGG: C00157 PG(18:4/20:4) ✓ ✓ ✓ 12.183 790.4734 C44 H71 O10 P + KEGG: C00344 O-decanoyl-R-carnitine ✓ ✓ 6.305 361.2453 C17 H33 N O4 - KEGG: C03299 Palmitoylglycine ✓ ✓ 9.931 335.2469 C18 H35 N O3 + HMDB13034 Arogenate ✓ ✓ 6.828 227.0775 C10 H13 N O5 - KEGG: C00826 α-Lipoic acida ✓ 5.666 206.0435 C8 H14 O2 S2 + KEGG: C00725 Ephedrinea ✓ 31.317 165.1154 C10 H15 N O + KEGG: C01575 3-Acetyl-8-methoxycoumarina ✓ 5.846 218.0579 C12 H10 O4 + HMDB34345 6-Hydroxymelatonina ✓ 1.184 248.1161 C13 H16 N2 O3 + KEGG: C05643 EBC was collected from 13 volunteers and pooled into four groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. EBC was collected from 13 volunteers and pooled into four groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. ✓indicates that a compound was detected. a indicates tandem MS fragmentation patterns were matched to the NIST14 Mass Spectral library using the NIST MS Search v.2.2 g program. MF: match factor; RMF: reverse match score. The fragmentation spectra for the listed compounds are available in the Additional file 7 Discussion Samples were analyzed on a Bruker Impact HD Q-TOF and proteins were search using Mascot. ✓indicates that a protein was detected within a particular group Table 4 Proteins detected in healthy non-smoker and healthy smoker EBC EBC was collected from 13 volunteers and pooled into 4 groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were analyzed on a Bruker Impact HD Q-TOF and proteins were search using Mascot. ✓indicates that a protein was detected within a particular group EBC was collected from 13 volunteers and pooled into 4 groups; healthy smokers, healthy non-smokers, non-smokers with nasal con with the common cold. Samples were analyzed on a Bruker Impact HD Q-TOF and proteins were search using Mascot. ✓indicates th within a particular group atrazine. Atrazine is an herbicide used to prevent weeds on golf courses and residential lawns. as high as the previously reported high value in healthy subjects of 51.9 ng/mL [19] and may be the result of in- dividual subject differences, sample preparation, or de- tection method. Reported EBC urea values have similar ranges within the same order of magnitude to our calcu- lated values. Effros et al [39] reported values in the range 0.33 to 0.39 μmol/L (19.8 to 23.4 ng/mL). Folesani et al [40] reported EBC urea concentrations found in healthy controls in the range 0.7-1.3 μM (42.0 to 78.1 ng/mL). Dwyer et al [41] reported EBC urea averaged 0.52 +/- 0.12 μmol/L (31.2 ng/mL) in 18 individuals. Our detected levels ranged from 1.56 ng/mL (clean-EBC) to 65.1 ng/mL (saliva-EBC) which falls within the same order of magnitude of these previously reported values. The differences among studies could be accounted for by variations in sample preparation procedures such as lyophilization, as well as differences in methods of collecting EBC. g Other metabolites found in EBC were tentatively identified as endogenous compounds; for example, 3- oxooctanoic acid is an endogenous keto acid involved in fatty acid biosynthesis. It is formed by the action of acid synthases from acetyl-CoA and malonyl-CoA pre- cursors. PE(44:7) is an endogenous glycerophospholipid associated with cell signaling and membrane integrity and also serves as an energy source [42]. Ganglioside GM3 (d18:0/20:0) is an endogenous sphingolipid. Discussion Gangliosides, including GM3 and GM2, have been shown to be down- regulated in the hyper-reactive lung and trachea compared to the normal lung and trachea in a guinea pig model of bronchial asthma [44]. Gangliosides have also been shown to be inversely associated with severe emphysema in COPD human plasma [45]. Dipeptides were also annotated. These included glutamyl-glycine, methionyl-arginine, tyrosyl-histidine, and phenylalanyl-histidine. Dipeptides are incomplete breakdown products of protein digestion or protein catabolism. Many dipeptides are short-lived in- termediates toward specific amino acid degradation path- ways while others have physiological [46] or cell-signaling effects [42]. Our second goal was to characterize the constituents of EBC using an untargeted metabolomics approach. We further determined the effect of saliva contamination on small molecules detection in EBC. The 77 small mole- cules that were detected in the clean-EBC and the saliva-EBC were undetected in the saliva samples; this suggests that these compounds may be specific to EBC. The 40 out of 77 that were database annotated using Human Metabolome Database (HMDB) [42] and the Kyoto Encyclopedia and Genes and Genomes (KEGG) [43] included the following: vitamin D metabolites, lipids, herbs, spices, food, plants, insecticides, herbicides, dipeptides, and PAH degradants. Five out of fourteen food metabolites were related to citrus fruit or tea, three were herbs and spices, and the remaining were vegetables, food flavorings, or oils. This is consistent with participants’ reports of drink- ing green tea, coffee, and chocolate milk, and eating pump- kin cake, green beans, club sandwich, and potato chips. Note that participants were not allowed to eat or drink at least two hours prior to sample collection. Other matches for two insecticides and three herbicides were also plausible since these samples were collected when subjects may have applied insect repellants such as diethyltoluamide (DEET) to their skin, and institutions were applying herbicides and pesticides to their lawns. For example, N-cyclopropylammelide is a degradation product of [ ] 3,4-dihydroxyfluorene is a polycyclic aromatic hydro- carbon (PAH) degradation metabolite. PAH’s have been associated with childhood asthma [47] and are found in oil, coal, and tar. They are the result of combustion in engines, and incinerators; sources include forest fires, vehicle exhaust, grilling or barbecuing meat, and smoked fish [48–50]. Lastly, there were two annotated vitamin D metabolites (25-hydroxyvitamin D2-25-glucuronide and 3-deoxy-3-azido-25-hydroxyvitamin D3). Discussion ✓indicates that a compound was detected. a indicates tandem MS fragmentation patterns were matched to the NIST14 Mass Spectral library using the NIST MS Search v.2.2 g program. MF: match factor; RMF: reverse match score. The fragmentation spectra for the listed compounds are available in the Additional file 7 Table 3 Compounds detected in healthy, sick, and smoker EBC EBC was collected from 13 volunteers and pooled into four groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. ✓indicates that a compound was detected. a indicates tandem MS fragmentation patterns were matched to the NIST14 Mass Spectral library using the NIST MS Search v.2.2 g program. MF: match factor; RMF: reverse match score. The fragmentation spectra for the listed compounds are available in the Additional file 7 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 17 of 22 Page 17 of 22 Table 4 Proteins detected in healthy non-smoker and healthy smoker EBC Protein Accession ID Smoker Nasal Congestion Cold/Flu Healthy Zinc finger protein 800 UniProtKB Q2TB10 ✓ Myoneurin UniProtKB Q9NPC7 ✓ Keratin, type 1 cytoskeleton 9 (Cytokeratin 9) UniProtKB P35527 ✓ EBC was collected from 13 volunteers and pooled into 4 groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were analyzed on a Bruker Impact HD Q-TOF and proteins were search using Mascot. ✓indicates that a protein was detected within a particular group Table 4 Proteins detected in healthy non-smoker and healthy smoker EBC Protein Accession ID Smoker Nasal Congestion Cold/Flu Healthy Zinc finger protein 800 UniProtKB Q2TB10 ✓ Myoneurin UniProtKB Q9NPC7 ✓ Keratin, type 1 cytoskeleton 9 (Cytokeratin 9) UniProtKB P35527 ✓ EBC was collected from 13 volunteers and pooled into 4 groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were analyzed on a Bruker Impact HD Q-TOF and proteins were search using Mascot. ✓indicates that a protein was detected within a particular group Table 4 Proteins detected in healthy non-smoker and healthy smoker EBC Protein Accession ID Smoker Nasal Congestion Cold/Flu Healthy Zinc finger protein 800 UniProtKB Q2TB10 ✓ Myoneurin UniProtKB Q9NPC7 ✓ Keratin, type 1 cytoskeleton 9 (Cytokeratin 9) UniProtKB P35527 ✓ EBC was collected from 13 volunteers and pooled into 4 groups; healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Discussion Therefore, detection of specific molecules or classes, may require derivatization, solid phase extraction, or enzymatic techniques, as described by Chérot-Kornobis et al [17] for nitrogen oxides, Esther Jr. et al [55] for purines, or Rossi et al [56] for glutathione. shikimic acid, succinic acid, and citric acid (Additional file 5). With additional sample, targeted MSMS can be performed on additional EBC metabolites to fur- ther explore their identities. Other metabolites present may be below our limit of detection or require more specialized sample preparation techniques. Therefore, detection of specific molecules or classes, may require derivatization, solid phase extraction, or enzymatic techniques, as described by Chérot-Kornobis et al [17] for nitrogen oxides, Esther Jr. et al [55] for purines, or Rossi et al [56] for glutathione. A major objective of the current study was to determine if eicosanoid detection in EBC was the result of saliva con- tamination. With the exception of one molecule in one subject, we were unable to detect eicosanoids in EBC of over 100 asthmatic subjects, a group in which higher con- centrations of these molecules have been reported (Fig. 4). Our EBC sample preparation step included diluting the EBC volume to 1 mL; this may have diluted levels to below the limit of detection for the targeted eicosanoid panel. However, the concentration of eicosanoids ranged from 10-fold to 100-fold lower in EBC compared to saliva from matched asthmatic subjects (Fig. 4). Since our sam- ple preparation methods, including low starting volumes, were consistent with previous investigations reporting the detection of eicosanoids in EBC, this suggests that previ- ously reported values could possibly be due to salivary contamination. This is further supported by the detection of some eicosanoids when a very highly concentrated (13 ml) EBC sample is used. Within the asthmatic saliva samples, the concentrations varied in the range 58-187% CV for the samples with detectable levels of eicosanoids. These results show that eicosanoid concentrations in saliva vary widely amongst asthmatic subjects. A major difference between our study and others is that our aim was specifically to determine the potential for and extent of saliva contamination in EBC sampling. Therefore, our studies incorporated a well-controlled set of experiments that included mimicking EBC collection. Overall, we required greater than 1 mL EBC for detec- tion of eicosanoids; preparation included concentrating samples in a lyophilizer. Other investigators have also concentrated their EBC samples prior to analysis. Discussion Mon- tuschi et al [57] collected 1.5 mL EBC per subject over 15 min using the ECoScreen and concentrated the EBC 40-fold. 20 μL of sample was injected and analyzed for LTB4 using LC-MS & LC-MS/MS. Our methods were replicated in an independent co- hort. EBC was collected from 13 volunteers and grouped in four categories based on health and smoking status: healthy non-smoker, healthy smoker, non-smoker with common cold, and non-smoker with nasal congestion. Saliva contamination was not present, as measured by a proteomic approach. Using untargeted LC-MS metabo- lomics, 172 metabolites were detected in EBC, 81 of which were present in all 4 sample groups (Fig. 5a). Fold changes were observed (Fig. 5b) but no statistical infer- ences could be made. Qualitatively however, the healthy EBC contained fewer compounds compared to the EBC of subjects with nasal congestion, the common cold, or the smoker (Fig. 5a). This may be due to differences in diet, or because subjects with signs of illness may ingest cold, flu, or nasal decongestion medication which may artificially increase the number of detected compounds in their EBC. y y g j Due to the poor detection of eicosanoids in EBC using targeted analysis and low starting volumes, we investigated whether any other small molecules could be detected in EBC when larger starting volumes are used. Leftover EBC from asthmatic subjects (n = 107) was pooled into a 13 mL aliquot, lyophilized, and reconstituted in 20 μL of HPLC buffer. Untargeted LC-MS revealed 97 metabolites, of which four were eicosanoid derivatives: LTE3, thromb- oxane, 11-trans-LTE4, 11-trans-LTC4, and 12-oxo-LTB4 (Additional file 6). Results suggest that although eicosa- noid metabolites are present in EBC, samples require a drastic pre-concentration step prior to LC-MS analysis in order for these molecules to be detected. In addition, some of these detected metabolites may be breakdown products of the deuterated standards spiked during sample preparation, particularly during the lyophilization step. Some of the detected metabolites in the smoker, healthy, common cold, and nasal congestion EBC (Table 3 and Additional file 7) were markers of environmental exposure such as 1-hydroxy-2-naphthoic acid which is a PAH and naphthalene degradation metabolite. This metabolite also mapped to the “degradation of aromatic compounds” pathway, as did 6-hydroxy caproic acid and p-cymene. Ephedrine, used as a decongestant, was only detected in the common cold group. Discussion Vitamin D and its metabolites are associated with asthma [51, 52] and vitamin D deficiency has been shown to be a risk factor for developing asthma [53, 54]. Because these com- pounds have previously reported associations with lung disease, they could either be used as diagnostic markers of health or disease state, or may be novel molecules re- quiring further interrogation. Due to low sample volumes, tandem mass spectrometry was only performed in negative mode and resulted in six spectral library matches corresponding to acetylsalicylic acid, 4-chloro-L-phenylalanine, 3,4-furandicarboxylic acid, Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 Page 18 of 22 EBC of 82 patients with occupational lung diseases & 27 controls were analyzed using SPE followed by LC-ESI- MS/MS. Results showed elevation of LTB4, LTC4, and LTE4 in asbestos-exposed and silica-exposed patients compared to controls. Conventz et al [19] collected 1-6 mL EBC from 27 healthy adults also using the ECoScreen. 1 mL EBC was used to quantify proline, hydroxyproline, and tyrosine using LC-ESI-MS/MS. Fritscher et al [6] col- lected at least 1 mL of EBC from 87 subjects for over 10 min and performed targeted analysis on a QQQ-MS. They examined twenty-three eicosanoids in the EBC from asthma and COPD individuals, five of which overlapped with our study. Our study examined an additional eleven molecules that were not present in their study. EBC of 82 patients with occupational lung diseases & 27 controls were analyzed using SPE followed by LC-ESI- MS/MS. Results showed elevation of LTB4, LTC4, and LTE4 in asbestos-exposed and silica-exposed patients compared to controls. Conventz et al [19] collected 1-6 mL EBC from 27 healthy adults also using the ECoScreen. 1 mL EBC was used to quantify proline, hydroxyproline, and tyrosine using LC-ESI-MS/MS. Fritscher et al [6] col- lected at least 1 mL of EBC from 87 subjects for over 10 min and performed targeted analysis on a QQQ-MS. They examined twenty-three eicosanoids in the EBC from asthma and COPD individuals, five of which overlapped with our study. Our study examined an additional eleven molecules that were not present in their study. shikimic acid, succinic acid, and citric acid (Additional file 5). With additional sample, targeted MSMS can be performed on additional EBC metabolites to fur- ther explore their identities. Other metabolites present may be below our limit of detection or require more specialized sample preparation techniques. Discussion Additional detected metabolites In a few studies, investigators increased the amount of EBC used to 1 mL and obtained detectable metabolite signal. For example, Pelclová et al [11] collected EBC over 15-20 min using the ECoScreen. They analyzed Page 19 of 22 Page 19 of 22 Page 19 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 such as 2-Oxo-4-hydroxy-5-aminovalerate, homoserine, arogenate, tetrahydrodipicolinate, and 2-amino-3,7- dideoxy-D-threo-hept-6-ulosonic acid play roles in amino acid metabolism and/or biosynthesis [58]. Many of these compounds were absent in the healthy EBC but were present in the EBC of individuals who smoked, had nasal congestion, or had the common cold. Other detected metabolites were part of purine metabolism [ureidogly- cine], sphingolipid signaling pathway [C14 sphingosine], glycerolipid metabolism [PA(22:2), MG(18:1)], and glycer- ophospholipid metabolism [PC(40:7), PG(38:8), PA(22:2), PI(24:0)]. The lipid compounds PA(22:2) and PI(24:0) were undetected in the healthy EBC but were detected in the other three groups. These biological pathways have been implicated in lung diseases such as asthma [59] and COPD [45, 60]. replication study were not contaminated with saliva. A lar- ger, more diverse cohort encompassing multiple lung dis- eases may be required to explore the diversity of exhaled proteins. This study is particularly significant to EBC researchers because it emphasizes the issues of compound adsorption, saliva contamination, and high volumes of EBC required for biomarker discovery studies. The strengths of this study lie in the precise methods used and the large sample size of Cohort 2 in the asthmatic study. We recognize that some limitations exist. First, the sample number is limited for Cohorts 1 and 3. Second, subjects in these cohorts only refrained from food intake for 2-3 h rather than 12 h, which could explain some differences in metabolites de- tected. Lastly, due to limited sample volumes, additional analyses could not be performed to compare the three co- horts across all mass spectrometry technologies. Future studies could be aimed at rectifying these limitations. Targeted eicosanoid analysis was also performed on the healthy, smoker, common cold, and nasal congestion EBC. Nineteen eicosanoids were detected in the smoker EBC which were undetected in the other groups. Ele- vated levels of 13-HODE, 13-OxoODE, 9-HODE, and 9-OxoODE were observed in both the smoker and nasal congestion EBC compared to the healthy and common cold EBC. This increase in eicosanoids in smoking samples has been observed in previous studies. Conclusions We conclude that measureable levels of small molecules, including amino acids and eicosanoids, are present in healthy EBC; however, the dilute nature of EBC requires larger volumes of starting material than currently re- ported in the literature. We suggest the collection of at least 15 mL of EBC per subject and pre-concentrating by at least 20-fold to as much as 500-fold prior to LC-MS analysis in order to confidently and reproducibly detect metabolites of interest. Secondly, although α-amylase assays can test for the presence of saliva in EBC, small volumes of saliva may still be present but be below the detection limits of the assay. Since saliva can be responsible for contaminating EBC samples, proper sample collection and handling is necessary, particularly the use of a saliva trap during sample collection. Thirdly, eicosanoid concen- trations in saliva vary widely amongst asthmatic subjects and this should be considered when designing experiments. Here, we provide a general presentation of EBC constitu- ents from which investigators can probe more specialized techniques to detect additional or lower abundant com- pounds of interest. These results suggest that large volumes of samples and a more targeted approach are needed when using EBC to study asthma and other lung diseases. Proteomics analysis of these four groups detected few proteins in the EBC samples using both human and bac- terial searches (Table 4). In a review in 2014, Harshman et al. [18] summarized 80 detected proteins in EBC from the current literature including one detected in our study. Although no proteins were detected in our nasal congestion or common cold EBC samples, keratin type I cytoskeleton 9 (Cytokeratin-9) was detected in healthy EBC. Cytokeratin-9 has been previously been identified in asthmatic EBC [62], and in the pooled EBC of non- smokers and healthy smokers [63]. However, others have suggested that cytokeratin in EBC is the result of ambi- ent air rather than the airways [64]. We detected zinc finger protein 800 and myoneurin in the smoker EBC. Zinc finger protein 800 and myoneurin (also a zinc finger protein) have not been previously reported in EBC. Other zinc finger proteins have been detected; zinc finger CCCH domain-containing protein 4 (ZC3H4) has been reported in healthy non-smoker and healthy smoker EBC [63]. In our study, no salivary proteins were detected, indicating that the EBC samples from the Discussion Sanak et al [61] analyzed EBC from 17 healthy smokers and 41 healthy non-smokers collected using the ECoScreen. Results showed an increase in 5-HETE and 8-iso-PGF2α in the current smokers compared to the non-smokers. We conclude that cigarette smoking increases the inflammatory and oxidative stress markers observed in our study to levels that were at least 3-fold higher compared to the healthy EBC. Additional file 1: Targeted mass spectrometry parameters for eicosanoid analysis. MRM parameters, retention times, associated internal standards, and ionization modes used for lipid mediators by LC-MS/MS. IS: internal standard. (PDF 23 kb) Additional file 2: Peak areas of selected amino acids and eicosanoids detected in EBC and/or saliva samples. (A) Peak areas of selected eicosanoids using untargeted metabolomics; (B) Peak areas of selected amino acids in spiked water (green), clean-EBC (black), saliva-EBC (blue), and saliva (red) using untargeted metabolomics. A control water sample Ethics approval and consent to participate Studies were approved by the Western Institutional Review Board or National Jewish Health IRB. All participants gave informed consent prior to the start of the study. References 1. Marteus H, Törnberg D, Weitzberg E, Schedin U, Alving K. Origin of nitrite and nitrate in nasal and exhaled breath condensate and relation to nitric oxide formation. Thorax. 2005;60(3):219–25. doi:10.1136/thx.2004.030635. 1. Marteus H, Törnberg D, Weitzberg E, Schedin U, Alving K. Origin of nitrite and nitrate in nasal and exhaled breath condensate and relation to nitric oxide formation. Thorax. 2005;60(3):219–25. doi:10.1136/thx.2004.030635. Additional file 9: Pathway analysis based on sample type. The compounds which were detected in each sample type were mapped to KEGG pathways using the online freeware pathway analysis software MBROLE. The compound names were based on database annotations using exact mass, isotope ratios and/or MSMS. Only pathways with hits ≥2 are listed. (PDF 278 kb) Additional file 9: Pathway analysis based on sample type. The compounds which were detected in each sample type were mapped to KEGG pathways using the online freeware pathway analysis software MBROLE. The compound names were based on database annotations using exact mass, isotope ratios and/or MSMS. Only pathways with hits ≥2 are listed. (PDF 278 kb) 2. Brzozowska A, Majak P, Jerzyńska J, Smejda K, Bobrowska-Korzeniowska M, Stelmach W, et al. Exhaled nitric oxide correlates with IL-2, MCP-1, PDGF-BB and TIMP-2 in exhaled breath condensate of children with refractory asthma. Adv Dermatol Allergol. 2015;32(2):107–13. doi:10.5114/pdia.2014.40953. are listed. (PDF 278 kb) 3. Carraro S, Giordano G, Piacentini G, Kantar A, Moser S, Cesca L, et al. Asymmetric dimethylarginine in exhaled breath condensate and serum of children with asthma. Chest. 2013;144(2):405–10. doi:10.1378/chest.12-2379. Funding Thi bl g This publication was supported by NIH-NCRR grant 1S10OD010366-01A1 to N.R. and NIH/NCATS Colorado CTSA Grant Number UL1 TR001082. Its contents are the authors’ sole responsibility and do not necessarily represent official NIH views. Additional file 4: Molecular formula annotated metabolites and unannotated metabolites detected in exhaled breath condensate (EBC). These 37 out of 77 unique compounds were not matched to a database compound. Samples were analyzed in positive and negative ionization mode using LC-MS untargeted metabolomics on an SB-AQ analytical column. + indicates detected in positive ionization mode, - indicates detected in negative ionization mode. (PDF 38 kb) Additional file 4: Molecular formula annotated metabolites and unannotated metabolites detected in exhaled breath condensate (EBC). These 37 out of 77 unique compounds were not matched to a database compound. Samples were analyzed in positive and negative ionization mode using LC-MS untargeted metabolomics on an SB-AQ analytical column. + indicates detected in positive ionization mode, - indicates detected in negative ionization mode. (PDF 38 kb) Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional files. Additional file 5: Tandem MS fragmentation patterns for six EBC metabolites. The mass spectral fragment peaks in red indicate the experimental results. The peaks in blue indicate the database matches based on standards. (PDF 36 kb) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 8: Targeted eicosanoid analysis of EBC. EBC was collected from four groups of volunteers: healthy smokers, healthy non- smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were pooled, lyophilized, reconstituted in LC-MS buffer, and analyzed using targeted LC-MS on a triple quadruple mass spectrometer. (PDF 40 kb) Additional file 8: Targeted eicosanoid analysis of EBC. EBC was collected from four groups of volunteers: healthy smokers, healthy non- smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were pooled, lyophilized, reconstituted in LC-MS buffer, and analyzed using targeted LC-MS on a triple quadruple mass spectrometer. (PDF 40 kb) Received: 3 November 2016 Accepted: 18 March 2017 Received: 3 November 2016 Accepted: 18 March 2017 Additional files Additional file 1: Targeted mass spectrometry parameters for eicosanoid analysis. MRM parameters, retention times, associated internal standards, and ionization modes used for lipid mediators by LC-MS/MS. IS: internal standard. (PDF 23 kb) Additional file 2: Peak areas of selected amino acids and eicosanoids detected in EBC and/or saliva samples. (A) Peak areas of selected eicosanoids using untargeted metabolomics; (B) Peak areas of selected amino acids in spiked water (green), clean-EBC (black), saliva-EBC (blue), and saliva (red) using untargeted metabolomics. A control water sample Page 20 of 22 Page 20 of 22 Cruickshank-Quinn et al. Respiratory Research (2017) 18:57 eicosatetraenoic acid; HILIC: Hydrophilic interaction chromatography; HMDB: Human metabolome database; KEGG: Kyoto encyclopedia of genes and genomes; LC: Liquid chromatography; LC-MS: Liquid chromatography mass spectrometry; LC-MS/MS: Liquid chromatography tandem mass spectrometry; LOD: Limit of detection; LOQ: Limit of quantitation; LTB4: Leukotriene B4; LTC4: Leukotriene C4; LTD4: Leukotriene D4; LTE4: Leukotriene E4; LXA4: Lipoxin A4; MS: Mass spectrometry; MSMS: Tandem mass spectrometry; PGE2: Prostaglandin E2; PGF2α: Prostaglandin F2 alpha; Protectin DX: 10(S),17(S)-DiHDoHE; QQQ: Triple quadrupole; Q-TOF: Quadrupole time-of-flight; RVD1: Resolvin D1; RVD2: Resolvin D2; SB-AQ: Stable bond (diisopropyl side chain group); SRM: Selected reaction monitoring; TxB2: Thromboxane B2 which was spiked with known concentrations of amino acids and eicosanoids was used to confirm the identities of these compounds in the saliva and EBC samples using exact mass, isotope ratios and retention time. (C) Peak area of LTB4 in internal standard, EBC and saliva using targeted analysis. (D) Peak area of LTE4 in internal standard and EBC using targeted analysis. Peak areas were extracted using MassHunter Quantitative Analysis software (Agilent). y-axis: mass spectral counts; x-axis: retention time. Starting volumes for untargeted metabolomics was 11.5 mL (clean-EBC) and 7.5 mL (saliva-EBC) with final volume of 20 μL and injection volume of 5 μL. Starting volume for targeted analysis was 1 mL saliva or EBC with an injection volume of 100 μL. (TIF 1669 kb) Additional file 3: Separation of PGF2α isomers in spiked control water. Samples were injected onto an SB-AQ analytical column. Since the four isomers could not be differentiated using untargeted analysis, multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer (QQQ-MS) with a C18 column was used to determine their elution order. (TIF 710 kb) Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Additional file 7: Metabolite annotations and tandem MS fragmentation patterns of compound detected in EBC. EBC was collected from four groups of volunteers: healthy smokers, healthy non-smokers, non-smokers with nasal congestion, and non-smokers with the common cold. Samples were pooled, lyophilized, reconstituted in 20 μL of buffer, and analyzed using LC-MS based metabolomics. Metabolite peaks were extracted with Mass Hunter Profinder software (Agilent) using exact mass and isotope ratios. Tandem MS was performed, spectra was exported to NIST MS Search v2.2, and matched to the NIST14 Mass Spectral library. Fragments in red indicate EBC sample, fragments in blue indicate NIST standard reference spectra. (PDF 545 kb) Authors’ contributions NR, MLA, and CCQ conceived and designed the experiments; CCQ, MLA, RP, and JG performed the experiments; CCQ, MLA, RP, and JG analyzed the data; CCQ wrote the manuscript; NR and ME edited the manuscript; all authors reviewed and approved the final manuscript. Additional file 6: Putatively identified eicosanoids in EBC. 13 mL of pooled EBC from 107 asthmatic subjects was lyophilized, reconstituted in 20 μL of buffer, and analyzed using LC-MS based metabolomics. Metabolite peaks were extracted using Profinder and MassHunter software using exact mass and isotope ratios (Agilent). Detected peaks are indicated by single colored lines. Database isotope pattern and distribution is indicated by a circled red box. Matches with multiple adducts are indicated. (PDF 55 kb) Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 5. 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Abbreviations 11β PGF 11b • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2234524225	http://www.bioline.org.br/pdf?rc15017	English	null	Adrenal venous sampling in a patient with adrenal Cushing syndrome	Colombia medica	2,015	cc-by	2,391	Builes-Montaño CE/et al/Colombia Médica - Vol. 46 Nº2 2015 (Apr-Jun) Builes-Montaño CE/et al/Colombia Médica - Vol. 46 Nº2 2015 (Apr-Jun) Builes-Montaño CE/et al/Colombia Médica - Vol. 46 Nº2 2015 (Apr-Jun) Case report colombiamedica.univalle.edu.co Article history La hiperplasia macro nodular bilateral o hiperplasia adrenal nodular bilateral independiente de la hormona adrenocorticotrópica es una de las causas menos frecuentes de hipercortisolismo, su diagnóstico supone un reto y no se tiene claridad de cuál es la mejor aproximación terapéutica. El muestreo venoso adrenal que frecuentemente se utiliza para hacer la distinción del sitio de producción hormonal en el hiperaldosteronismo primario podría ser una herramienta útil en este contexto ya que podría brindar información que pudiera guiar el tratamiento. Presentamos el caso de una paciente con síndrome de Cushing ACTH independiente en quien el uso del muestreo venoso adrenal con algunas modificaciones cambio de manera radical el tratamiento y permitió confirmar una hiperplasia adrenal macro nodular. The primary bilateral macronodular adrenal hyperplasia or the independent adrenocorticotropic hormone bilateral nodular adrenal hyperplasia is a rare cause hypercortisolism, its diagnosis is challenging and there is no clear way to decide the best therapeutic approach. Adrenal venous sampling is commonly used to distinguish the source of hormonal production in patients with primary hyperaldosteronism. It could be a useful tool in this context because it might provide information to guide the treatment. We report the case of a patient with ACTH independent Cushing syndrome in whom the use of adrenal venous sampling with some modifications radically modified the treatment and allowed the diagnosis of a macronodular adrenal hyperplasia. Received: 20 April 2015 Revised: 13 May 2015 Accepted: 09 June 2015 Received: 20 April 2015 Revised: 13 May 2015 Accepted: 09 June 2015 Adrenal venous sampling in a patient with adrenal Cushing syndrome Uso del muestreo venoso adrenal en un paciente con un síndrome de Cushing adrenal Carlos Esteban Builes-Montaño1, Carlos Andrés Villa-Franco2, Alejandro Román-Gonzalez3, Alejandro Velez-Hoyos4, Santiago Echeverri-Isaza5 Carlos Esteban Builes-Montaño1, Carlos Andrés Villa-Franco2, Alejandro Román-Gonzalez3, Alejandro Velez-Hoyos4, Santiago Echeverri Isaza5 1. Médico Internista Endocrinólogo. Sección de Endocrinología, Departamento de Medicina Interna. Hospital Pablo Tobón Uribe - Universidad de Antioquia. Medellín, Colombia. o Internista. Departamento de Medicina Interna, Hospital Pablo Tobón Uribe. Medellín, Colombia. 2. Médico Internista. Departamento de Medicina Interna, Hospital Pablo Tobón Uribe. Medellín, Colombia. 2. Médico Internista. Departamento de Medicina Interna, Hospital Pablo Tobón Uribe. Medellín, Colombia. 3. Residente de Endocrinología. Sección de Endocrinología, Departamento de Medicina Interna. Universidad de Antioquia. Medellín, Colombia. 4. Patólogo. Departamento de Patología. Hospital Pablo Tobón Uribe. Medellín, Colombia. 3. Residente de Endocrinología. Sección de Endocrinología, Departamento de Medicina Interna. Universidad de Antioquia. Medellín, Colombia. 4. Patólogo. Departamento de Patología. Hospital Pablo Tobón Uribe. Medellín, Colombia. 5. Médico Radiólogo Intervencionista. Departamento de Radiología. Hospital Pablo Tobón Uribe. Medellín, Colombia. Builes-Montaño CE, Villa-Franco CA, Román-Gonzalez A, Velez-Hoyos A, Echeverri-Isaza S. Adrenal venous sampling in a patient with adrenal Cushing syndrome. Colomb Med. 2015; 46(2): 84-7. © 2015. Universidad del Valle. 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. © 2015. Universidad del Valle. This is an Open Access article distributed under the terms of the Creative Commons Attributi distribution, and reproduction in any medium, provided the original author and source are credited. Keywords Cushing syndrome; adrenal cortex diseases; adrenal cortex function tests Síndrome de cushing; enfermedades de la corteza suprarrenal; pruebas de función de la corteza suprarrenal Palabras clave Síndrome de cushing; enfermedades de la corteza suprarrenal; pruebas de función de la corteza suprarrenal Corresponding author: Corresponding author: Carlos Esteban Builes Montaño, Hospital Pablo Tobón Uribe, Universidad de Antioquia. Medellín, Colombia. Telephone: +57 4 4459000. E-mail: cbuiles@ hptu.org.co. 84 Table 1. Main laboratory results Introduction Parameter Result Reference value Cortisol 11 pm (µg/dL) 19 5-18 Cortisol after suppression with dexa- methasone 1mg (µg/dL) 13.3 <1.8 12 Cortisol after suppression with dexa- methasone 8 mg (µg/dL) 15 ACTH (pg/mL) <1.0 7.2-63.3 Urinary cortisol (µg/24Horas) 212 32-243 Plasmatic renin activity (ng/mL/hora) 0.7 0.25-5.82 Aldosterone (ng/dL) 9.9 10-32 Creatinine (mg/dL) 0.64 0.6-1.1 Table 1. Main laboratory results Cushing syndrome is a rare disease caused in most cases (after steroid use has been excluded) by a pituitary ACTH-producing adenoma1. However, sometimes it is caused by an adrenal condition such as a unilateral adrenal adenoma, adrenal carcinoma or bilateral adrenal hyperplasia either pigmented nodular adrenocortical disease, also known as bilateral micronodular hyperplasia, or bilateral adrenal macronodular hyperplasia2-4. The standard of care in the adrenal Cushing’s syndrome is the resection of the affected gland, unilateral adrenalectomy in the case of an adenoma or bilateral in the case of a hyperplasia. In the latter case it implies a lifetime glucocorticoid and mineralocorticoid supplement. Some patients can present with a bilateral adrenal adenoma, in the case of primary hyperaldosteronism most of the time only one of the lesions is responsible of the hormonal production and the other one is simply a non-producing adenoma. In these patients with clearly identified hyperaldosteronism a bilateral adrenal veins catheterization with hormonal sampling is sometimes necessary in order to locate the source of aldosterone production, which may be unilateral or bilateral regardless of history and imaging findings5,6. This procedure has been described very rarely to differentiate cases of adrenal Cushing syndrome. In this report we describe the first case of adrenal vein catheterization for the study of an adrenal Cushing’s syndrome and a review of the bilateral adrenal hyperplasia as a rare cause of this disease. With these results an ACTH (adrenocorticotropic hormone) independent form of hypercortisolism was diagnosed and because the scan of the abdomen had documented the presence of two adenomas, one in each adrenal gland larger than 10 mm, we found ourselves with a therapeutic challenge since the resection of one of the glands may not cure the hypercortisolism in case that the hormonal production came from both glands and the resection of both glands with leave the patient with a permanent hypoadrenalism. It was decided then to perform an adrenal venous sampling to try to determine the origin of the of cortisol production. Conclusion To our knowledge this is the first report of Adrenal vein sampling in a patient with hypercortisolism and bilateral adrenal adenomas in our country. Own resources Own resources Discussion The macronodular bilateral adrenal hyperplasia or ACTH- independent macronodular adrenal hyperplasia (AIMAH), is one of the rarest causes of hypercortisolism and its true frequency has not been established. Less than 2% of all cases of hypercortisolism are explained by any form of bilateral adrenal hyperplasia either macro or micro nodular4. AIMAH usually presents with bilateral lesions that are larger than 10 mm and it is more frequent in women. The onset is usually later when compared to other forms of hypercortisolism8 (usually after the fifth decade of life) and the clinical manifestations are generally an autonomous subclinical glucocorticoids hypersecretion or subclinical Cushing syndrome (SCS). Our patient met the two criteria proposed for the diagnosis of this entity9. The SCS is a diagnostic challenge due to the lack of usual clinical features of this condition, the occasional small elevations of cortisol levels, the great amount of tests, the different cutoff points proposed by different authors for different tests and the lack of specificity of radiological characteristics that can occur in different conditions besides AIMAH like adrenal metastases. Our patient had lost of the circadian rhythm of cortisol production evidenced by high levels of cortisol at 23:00 h and autonomy in the production shown by the ACTH levels and lack of suppression in the low-dose dexamethasone test. As has been reported by other authors urinary free cortisol has a poor diagnostic yield in patients with SCS. Our patient had completely normal levels in several measurements10. We made some changes to the procedure reported in the literature, given the difficulty with the measurement of catecholamines we chose to perform verification of proper positioning of the catheter tip by the image from the venography and to correct the dilution difference by measuring aldosterone, the biochemical diagnosis was confirmed histological which allows us to conclude that the verification by venography was adequate. This technique allowed an accurate diagnosis and avoids the need for re-intervention in the case that the glandular size guided the treatment. Case report Place Cortisol (µg/dL) Aldosterone (pg/mL) Cortisol/aldosterone Ratio Cortisol ratio Cortisol lateralization ratio Adrenal vein/peripheral vein Right side/left side Right adrenal vein 63.6 160.5 3.9 5.0 3.1 Left adrenal vein 20.4 57.7 3.6 1.6 Inferior cava vein 12.6 41.3 3.1 3.1 The patient did not have any postoperative complication and received replacement therapy with hydrocortisone 50 mg every 8 h during the first 48 h and subsequently received prednisolone 10 mg and 0.1 mg of fludrocortisone replacement with resolution of the hypokalemia. Based on the above it was decided to perform the extraction of both adrenal glands and the result of the pathology confirmed the diagnosis of AIMAH. Based on the above it was decided to perform the extraction of both adrenal glands and the result of the pathology confirmed the diagnosis of AIMAH. Conflict of interest: l All authors do not have any possible conflicts of interest. Case report A previously described protocol was used7 with some changes. The authors of the original protocol propose the measurement of epinephrine as a method to determine the proper location of the catheters when performing the sampling. Because we do not have readily available the measurement of plasma catecholamines the test was performed using radiographic documentation of the tip of the catheter and aldosterone levels were used to make the corrections in the dilution between both sides2. Samples of both adrenal veins and inferior cava vein were taken (the procedure is shown in Figure 2 and the results are shown in Table 2). Based on the model of interpretation of adrenal venous sampling proposed by Young3 the test suggest that the patient has an adrenal hyperplasia with predominance in the production of cortisol from the right side, the results were discussed with the patient and the surgical team and it was then decided to perform a bilateral adrenalectomy. The result of the histological study of the glands was consistent with a bilateral adrenal macronodular hyperplasia. A 76 years-old woman with history of controlled hypertension (with losartan and amlodipine) presented to our hospital referring weight loss of 4 kg in two months, edema that progressed to anasarca and back lumbar pain. Besides the edema her physical examination was completely normal with no clinical signs suggestive of hypercortisolism. Among the studies requested, an abdominal tomography showed a right adrenal gland nodule of 14x9 mm and another one in the left adrenal gland of 23x18 mm (Fig. 1), additionally multiple vertebral fractures were reported. As part of the study of adrenal adenomas the patient had an abnormal value of cortisol after a low dose suppression test with dexamethasone (13.3 µg/dL (normal value: <1.8 µg/dL), with a normal value of free urinary cortisol and her potassium was low (she was not on diuretics). The results of the patient laboratory test are shown on Table 1. Figure 1 Abdominal tomography Figure 2. Adrenal vein sampling Figure 2. Adrenal vein sampling Figure 2. Adrenal vein sampling Figure 2. Adrenal vein sampling Figure 1. Abdominal tomography Figure 1. Abdominal tomography 85 Builes-Montaño CE/et al/Colombia Médica - Vol. 46 Nº2 2015 (Apr-Jun) Table 2. Adrenal venous sampling results. Table 2. Adrenal venous sampling results. Funding: Own resources References 1. Montoya-Escobar J, Builes-Montaño C, Johnayro G-R, Campuzano MG. Muestreo de senos petrosos inferiores en el diagnóstico de pacientes con síndrome de Cushing dependiente de hormona adrenocorticotrópica. Med Lab. 2013; 19(9-10): 411- 50. Adrenal vein sampling is used mainly in patients with hyperaldosteronism. Even in high volume centers with experienced radiologist the success rates are around 70 to 90%6. Adrenal vein sampling is rarely used for the study of hypercortisolism and this is probably due to the rarity of cases in which adrenal cortisol secretion and bilateral nodules present together, but it can be a useful test in this group of patients. It is proposed that some patients could be treated with a unilateral adrenalectomy as production of cortisol could be related to the size of the nodule11. But in the case of our patient catheterization revealed something else entirely, the nodule of the left adrenal gland was much larger but the production of cortisol came predominantly from the right adrenal gland nodule, although it has been reported that the 85% of people may have an increased production of cortisol in the right adrenal gland patient values exceeded the gradient described5. 2. Dinneen SF, Carney JA, Carpenter PC, Grant CS, Young WF. Acth-independent Cushing’s syndrome: bilateral cortisol- producing adrenal adenomas. Endocr Pract. 1995; 1(2): 77-81. 3. Young WF Jr, du Plessis H, Thompson GB, Grant CS, Farley DR, Richards ML, et al. The clinical conundrum of corticotropin- independent autonomous cortisol secretion in patients with bilateral adrenal masses. World J Surg. 2008; 32(5): 856-62. 4. De Venanzi A, Alencar GA, Bourdeau I, Fragoso MC, Lacroix A. Primary bilateral macronodular adrenal hyperplasia. Curr Opin Endocrinol Diabetes Obes. 2014; 21(3): 177-84. 5. Young WF, Stanson AW, Thompson GB, Grant CS, Farley DR, van Heerden JA. Role for adrenal venous sampling in primary aldosteronism. Surgery. 2004; 136(6): 1227-35. 86 Builes-Montaño CE/et al/Colombia Médica - Vol. 46 Nº2 2015 (Apr-Jun) 6. Stewart PM, Allolio B. Adrenal vein sampling for Primary Aldosteronism: time for a reality check. Clin Endocrinol (Oxf). 2010; 72(2): 146-8. 9. Akehi Y, Kawate H, Murase K, Nagaishi R, Nomiyama T, Nomura M, et al. Proposed diagnostic criteria for subclinical Cushing’s syndrome associated with adrenal incidentaloma. Endocrine J. 2013;60(7):903-12. 7. Maghrabi A, Yaqub A, Denning KL, Benhamed N, Faiz S, Saleem T. Challenges in the diagnostic work-up and management of patients with subclinical Cushing’s syndrome and bilateral adrenal masses. Endocrine Practice. References 2013; 19(3): 515-21. 10. Kidambi S, Raff H, Findling JW. Limitations of nocturnal salivary cortisol and urine free cortisol in the diagnosis of mild Cushing’s syndrome. European J Endocrinol. 2007; 157(6): 725- 31. 8. Lieberman SA, Eccleshall TR, Feldman D. ACTH-independent massive bilateral adrenal disease (AIMBAD): a subtype of Cushing’s syndrome with major diagnostic and therapeutic implications. European J Endocrinol. 1994; 131(1): 67-73 11. Lacroix A. ACTH-independent macronodular adrenal hyperplasia. Best Prac Res Clin Endocrinol Metab. 2009; 23(2): 245-59. Colomb Med. 2015; 46(2): 84-7 87
https://openalex.org/W4230110844	https://www.researchsquare.com/article/rs-45949/latest.pdf	English	null	Triboelectric Touch Sensor for Position Mapping During Total Hip Arthroplasty	Research Square (Research Square)	2,020	cc-by	2,920	Triboelectric touch sensor for position mapping during total hip arthroplasty Jae Bum Jeong Gyeongsang National University Hyeok Kim University of Seoul JUN-IL YOO (  furim@daum.net ) Gyeongsang National University Hospital JUN-IL YOO (  furim@daum.net ) Gyeongsang National University Hospital Introduction Total hip arthroplasty (THA) has become a common treatment for end-stage osteoarthritis of the hip.[1] However, despite improved implant designs and surgical techniques, bearing surface wear and the resultant wear-induced osteolysis have been major limitations to long-term prosthesis survival.[1–3] In an attempt to avoid the problems caused by wear debris, hard bearing surfaces, such as ceramic-on-ceramic (CoC) have been developed. In the decades since the 1970s, CoC bearings have made many advances. COC bearings have improved significantly in terms of wear reduction. However, concerns such as ceramic fracture, have not been resolved yet.[4] One of the biggest reasons for ceramic fracture is mal-seating of the ceramic liner.[5,6] Yoshitoshi et al. reported that 20% of the liners were observed to be mal-seated in imaging studies.[5] The clinical outcome of liner mal-seating, however, was not determined because there was no long-term follow-up. Nevertheless, negative outcomes, such as osteolysis, may occur. In addition, an intraoperative range of motion (ROM) check in the surgical field can predict postoperative impingement. And soft tissue tension and balance are measured by the Shuck test. All of the various postoperative problems depend on the experience and judgement of the surgeon in the operative field. Recently, Mecdessay et al. reported that the method for determining soft tissue balance used in total knee replacement arthroplasty (TKRA) was very inaccurate.[7] Moreover, they also noted that the use of pressure-based sensors increased the accuracy of knee balance determinations. In addition, an intraoperative range of motion (ROM) check in the surgical field can predict postoperative impingement. And soft tissue tension and balance are measured by the Shuck test. All of the various postoperative problems depend on the experience and judgement of the surgeon in the operative field. Recently, Mecdessay et al. reported that the method for determining soft tissue balance used in total knee replacement arthroplasty (TKRA) was very inaccurate.[7] Moreover, they also noted that the use of pressure-based sensors increased the accuracy of knee balance determinations. Inertial Measurement Units (IMU) based THA surgery has been reported in the experimental setting. However, there are few reports about sensor-based total hip implants in the real-life clinical field. This is because it is difficult to measure pressure and soft tissue balancing in spherical ball and socket joints. The triboelectric nanogenerator (TENG) was recently been developed as an effective tool for converting mechanical energy generator by an organic/polymer nanogenerator (NG) into electricity. Research note Research note Keywords: Triboelectric touch sensor, position mapping, total hip arthroplasty Posted Date: August 18th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-45949/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on August 26th, 2020. See the published version at https://doi.org/10.1186/s13104-020-05238-4. Page 1/10 Abstract Objective: In this research, a triboelectric nanogenerator (TENG) was utilized to determine if a pressure- based sensor could detect bearing friction in a total hip arthroplasty (THA) and detect the contact of specific areas during ROM checks. Results : The pressure-based sensor shows capability to sense bearing friction. In more detail, the TENG embedded in four different sides of the trial exhibits up to 1 V from peak-to-peak. Moreover, these flexible touch sensors with TENG describes a peak signal in output voltage which should lead to extremely sensitive detection of bearing friction induced by the THA. Methods To fabricate the flexible switch sensor, polydimethylsiloxane (PDMS) was mixed well with base and hardener at a ratio of 10: 1. Bubbles are formed in the PDMS during mixing, so treatment with a vacuum desiccator was performed to remove all bubbles from the PDMS. When all the bubbles were removed, the PDMS was poured onto a 4-inch Si wafer and spin-coated at 300 rpm for 30 seconds. Then, the PDMS was cured at 65 ° C for 2 hours in an oven. The PDMS film was then removed from the Si wafer. Copper tape, which is made of metal, was attached to the produced PDMS film to make an electrode and connection was made with a wire. The device structure of the TENG sensor described above is schematically shown in Fig 1a. In this configuration, the TENG worked through frictional triboelectricity by touching/untouching induced from deformation (Figs. 1b, c). To demonstrate the output characteristics of a TENG, a TENG-embedded trial was designed as depicted in Fig. 2a. Figs. 2b - e show the results obtained by measuring the output voltage with respect to time evolution by the application of touch between the TENG and the trial in all four directions. While the trial moved, a deformation appeared in the TENG and the bottom surface of the PDMS attached to the copper tape produced and lost triboelectric surface charges repeatedly. As a result, the output voltage emerged at the external circuit of the TENG. We measured these output voltages with an oscilloscope (KEYSIGHT DSOX2014A). Introduction The TENG has attracted considerable interest in the field of conventional piezoelectric devices and has been applied to various applications, such as wearable devices, wireless, stretchable devices, sensors, and flexible electronics.[8–13] The TENG can also play roles as pressure and touch sensors by sensitive reactions Page 2/10 Page 2/10 Page 2/10 based, not only on the large capacity for voltage generation but also the amount of electricity generated due to friction or pressure.[9] Pressure sensors can be used in a wide variety of applications, such as smart medical devices, real-time health status analysis, and operation modules for virtual reality control. [12] Moreover, most sensors require an external power supply, however, the TENG aims for wearable sensing devices because it can generate electricity and drive the sensor without any external power sources. [14–16] based, not only on the large capacity for voltage generation but also the amount of electricity generated due to friction or pressure.[9] Pressure sensors can be used in a wide variety of applications, such as smart medical devices, real-time health status analysis, and operation modules for virtual reality control. [12] Moreover, most sensors require an external power supply, however, the TENG aims for wearable sensing devices because it can generate electricity and drive the sensor without any external power sources. [14–16] In this research, a TENG was utilized to determine if a pressure-based sensor could detect bearing friction in a THA and detect the contact of specific areas during ROM checks. Results And Discussion The pressure-based sensor was able to sense bearing friction. In more detail, the TENG embedded in four different sides of the trial showed up to 1 V from peak-to-peak which was large enough to differentiate the detected signal from a noise level less than 0.1 V. Moreover, these flexible touch sensors with TENG exhibited a peak signal in output voltage which should lead to extremely sensitive detection of bearing friction induced by the THA. In general, pressure sensors, which are based on piezo-resistive active material or semiconductor materials fabricated by micro-electromechanical systems (MEM) technology, describe a continuous signal from external pressure. This may lead to insensitive detection between Page 3/10 Page 3/10 Page 3/10 diverse motions on the THA. In contrast, the TENG sensor system enabled comparably high sensitivity due to its capability to produce an output voltage with peaks. The pressure-based sensor was able to detect contact in certain areas while the COC bearing was in ROM. To investigate how the other sensors were affected when we applied pressure to one of four sensors in the THA, we measured the noise signals of the other three sensors, as depicted in the insets of Fig. 2 (b– e). The insets show the noise signals in the output voltage from the three other sensors when pressure was applied to the other sensor. The noise level rose as high as 0.2 V, which was negligible compared to the measured voltage output of the sensor in the target position (up-side) in Fig 2 b. The same phenomenon observed in Fig 2 (c)-(e) was seen for the right, left, and down sides respectively. These results show that sensing orthogonality was completely guaranteed by this device. The principal findings were that the TENG pressure-based sensor was able to detect bearing friction in the THA and that it was able to detect the contact area of the bearing surface during ROM. Soft tissue balancing is a very important test to prevent hip dislocation and to decrease postoperative pain in THA.[17] Until now, it has been evaluated subjectively by the operator using such tools as the Shuck test.[17] However, an objective evaluation of the pressure sensor used in this study will increase the success rate of the surgery. Results And Discussion Soft tissue balancing is a very important test to prevent hip dislocation and to decrease postoperative pain in THA.[17] Until now, it has been evaluated subjectively by the operator using such tools as the Shuck test.[17] However, an objective evaluation of the pressure sensor used in this study will increase the success rate of the surgery. In the case of total knee arthroplasty, a soft tissue balance check using a pressure-based sensor was conducted in the clinical field and was shown to be highly reproducible compared to the hand check.[7] If such sensor base data accumulates, it will be possible to explain the post-operative dislocation or complications that are unknown. In addition, better postoperative results can be expected by using pressure data, as well as imaging data, to determine the length of legs during surgery. When the sensor is inserted into the body, real-time wear monitoring becomes possible and the data can be used to analyze the cause of the revision timing and the pain of the patient. Furthermore, it is expected that research on sensors will be carried out at various implant development stages. In the range of motion evaluation during surgery, it is possible to predict the risk of impingement and dislocation by observing an increase in pressure at a specific area of the bearing surface. That is, it will be possible to immediately change the implant location within the surgical field. In addition, pressure sensing, and specific area mapping techniques will be used to determine the position of the ceramic liner and reduce the risk of mal-seating. In the future, the TENG sensor in THA will help soft tissue balancing and intraoperative ROM measurement. In particular, as the material science develops due to the characteristic of the self-powered TENG sensor, direct implantation into the articulation will be possible. In addition, the future integration of sensors that quantify the patient’s soft tissue tension, and hip stability through a full range of motion, enables the robot to make incremental implant and bone readjustments to allow true customization of a patient’s total hip soft tissue balance and alignment. Page 4/10 The conclusion of this study is that the TENG pressure-based sensor was able to detect friction in the THA bearing and detect the contact area of the bearing surface in the ROM. Further research will be carried out to develop biocompatible sensors and to enable precise pressure- sensing. Consent for publication Not applicable Availability of data and materials The dataset supporting this article is available upon request; please contact the corresponding author. The dataset supporting this article is available upon request; please contact the corresponding author. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable Limitations There were several limitations to this study. First, finer resolution was not able to scale the pressure. Second, we could not fine-tune the sensor mapping while guaranteeing orthogonality. Third, biotoxicity and biocompatibility studies should be performed to develop biodegradable formulations. And last, the accuracy and cost-effectiveness should be compared with existing navigation equipment. List of abbreviations TENG: triboelectric nanogenerator, THA: total hip arthroplasty, PDMS: polydimethylsiloxane, ROM: range of motion, CoC: ceramic-on-ceramic, TKRA: total knee replacement arthroplasty, IMU: Inertial Measurement Units Authors' Contributions All authors participated in the design, interpretation of the studies, analysis of the data, and review of the manuscript. HK and JIY did the design of the study and drafted the manuscript. HK and JBJ performed the exam and analyzed the data. JBJ wrote the manuscript. All authors read and approved the final manuscript. Affiliations Jae Bum Jeong; Department of Electrical Engineering, RIGET, Gyeongsang National University, Jinju 52828, Korea Hyeok Kim; Department of Electrical Engineering, ERI, Gyeongsang National University, Jinju 52828, Republic of Korea Hyeok Kim; Department of Electrical Engineering, ERI, Gyeongsang National University, Jinju 52828, Republic of Korea Jun-Il Yoo: Department of Orthopaedic Surgery, Gyeongsang national university hospital, Jinju, Republic of Korea Acknowledgements The authors would like to acknowledge Dr. Kang, Dr. Kim for their contributions to the content validation of the TENG sensor. Competing interests All authors have no conflict of interest to declare. Page 5/10 Funding This work was funded by the by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1F1A1059208). The National Research Foundation of Korea had no role in design or analysis of the project, or the writing of this article. References [1] Karachalios T, Komnos G, Koutalos A. Total hip arthroplasty: Survival and modes of failure. EFORT Open Rev. 2018 May 21;3(5):232-239. Page 6/10 Page 6/10 Self-powered, ultrasensitive, flexible tactile sensors based on contact electrification. Nano Lett. 2014 June;14(6):3208-3213. [16] Chen J, Zhu G, Yang W, Jing Q, Bai P, Yang Y, Hou TC, Wang ZL. Harmonic-resonator-based triboelectric nanogenerator as a sustainable power source and a self-powered active vibration sensor. Adv Mater Weinheim. 2013 Nov;25(42):6094-6099. [16] Chen J, Zhu G, Yang W, Jing Q, Bai P, Yang Y, Hou TC, Wang ZL. Harmonic-resonator-based triboelectric nanogenerator as a sustainable power source and a self-powered active vibration sensor. Adv Mater Weinheim. 2013 Nov;25(42):6094-6099. [17] Department of orthopedics and traumatology, MHAT Ruse, Ruse, Bulgaria., Kosev P, Valentinov B, Andonov Y, Sokolov C. SOFT TISSUE BALANCING IN TOTAL HIP ARTHROPLASTY. Journal of IMAB - Annual Proceeding (Scientific Papers). 2015 Jan;21(1):752-756. Page 6/10 [2] Kumar N, Arora GNC, Datta B. Bearing surfaces in hip replacement - Evolution and likely future. Med J Armed Forces India. 2014 Oct;70(4):371-376. [3] Gopinathan P. The Hard on Hard Bearings in THA - Current concepts. J Orthop. 2014 Sep;11(3):113- 116. [4] Howard DP, Wall PDH, Fernandez MA, Parsons H, Howard PW. Ceramic-on-ceramic bearing fractures in total hip arthroplasty: an analysis of data from the National Joint Registry. Bone Joint J. 2017 Aug;99- B(8):1012-1019. [5] Higuchi Y, Hasegawa Y, Komatsu D, Seki T, Ishiguro N. Incidence of Ceramic Liner Malseating After Ceramic-on-Ceramic Total Hip Arthroplasty Associated With Osteolysis: A 5- to 15-Year Follow-Up Study. J Arthroplasty. 2017 May;32(5):1641-1646. [6] Miller AN, Su EP, Bostrom MPG, Nestor BJ, Padgett DE. Incidence of ceramic liner malseating in Trident acetabular shell. Clin Orthop Relat Res. 2009 April;467(6):1552-1556. [7] MacDessi SJ, Gharaibeh MA, Harris IA. How Accurately Can Soft Tissue Balance Be Determined in Total Knee Arthroplasty? J Arthroplasty. 2019 Feb;34(2):290-294.e1. [8] Someya T, Sekitani T, Iba S, Kato Y, Kawaguchi H, Sakurai T. A large-area, flexible pressure sensor matrix with organic field-effect transistors for artificial skin applications. Proc Natl Acad Sci USA. 2004 July;101(27):9966-9970. [9] Hu Y, Zhang Y, Lin L, Ding Y, Zhu G, Wang ZL. Piezo-phototronic effect on electroluminescence properties of p-type GaN thin films. Nano Lett. 2012 June;12(7):3851-3856. [10] Sekitani T, Yokota T, Zschieschang U, Klauk H, Bauer S, Takeuchi K, Takamiya M, Sakurai T, Someya T. Organic nonvolatile memory transistors for flexible sensor arrays. Science. 2009 Dec;326(5959):1516- 1519. [11] Schwartz G, Tee BC-K, Mei J, Applceton AL, Kim DH, Wang H, Bao Z. Flexible polymer transistors with high pressure sensitivity for application in electronic skin and health monitoring. Nat Commun. 2013 May;4:1859. [12] Niu S, Wang S, Lin L, Liu Y, Zhou YS, Hua YF, Wang ZL. Theoretical study of contact-mode triboelectric nanogenerators as an effective power source. Energy Environ Sci. 2013 Jul;6(12):3576-3583. [13] Hu Y, Zhang Y, Xu C, Lin L, Snyder RL, Wang ZL. Self-powered system with wireless data transmission. Nano Lett. 2011 May;11(6):2572-2577. [14] Wang S, Xie Y, Niu S, Lin L, Wang ZL. Freestanding Triboelectric-Layer-Based Nanogenerators for Harvesting Energy from a Moving Object or Human Motion in Contact and Non-contact Modes. Advanced Materials. 2014 May;26(18):2818-2824. Page 7/10 Page 7/10 [15] Zhu G, Yang WQ, Zhang T, Jing Q, Chen J, Zhou YS, Bai P, Wang ZL.. Figures Page 8/10 gure 1 ENG sensor (A) Device Structure (B) PDMS (C) Operational mechanism of tribo Figure 1 TENG sensor (A) Device Structure (B) PDMS (C) Operational mechanism of triboelectric switch sensor Figure 1 TENG sensor (A) Device Structure (B) PDMS (C) Operational mechanism of triboelectric switch sensor TENG sensor (A) Device Structure (B) PDMS (C) Operational mechanism of triboelectric switch sensor TENG sensor (A) Device Structure (B) PDMS (C) Operational mechanism of triboelectric switch sensor TENG sensor (A) Device Structure (B) PDMS (C) Operational mechanism of triboelectric switch sensor Page 9/10 Figure 2 (A) Photograph of flexible touch sensors-embedded trial (B) Output voltage of measured signal on upsid Output voltage of measured signal on (C) right (D) left (E) down sides. Figure 2 (A) Photograph of flexible touch sensors-embedded trial (B) Output voltage of measured signal on upside; Output voltage of measured signal on (C) right (D) left (E) down sides. (A) Photograph of flexible touch sensors-embedded trial (B) Output voltage of measured signal on upside; Output voltage of measured signal on (C) right (D) left (E) down sides. Page 10/10
https://openalex.org/W2019759253	https://icce-ojs-tamu.tdl.org/icce/article/download/6662/pdf_641	English	null	EVALUATION OF A PARAMETRIC-TYPE WAVE TRANSFORMATION MODEL AGAINST FIELD AND LABORATORY DATA	Proceedings of Conference on Coastal Engineering/Proceedings of ... Conference on Coastal Engineering	2,012	cc-by	7,602	INTRODUCTION Parametric wave propagation models are broadly used in many coastal engineering applications. Depending on the parameter fitting, they mainly reflect 80%-85% accuracy, which is desirable in most coastal area problems (Ruessink et al., 2003; van Rijna et al., 2003). However, errors in wave prediction normally add up the amount of computation errors in hydrodynamic related parameters such as wave set up, sediment transport, and radiation stress (Guard and Baldock, 2007). Therefore, numerous researchers have put effort into this issue to increase the accuracy of parametric wave propagation models (e.g. Alsina and Baldock, 2007; Baldock et al., 1998; Battjes and Janssen, 1978; Ruessink et al., 2003; Thornton and Guza, 1983). Alsina and Baldock(2007) proposed a modified form of the parametric wave propagation model for non-saturated surfzone based on Baldock et al. (1998). However, they presented the results against laboratory data. In the present papert, Alsina and Baldock's (2007) model, hereafter referred to asAB07, is applied to data collected in South-East Queensland under stormy and calm conditions (Jafari et al., 2011), as well as laboratory data. Meanwhile, the model of Thornton and Guza (1983), hereafter referred to as TG83, and also Baldock et al. (1998), hereafter referred to as B98, were compared to the AB07 results. 1 Griffith School of Engineering, Gold Coast Campus, Griffith University, QLD 4222, Australia, (a.jafari@griffith.edu.au & n.cartwright@griffith.edu.au) Alireza Jafari1 and Nick Cartwright1 Alireza Jafari1 and Nick Cartwright1 Predicting wave properties via parametric wave propagation models are broadly used in many coastal engineering applications. Numerous researchers have refined these types of models to increase their accuracy including; Battjes and Janssen (1978), Thornton and Guza (1983), Baldock et al. (1998), and Alsina and Baldock (2007). Alsina and Baldock (2007), proposed an improved parametric wave propagation models for a non-saturated surfzone which returns relatively more accuracy in comparison to others. In this paper, the Alsina and Baldock (2007) model along with Baldock et al. (1998) and Thornton and Guza (1983), are applied to data collected in South-East Queensland under stormy and calm conditions as well as laboratory data. Some of the comparisons indicate the need to incorporate some additional energy loss at the break point to account for plunging type breakers where the existing bore dissipation model is insufficient. Keywords: Wave transformation model; surfzone hydrodynamics; wave energy dissipation; field data; storm condition PARAMETRIC WAVE MODELS Batttjes and Janssen (1978) took the very first step in this field and introduced their pioneer model which later modified and refined by others (e.g. TG83, B98, and AB07). Parametric models evaluate the wave height across the surfzone using energy flux equilibrium, ( ) ( ) E g D x EC − = ∂ ∂ (1) (1) where, Cg and E are respectively the group velocity and wave energy which can be estimated using linear wave theory, where, Cg and E are respectively the group velocity and wave energy which can be estimated using linear wave theory, 2 rms gH 8 1 E ρ = (2) θ cos 2 sinh 2 1 2 1 g ⎟ ⎠ ⎞ ⎜ ⎝ ⎛+ = kh kh C C (3) (3) where ρ is water density, C is wave phase velocity, k is wave number, h is water depth, and θ is approaching wave angle. DE denotes time averaged wave energy dissipation. In parametric models for determining DE, primarily, bore dissipation, DB, and secondarily dissipation due to bottom friction, Df, are considered (Thornton and Guza, 1983; Baldock et al., 1998; Alsina and Baldock, 2007). D D D where ρ is water density, C is wave phase velocity, k is wave number, h is water depth, and θ is approaching wave angle. DE denotes time averaged wave energy dissipation. In parametric models for determining DE, primarily, bore dissipation, DB, and secondarily dissipation due to bottom friction, Df, are considered (Thornton and Guza, 1983; Baldock et al., 1998; Alsina and Baldock, 2007). f B E D D D + = (4) (4) 1 COASTA TAL ENGINEEERING 2012 2 Bore Dissipation Model Bore Dissipation Model Bore e Dissipation Model Figure 1. Schematic sketch of wwave energy dissipation A wate (Batt and cons are l itself (LeM As a result of er surface. Th tjes and Janss can be treat servation of m located at unif f can be avo Mehaute, 1962 g f the wave bre he dissipation sen, 1978). Th ed as a trave mass and mom form flows bo oided. PARAMETRIC WAVE MODELS Hence 2), eaking, the wa rate due to br he turbulence elling hydrau mentum on the oth upstream a , the average ave crest gene reaking depen e on the spillin ulic jump (Th e control volu and downstrea e bore dissip gy erally curls ov nds on the siz ng breaker, q hornton and ume shown in am, the detail pation per un p ver and gener ze and strength qualitatively, i Guza, 1983). Figure 1, wh ls associated w nit area can b ates vortices a h of these vo s similar to a . By applying here the bound with the turbu be determine at the rtices a bore g the daries ulence ed by D B 4 1 ρ = ( ) h h h h g 1 2 3 1 2 ρ − ( g q 3 4 1 ρ ≈ ) q h BH 2 3 (5) (5) w facto defin where, H is th or describing t ned as ratio of he wave heigh the ration of f f phase velocit ht, q is the vo foam region o ty times water olume dischar over total wav r depth over w rge per unit w ve height. For wave length (H width across th a linear perio Hwang and Di he bore, and B odic bore, q c ivoky, 1970), B is a an be w facto defin where, H is th or describing t ned as ratio of he wave heigh the ration of f f phase velocit ht, q is the vo foam region o ty times water olume dischar over total wav r depth over w rge per unit w ve height. For wave length (H width across th a linear perio Hwang and Di he bore, and B odic bore, q c ivoky, 1970), B is a an be L Ch q = h f p = L Ch q = h f p = (6) (6) si peak frequen ingle breaking ncy. Substitut g wave as foll ting equation lows, (6) back into equation ((5) gives the bore w dissi where, fp is ipation for a si peak frequen ingle breaking ncy. Substitut g wave as foll ting equation lows, (6) back into equation ((5) gives the bore gf D B 4 1 ρ = ( ) h BH gf p 3 (7) (7) Bore dissipation model applied to a wave distribution equa the p over wave deter In order to o ation (7)needs product of eq r the total num e transformati rmining the fr obtain the av s to be multipl quation (7) an mber of waves ion across the raction of brea verage bore e lied by the nu nd the probabi s (see Figure e surfzone. Th aking waves in energy dissip umber of break ility density f 2), parametric he main differ n an irregular ation for a d king waves in function, pdf, c models eval rence between sea wave stat distribution o n the distributi of the ratio o luate the ener n the various te. f irregular w ion. By integr of breaking w rgy dissipation models rises waves, rating waves n and from COASTAL ENGINEERING 2012 3 Figure 2.Theoretical Rayleigh distribution of wave height (solid line) and breaking wave height distribution (shaded are) where H/Hrms≥ Hb/Hrms Figure 2.Theoretical Rayleigh distribution of wave height (solid line) and breaking wave height distribution (shaded are) where H/Hrms≥ Hb/Hrms After Batttjes and Janssen (1978),TG83 proposed an empirical weighted Rayleigh distribution, ased on field data recorded from Torrey Pines beach, ⎪⎭ ⎪⎬ ⎫ ⎪⎩ ⎪⎨ ⎧ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ − × ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ = = 2 rms 2 rms rms b H H exp H H 2 h H ) H ( p ) H ( W ) H ( p γ (8) (8) where, γ is the ratio of wave height over water depth and n is a variable determined equal to 4 based on observation. Therefore, by integrating the product of equation (7) and equation (8), TG83 proposed the overall energy dissipation as follows (Thornton and Guza, 1983), ⎤ ⎡ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ + − = − 2 5 2 rms 3 2 5 rms 3 p 83 TG B h H 1 1 1 h H B gf 16 3 D γ γ ρ π (9) (9) ⎦ ⎣ TG83 needs to be calibrated by means of determining the optimum value of coefficient B via iteration. Bore dissipation model applied to a wave distribution Consequently, achieving the best results from TG83 is limited to the availability of field data. TG83 needs to be calibrated by means of determining the optimum value of coefficient B via iteration. Consequently, achieving the best results from TG83 is limited to the availability of field data. B98 obtained the proportion of breaking waves, Qb, directly from the Rayleigh distribution. Qb is determined by integrating the Rayleigh distribution over all waves for which H/H ≥Hb/H resulting iteration. Consequently, achieving the best results from TG83 is limited to the availability of field data. B98 obtained the proportion of breaking waves, Qb, directly from the Rayleigh distribution. Qb is determined by integrating the Rayleigh distribution over all waves for which H/Hrms≥ Hb/Hrms resulting in (Baldock et al., 1998), B98 obtained the proportion of breaking waves, Qb, directly from the Rayleigh distribution. Qb is determined by integrating the Rayleigh distribution over all waves for which H/Hrms≥ Hb/Hrms resulting in (Baldock et al., 1998), , ), ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ − = ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ ⎪⎭ ⎪⎬ ⎫ ⎪⎩ ⎪⎨ ⎧ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ − = ∫ ∞ 2 rms b rms H 2 rms rms b H H exp H H d H H exp H H 2 Q * (10) (10) ⎦ ⎣ where, H* = Hb/Hrms and Hb is maximum wave height just before breaking. B98 applied Nairn's (1990) expression of Hb which is as follows, where, H* = Hb/Hrms and Hb is maximum wave height just before breaking. B98 applied Nairn's (1990) expression of Hb which is as follows, ( ) 0 33 tanh 56 . 0 39 . 0 S h H b + = (11) (11) where, S0 is offshore wave steepness. B98 assumed that the relationship of H/h in equation (7) is close to 1. Also, they suggested that factor B can be considered 1 for simplification purposes. Bore dissipation model applied to a wave distribution Thus, the time averaged rate of energy dissipation proposed by B98 does not assume prior knowledge of the surfzone condition, which is given by (Baldock et al., 1998), ( ) 2 rms 2 b 2 rms b p 98 B B H H H H exp B gf 4 1 D + × ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ − = − ρ (12) (12) 4 COASTA TAL ENGINEEERING 2012 A How (199 Henc dissi AB07 followe wever,AB07 m 96)showed the ce, the origina ipation formul ed the B98 ap mentioned that e assumption o al term of H3/h la (Alsina and proaches. The t the field and of considering hin equation ( d Baldock, 200 ey considered laboratory da g ratio of H/h e (7)was retaine 07), the Rayleigh ata presented b equal to 1 is n ed and AB07 p distribution fo by Raubenheim not the case clo proposed the f for evaluating mer et al. ose to shorelin following bore Qb. ne. e ⎪ ⎪ ⎪ ⎩ ⎪⎪ ⎪ ⎨ ⎧ ⎢ ⎢ ⎣ ⎡ ⎜⎜ ⎝ ⎛ − ⎢ ⎢ ⎣ ⎡ ⎠ ⎞ ⎜⎜ ⎝ ⎛ − rms b 07 AB B H exp H H D + ⎥ ⎥ ⎦ ⎤ ⎟⎟ ⎠ ⎞ ⎥ ⎥ ⎦ ⎤ + ⎟⎟ ⎠ ⎞ = 2 rms b rms b 3 p 4 3 H H H H 2 3 H B gf 4 1 ρ ⎢ ⎢ ⎣ ⎡ ⎜⎜ ⎝ ⎛ − × × 3 rms H erf 1 h π ⎪ ⎪ ⎪ ⎭ ⎪⎪ ⎪ ⎬ ⎫ ⎥ ⎥ ⎦ ⎤ ⎟⎟ ⎠ ⎞ rms b H H (13) (13) where, erf denote the error function. Data collection and analysis F used wate show Field data wa d an array of p er levels (MW wn in Figure 3 as collected fr permanently de WL) and high f 3 (Jafari et al., rom a field si eployed mano frequency pre 2011). ite located at ometer tubes i essure fluctuat The Spit on n order to obs tions at 12 loc Gold Coast, A serve both tim cations throug Australia. Th me averaged (m gh the surf zo e site mean) one as offsh Wav to th of sp in Fi Fig The setup of hore. The pre ve height coul he recorded sig pectral analys igure 4. gure 3. Sketch f the manome essure transdu d then be calc gnals. The sta is and zero-cr of the array of eter tube syste ucer sensors w culated by app atistics of the w rossing approa f the manomete em enabled t were used to plying a series wave properti aches. The ba er tubes used t he wave train record the hig s of equations ies were then athymetry of e to record the fi ns to be mon gh frequency s specified by computed by each field con ield data nitored from 5 water fluctua Jafari et al. (2 the usual met ndition is pres 500m ation. 2011) thods ented Figure 3. Sketch of the array of the manometeer tubes used to record the fi ield data Figure 3. Sketch of the array of the manometeer tubes used to record the fiield data offsh Wav to th of sp in Fi The setup of hore. The pre ve height coul he recorded sig pectral analys igure 4. f the manome essure transdu d then be calc gnals. The sta is and zero-cr eter tube syste ucer sensors w culated by app atistics of the w rossing approa em enabled t were used to plying a series wave properti aches. The ba he wave train record the hig s of equations ies were then athymetry of e ns to be mon gh frequency s specified by computed by each field con nitored from 5 water fluctua Jafari et al. (2 the usual met ndition is pres 500m ation. 2011) thods ented COASTAL ENGINEERING 2012 COASTAL ENGINEERING 2012 COASTAL ENGINEERING 2012 5 Figure 4. Surfzone bathymetry based on the Australian height datum (AHD) used in the modelling Figure 4. Data collection and analysis Surfzone bathymetry based on the Australian height datum (AHD) used in the modelling Consistent with data obtained from the nearby Gold Cost wave rider buoy, the manometer tube wave data was divided into thirty minute time blocks and wave characteristics were extracted using both spectral and zero-crossing approaches. Result are compared and verified against Gold Cost wave rider buoy. The buoy data obtained from the Queensland Department of Environment and Resource Management. Results shows a very good agreement between the recorded data and the buoy data (Jafari et al., 2011). Table 1 presents the field condition and wave statistical data sets used in the comparison against the AB07, B98, and TG83 models. These wave properties are extracted from zero up crossing method. Also these data are just extracted from one block of data sets (thirty minutes of recording). The tide condition of each block of data presented in Figure 5. In order to fairly evaluate the Rayleigh distribution based models, the recorded data of each event were first analysed to check that they indeed conformed to the Rayleigh distribution. Figure 6 displays the comparison of the recorded data by offshore sensor against Rayleigh distribution for all events. This comparison reveals that the recorded field data do conform to the Rayleigh distribution (the poorest R2 is 0.97). Table 1. Field Condition tested against model prediction, where So is offshore wave steepness Event Offshore Boundary (m) Offshore Depth (m) Hmax Hrmso Tp Tave So TC Hamish (11/03/2009) 500 7.6 4.8 2.3 8.2 8.2 0.022 East Coast Lows (21/05/2009) 500 7.8 5.4 3.2 9.4 10.0 0.023 East Coast Lows (21/05/2009) 300 5.6 2.7 1.1 9.7 12.0 0.008 Calm Condition (11/11/2009) 500 5.4 0.9 0.6 7.9 7.2 0.008 TC Ului (20/03/2010) 300 4.4 3.0 1.4 11.0 7.6 0.015 Table 1. Field Condition tested against model prediction, where So is offshore wave steepness COASTA TAL ENGINEEERING 2012 6 Figure 5. Tide condition for each event thhat field data were collected Figure 5. Tide condition for each event thhat field data were collected Figure 5. Tide condition for each event thhat field data were collected Results and Discussion in pr The data-mod rediction, ε, as del comparison s used by Alsi n was quantif ina and Baldo fied based on t ck (2007) and the coefficien d calculated as nt of determina s follows: ation, R2, and erro in pr The data-mod rediction, ε, as del comparison s used by Alsi n was quantif ina and Baldo fied based on t ck (2007) and the coefficien d calculated as nt of determina s follows: ation, R2, and error ε ( co H N 1 ∑ = ) o 2 meas omp H H − (14) (14) wher the s re, the N is th statistical wav he total numb ve property tha er of samples at is used in th s and Ho is of his calculation 2 ffshore bound n. ary wave heigght. Also, ܪ௥௠ ௠௦, i A acro pred amou As it depicted ss the surfzon dict the wave unt of the en d in Figure 7 b ne is in good a shoaling that nergy dissipati based on calcu agreement wit t took places ions calculate ulated R2, and th the data of in recorded d ed by the mo d ε, the model f TC Hamish. data in about odels (second ls prediction o However, the t 400m offsho panel of Figu of the wave p ey cannot prec ore tube. The ure 7) from 4 rofile cisely en the 400m A acro pred amou As it depicted ss the surfzon dict the wave unt of the en d in Figure 7 b ne is in good a shoaling that nergy dissipati based on calcu agreement wit t took places ions calculate ulated R2, and th the data of in recorded d ed by the mo d ε, the model f TC Hamish. data in about odels (second ls prediction o However, the t 400m offsho panel of Figu of the wave p ey cannot prec ore tube. The ure 7) from 4 rofile cisely en the 400m 7 COASTAL ENGINEERING 2012 offshore tube to 300m are about half of what the recorded data showed. Also, it should be noted that as TG83 results numerically fit to the data by varying the B coefficient returns slightly better coefficient of determination (R2) with data and a bit less error comparing with AB07 and B98. Results and Discussion In East Coast low if the offshore boundary set on 500m tube length, the results of the models predictions are over predicted (see Figure 8). The main reason is that the models cannot capture the first breaking point where was happened somewhere around 400m offshore. Accordingly, when the 300m tube length set as the offshore boundary of the the models, i.e. Figure 9, they still cannot clearly capture the second breaking point; however the results are not over predicted. In both events, TG83 showed better prediction than AB07 and B98 base on the fact that the coefficient factor B in equation (9) should be optimize via recorded data, however it fixed as unity in AB07 and B98. By increasing the value of B coefficient in the dissipation formula, literally, the ratio of the vortices area of the bore to the wave height increases. The B values over one conveys the fact that the ration of vortices penetrated into mean water level below the trough of the wave. Also, in second panel of Figure 9, which shows the amount of energy dissipation, the TG83 model shows a spike on the shoreline boundaryFigure 8. Therefore, it conveys that wave breaking occurs on that point which is not the case. Although the TG83 model prediction of stormy events, gives a better correlation with field data due to manipulating the B factor, it didn't physically picture a better result in comparison with B98 and AB07. Figure 6. Compare data series of offshore boundaries sensors (from top-left to bottom-right H1, E1-B, C1-B, and U1) against Rayleigh distribution. N is the total number of wave data and n represent the rank of corresponding wave height sorted from large to small. R 2 is Coefficients of determinations between the data and Rayleigh distribution. Figure 6. Compare data series of offshore boundaries sensors (from top-left to bottom-right H1, E1-B, C1-B, and U1) against Rayleigh distribution. N is the total number of wave data and n represent the rank of corresponding wave height sorted from large to small. R 2 is Coefficients of determinations between the data and Rayleigh distribution. As depicted in Figure 10 if the offshore boundary is set on 500m offshore in the calm condition data sets, the model results are under predicted. The main reason is that none of the models can accurately evaluate the shoaling which occurs about 400m offshore. Results and Discussion Also, the value of estimated energy dissipation per meter square by the AB07 and B98 models at second breaking point are almost as half of what really happened in the field. COASTAL ENGINEERING 2012 8 Figure 7. TC Hamish model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 8. ECL model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 7. TC Hamish model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 7. TC Hamish model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 8. ECL model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 8. ECL model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. COASTAL ENGINEERING 2012 9 Figure 9. ECL (offshore boundary set as 300m) model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 10. Results and Discussion TC Ului ipation per uni In the TC Ulu models cannot gy dissipation er than what f uce the error, i AB07 model fficient of dete Figure 12 0 5 10 15 20 25 30 35 % Error model-data co t area and 3rd the ui events, the t predict the n n calculated b field data sho it was shown yields a slig ermination of 2. Calculated e Hamish omparison; Top panel is the ba red dotted line offshore boun near shore brea by models at t ows. Also due a better predic ghtly better r the B98 mode rror of models ECL-500 E p panel is Hrms athymetry. Circ e and TG83 is t ndary was set aking. Second the near shore e to numerica ction in comp result in term el result is bet s prediction bas ECL-300 M distribution, 2 cles represent he green dash at 400m offsh d panel of the e breaking lin ally calibration pare to AB07 ms of predict ter than what sed on equatio Mild Ulu 2nd panel is dis data; AB07 is line. hore. As illus Figure 11 cle ne (x = -150m n of the TG3 and B98. Furt ion error rath AB07 returns on (14) against ui % AB B9 TG TG stribution of en the blue line; B trated in Figu arly shows th m) are conside model in ord thermore, alth her than B98 s. field data Error B07 98 G83 G83 (B=1) nergy B98 is ure 11 at the erably der to hough 8, the Figu dissi ure 11. TC Ului ipation per uni model-data co t area and 3rd the omparison; Top panel is the ba red dotted line p panel is Hrms athymetry. Circ e and TG83 is t distribution, 2 cles represent he green dash 2nd panel is dis data; AB07 is line. stribution of en the blue line; B nergy B98 is Figu dissi ure 11. TC Ului ipation per uni model-data co t area and 3rd the omparison; Top panel is the ba red dotted line p panel is Hrms athymetry. Circ e and TG83 is t distribution, 2 cles represent he green dash 2nd panel is dis data; AB07 is line. Results and Discussion stribution of en the blue line; B nergy B98 is the m ener lowe redu the A coef In the TC Ulu models cannot gy dissipation er than what f uce the error, i AB07 model fficient of dete ui events, the t predict the n n calculated b field data sho it was shown yields a slig ermination of offshore boun near shore brea by models at t ows. Also due a better predic ghtly better r the B98 mode ndary was set aking. Second the near shore e to numerica ction in comp result in term el result is bet at 400m offsh d panel of the e breaking lin ally calibration pare to AB07 ms of predict ter than what hore. As illus Figure 11 cle ne (x = -150m n of the TG3 and B98. Furt ion error rath AB07 returns trated in Figu arly shows th m) are conside model in ord thermore, alth her than B98 s. ure 11 at the erably der to hough 8, the the m ener lowe redu the A coef In the TC Ulu models cannot gy dissipation er than what f uce the error, i AB07 model fficient of dete ui events, the t predict the n n calculated b field data sho it was shown yields a slig ermination of offshore boun near shore brea by models at t ows. Also due a better predic ghtly better r the B98 mode ndary was set aking. Second the near shore e to numerica ction in comp result in term el result is bet at 400m offsh d panel of the e breaking lin ally calibration pare to AB07 ms of predict ter than what hore. As illus Figure 11 cle ne (x = -150m n of the TG3 and B98. Furt ion error rath AB07 returns trated in Figu arly shows th m) are conside model in ord thermore, alth her than B98 s. ure 11 at the erably der to hough 8, the the m ener lowe redu the A coef In the TC Ulu models cannot gy dissipation er than what f uce the error, i AB07 model fficient of dete ui events, the t predict the n n calculated b field data sho it was shown yields a slig ermination of offshore boun near shore brea by models at t ows. Results and Discussion Mild Condition model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 9. ECL (offshore boundary set as 300m) model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 9. ECL (offshore boundary set as 300m) model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 9. ECL (offshore boundary set as 300m) model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 10. Mild Condition model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. Figure 10. Mild Condition model-data comparison; Top panel is Hrms distribution, 2nd panel is distribution of energy dissipation per unit area and 3rd panel is the bathymetry. Circles represent data; AB07 is the blue line; B98 is the red dotted line and TG83 is the green dash line. COASTA TAL ENGINEEERING 2012 10 Figu dissi the m ener lowe redu the A coef ure 11. Results and Discussion Calculated f of model pred rder to being f and then the er ese figures, the 0 0 0 0 0 0 0 0 0 0 0 Hamish factor of determ diction against fair to all mod rror and R2 we e TG83 mode ECL-500 mination (R 2) o t field data are dels, the calibr ere recalculate el with conside ECL-300 of models pred e presented in ration factor, B ed and display ering B equal Mild Ul iction against f Figure 12 and B, is also cons yed on Figure to one genera lui R AB B9 TG TG field data d Figure 13 sidered as on 12 and Figu ally returned R2 B07 98 G83 G83 (B=1) Figure 13. Calculated factor of determination (R 2) oof models prediction against ffield data The respe the T As it error error and R2 o ectively. In or TG83 model a t shown in the rs and a bit les of model pred rder to being f and then the er ese figures, the ss R2. diction against fair to all mod rror and R2 we e TG83 mode t field data are dels, the calibr ere recalculate el with conside e presented in ration factor, B ed and display ering B equal Figure 12 and B, is also cons yed on Figure to one genera d Figure 13 sidered as one 12 and Figur ally returned h for e 13. higher The respe the T As it error error and R2 o ectively. In or TG83 model a t shown in the rs and a bit les of model pred rder to being f and then the er ese figures, the ss R2. diction against fair to all mod rror and R2 we e TG83 mode t field data are dels, the calibr ere recalculate el with conside e presented in ration factor, B ed and display ering B equal Figure 12 and B, is also cons yed on Figure to one genera d Figure 13 sidered as one 12 and Figur ally returned h for e 13. highe Results and Discussion Also due a better predic ghtly better r the B98 mode ndary was set aking. Second the near shore e to numerica ction in comp result in term el result is bet at 400m offsh d panel of the e breaking lin ally calibration pare to AB07 ms of predict ter than what hore. As illus Figure 11 cle ne (x = -150m n of the TG3 and B98. Furt ion error rath AB07 returns trated in Figu arly shows th m) are conside model in ord thermore, alth her than B98 s. ure 11 at the erably der to hough 8, the Figure 12 0 5 10 15 20 25 30 35 % Error 2. Calculated e Hamish rror of models ECL-500 E s prediction bas ECL-300 M sed on equatio Mild Ulu on (14) against ui % AB B9 TG TG field data Error B07 98 G83 G83 (B=1) Figure 12. Calculated error of models prediction bassed on equation (14) against field data COASTA TAL ENGINEEERING 2012 11 The respe the T As it error Figure 1 error and R2 o ectively. In or TG83 model a t shown in the rs and a bit les 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 R2 3. Calculated f of model pred rder to being f and then the er ese figures, the ss R2. 0 0 0 0 0 0 0 0 0 0 0 Hamish factor of determ diction against fair to all mod rror and R2 we e TG83 mode ECL-500 mination (R 2) o t field data are dels, the calibr ere recalculate el with conside ECL-300 of models pred e presented in ration factor, B ed and display ering B equal Mild Ul iction against f Figure 12 and B, is also cons yed on Figure to one genera lui R AB B9 TG TG field data d Figure 13 sidered as one 12 and Figur ally returned h R2 B07 98 G83 G83 (B=1) for e 13. higher Figure 1 ror and R2 o tively. In or 83 model a hown in the 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 R2 3. Experimental Setup Figure 15.1:57 scale of the Gold Coast profile made in the wave flume for the experiment The offshore wave height recorded by the first sensor immediately after the wave maker, at a depth of 0.188m, was defined as the offshore boundary of the model. 36 different wave conditions were generated in this experiment which Hrms varies from 0.02m to 0.058m and Tp varies from 0.623s to 4.101s, using JONSWAP spectrum with gamma equal to 3.3. The offshore wave height recorded by the first sensor immediately after the wave maker, at a depth of 0.188m, was defined as the offshore boundary of the model. 36 different wave conditions were generated in this experiment which Hrms varies from 0.02m to 0.058m and Tp varies from 0.623s to 4.101s, using JONSWAP spectrum with gamma equal to 3.3. Experimental Setup were irreg beac heigh poin and z first Laboratory ex e generated us gular waves. A ch profile was ht, 15 Pressur nts along the f zero-up cross sensor from t xperimentatio sing an electr A wave absor s constructed re Transducer flume (see Fig ing analysis. I the left hand s ns were perfo rically driven rption system in the scale r sensors with gure 14). The Incident wave ide (see Figur ormed on an wave maker was deploye of 1:57. In o h accuracy of e statistical wa e properties w re 14). 8m long and with the abil ed at the far e order to captu f order ±1 mm ave properties were extracted 0.5m wide w ity to produc end of the flum ure the high r m were instal s were calcula from an offsh wave flume. W e both regula me. A Gold C resolution of led in 12 diff ated using spe hore sensor, i. Waves ar and Coast wave ferent ectral e. the 12 COASTAL ENGINEERING 2012 Figure 14.Elevation (top) and plan (bottom) views of the experiment setup in the wave flume. The wave maker is located at the far end of the left side of the flume. The pressure transducer sensor spacing are shown in both the plan and the section views of the wave flume The bottom profile was surveyed by filling the flume up to 0.215m, measured at the offshore boundary, and then the profile was measured against the still water level at 0.1m intervals along the flume (see Figure 15). The survey was repeated on different days of testing in order to capture any changes. Also a test run was made prior to each experiment in order to find out the approximate breaking points. This information was used to determine the optimal position of the sensors in order to capture the wave breaking with more resolution. Figure 15.1:57 scale of the Gold Coast profile made in the wave flume for the experiment The offshore wave height recorded by the first sensor immediately after the wave maker, at a depth f 0.188m, was defined as the offshore boundary of the model. 36 different wave conditions were enerated in this experiment which Hrms varies from 0.02m to 0.058m and Tp varies from 0.623s to 101s, using JONSWAP spectrum with gamma equal to 3.3. Results and Discussion For all the test conditions the offshore surf similarity, ζo, was calculated along with R2 and ε. Results of model prediction against experimental data are plotted versus ζo and presented in Figure 16 and Figure 17. according to Coastal Engineering Manual (2002) ζo is defined as a wave breaker type criteria which ζo is less than 0.5, the breaker type is spilling and for ζo greater than 0.5, the breaker type would be plunging. Figure 16 clearly illustrated that for ζo greater than 0.5 the amount of calculated error in model prediction increases. Also in Figure 17 this fact in confirmed by decreasing the amount of R2 for ζo greater than 0.5. Hence, based on these results, the bore dissipation theory which deployed in the current parametric energy dissipation models is not good enough to predict the plunging breaker type with high accuracy. Therefore, it is require to consider the plunging breaking dissipation in this type of models. COASTAL ENGINEERING 2012 13 13 Figure 16. Calculated error of models prediction based on equation (14) against lab data versus ξo Figure 17. Calculated R 2 of models prediction based on equation (14) against lab data versus ξo NCLUSION 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 ε Offshore Surf Similarity (ξo) ξo vs ε AB07 B98 TG83 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 R2 Offshore Surf Similarity (ξo) ξo vs R2 AB07 B98 TG83 Figure 16. Calculated error of models prediction based on equation (14) against lab data versus ξo 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 ε Offshore Surf Similarity (ξo) ξo vs ε AB07 B98 TG83 Offshore Surf Similarity (ξo) Figure 16. Calculated error of models prediction based on equation (14) against lab data versus ξo Figure 16. Calculated error of models prediction based on equation (14) against lab data versus ξo Figure 17. CONCLUSION The parametric wave models result in predicting the wave profile across the surfzone are very precise in the case of swells. Also, in term of choosing a proper model, AB07 and B98 have privilege to TG83 in the cases that no data is available prior to modeling. TG83 leaves two free parameters (i.e. B and γ) in the model in case of calibration based on existing data. Free parameter, “B”, in TG83 model numerically improves the results in comparison against the field data (e.g. more than 140% improvement in ε in ECL). However, B can be considered as a tuning parameter in AB07 and B98 as well. Moreover, considering the field data comparison, models are not able to capture the sudden energy loss due to plunging breaker. Consequently, plunging dissipation should be considered in order to improve the existing parametric models. Hence, wave energy dissipation due to plunging is going to be considered in the next stage of this research. Results and Discussion Calculated R 2 of models prediction based on equation (14) against lab data versus ξo NCLUSION 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 R2 Offshore Surf Similarity (ξo) ξo vs R2 AB07 B98 TG83 Figure 17. Calculated R 2 of models prediction based on equation (14) against lab data versus ξo 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 R2 Offshore Surf Similarity (ξo) ξo vs R2 AB07 B98 TG83 Figure 17. Calculated R 2 of models prediction based on equation (14) against lab data versus ξo Alsina, J.M. and Baldock, T.E., (2007). Improved representation of breaking wave energy dissipation in parametric wave transformation models. Coastal Engineering 54 765–769. Baldock, T.E., Holmes, P., Bunker, S. and Van Weert, P., (1998). Cross-shore hydrodynamics within an unsaturated surf zone. Coastal Engineering, 34: 173–196. Battjes, J.A. and Janssen, J.P.F.M., (1978). Energy loss and set-up due to breaking of random waves, 16th Int. Conf. on Coastal Engineering ASCE, Hamburg, Germany, pp. 569–587. Alsina, J.M. and Baldock, T.E., (2007). Improved representation of breaking wave energy dissipation in parametric wave transformation models. Coastal Engineering 54 765–769. Baldock, T.E., Holmes, P., Bunker, S. and Van Weert, P., (1998). Cross-shore hydrodynamics within an unsaturated surf zone. Coastal Engineering, 34: 173–196. B j J A d J J P F M (1978) E l d d b ki f d f g g, Battjes, J.A. and Janssen, J.P.F.M., (1978). Energy loss and set-up due to breaking of random waves, 16th Int. Conf. on Coastal Engineering ASCE, Hamburg, Germany, pp. 569–587. Alsina, J.M. and Baldock, T.E., (2007). Improved representation of breaking wave energy dissipation in parametric wave transformation models. Coastal Engineering 54 765–769. REFERENCES Alsina, J.M. and Baldock, T.E., (2007). Improved representation of breaking wave energy dissipation in parametric wave transformation models. Coastal Engineering 54 765–769. p f g g Baldock, T.E., Holmes, P., Bunker, S. and Van Weert, P., (1998). Cross-shore hydrodynamics within an unsaturated surf zone. Coastal Engineering, 34: 173–196. f g g, Battjes, J.A. and Janssen, J.P.F.M., (1978). Energy loss and set-up due to breaking of random waves, 16th Int. Conf. on Coastal Engineering ASCE, Hamburg, Germany, pp. 569–587. COASTAL ENGINEERING 2012 14 Guard, P.A. and Baldock, T.E., (2007). The influence of seaward boundary conditions on swash zone hydrodynamics. Coastal Engineering, 54(4): 321-331. y y g g ( ) Hwang, L.S. and Divoky, D., (1970). Braking wave set up and decay on gentle slopes, In Proceeding of the 12th international conference on coastal engineering pp. 377-389. Jafari, A., Cartwright, N. and Nielsen, P., (2011). Stormy Wave Analysis Based on Recorded Field Data on South-East Coasts of Queensland, Australia. Journal of coastal Research, SI 64: 527- 533. LeMehaute, (1962). On Non-Saturated Breakers and the Wave Run-Up, 8th International Conference of Coastal Engineering, New York, pp. 77-92. Nairn, R.B., (1990). Prediction of cross-shore sediment transport and beach profile evolution University of London, London, England. Raubenheimer, B., Guza, R.T. and Elgar, S., (1996). Wave transformation across the inner surf zone. Journal of geophysical research, 101: 25,589-25,597. Ruessink, B.G., Walstraa, D.J.R. and Southgate, H.N., (2003). Calibration and verification of a parametric wave model on barred beaches. Coastal Engineering, 48: 139–149 Thornton, E.B. and Guza, R.T., (1983). Transformation of Wave Height Distribution. Journal o geophysical research, 88: 5925-5938. g p y US Army Corps Of Engineers, N., (2002). Coastal Engineering Manual. Analysis. rmy Corps Of Engineers, N., (2002). Coastal Engine y p g ( ) g g y van Rijna, L.C. et al., (2003). The predictability of cross-shore bed evolution of sandy beaches at the time scale of storms and seasons using process-based Profile models. Coastal Engineering, 47(295–327).
W3042667291.txt	https://journal2.um.ac.id/index.php/jabe/article/download/14367/6010	en		Individualized Excel-Based Exams to Prevent Students from Cheating	JABE (Journal of Accounting and Business Education)	2,020	cc-by-sa	5,132	A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 JOURNAL OF ACCOUNTING AND BUSINESS EDUCATION P-ISSN 2528-7281 E-ISSN 2528-729 E-mail: jabe.journal@um.ac.id http://journal2.um.ac.id/index.php/jabe/ Individualized Excel-Based Exams to Prevent Students from Cheating Ani Wilujeng Suryani Accounting Department, Faculty of Economics, Universitas Negeri Malang, Indonesia email: ani.suryani@um.ac.id DOI: http://dx.doi.org/10.26675/jabe.v5i1.14367 Abstract: The COVID-19 pandemic has brought a disruption to education, especially on how courses are delivered in higher education. Higher education providers are forced to go online, without considering the availability of IT infrastructure and capability of lecturers in delivering online courses. Since online learning is perceived as an opportunity for academic integrity breaches, academicians need to ensure that the learning processes prevent students from cheating activities, especially during the exams. This paper presents an innovative approach in delivering online, Excel-based exams which enable the lecturers to develop individualized exam questions. The accounting method and/or policy, as well as the accounting figures in the exams are automatically set based on the students’ identification numbers. This program also allows automatic scorings for such exams. Based on the students’ perceptions collected from the online survey, this type of exam was not considered complicated and somewhat reduced the cheating opportunities. Hence, it is suggested to use these individualized Excel-based exams in the future online exams to increase the exam’s scoring objectivity and reliability scoring. Keywords: excel-based exam, automatic scoring, accounting exam, individualized exam, accounting INTRODUCTION The Covid-19 outbreak with its massive challenges for human life brings disruptions which have never been predicted before. About 186 countries are affected, and one way to prevent further damage is by closing workplaces, shopping malls, and even schools/universities. Hence, like it or not, academicians are forced to adapt with the new ways of teaching through online learning. Consequently, teachers and lecturers should adopt the online flipping learning which combines the synchronous and/or asynchronous methods. These are expected creating flexibility and enjoyable learning experiences for students. However, some academicians are not ready to move their classes from offline into online learning environment. They merely change their classes from offline to online meetings via Zoom or any other video conference devices and in fact, this is a backward in education where the teacher-centered mode is re-used. An accounting class is generally large. Hence, when it comes to exams, not only designing (Ashworth, Bannister, and Thorne, 1997; Doran et al., 2011) but also scoring the paper-based exams bring pain to the lecturers (Bertheussen, 2014). For this reason, it is suggested to use an innovative way, such as using computer-aided assessment to reduce the workloads (Evans, 2013; Gikandi, Morrow, and Davis, 2011; Marriott and Lau, 2008). Although an accounting exam can be conducted online, many prefer to have a traditional type of hands-on exam for the security and surveillance issues (Rogers, 2006; Sindre and Vegendla, 2015) to minimize the students’ cheating activities during the exam. It is admitted by the 14 A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 students that cheating in an online exam is much easier (King, Guyette, and Piotrowski, 2009) unless the exam is well designed (Harmon, Lambrinos, and Buffolino, 2010). During the pandemic, exams must be given online since conducting exams in offline classes are strictly prohibited by the law. In this online environment, lecturers must be highly creative in designing an individualized exam (Blayney and Freeman 2008; Nnadi, 2003) that no rooms for students to collude (Nnadi and Rosser, 2014). This paper aims at explaining an innovative approach possibly developed by the accounting lecturers to create the individualized excel-based exams to maintain the online exam quality and minimize the academic integrity breaches. In the following sections, previous studies on online learning are presented, and exam designs are described. This also consists of problems, solutions, and scoring generating techniques. Next, students’ feedbacks collected from surveys are presented. Conclusion and suggestions for future research are provided in the last section. LITERATURE REVIEW Online learning is actually not a new concept, as massive open online courses have been introduced a decade ago, allowing distance education. A good online learning should support the learner-centered approach to enable the students to interact with the others; and digest information as active learners who can logically adapt the knowledge transfer (Bento and Schuster, 2003). This online learning is more flexible than the traditional one (Herliandry et al., 2020), where teaching and learning activities may be performed anytime and anywhere (Dewi, 2020) as less face-to-face interactions between students and facilitators (Sudiana, Fatah, and Khaerunnisa, 2017). Some argue than online learning increases students’ participations in the class (Saifuddin, 2018) because they are more confident and comfortable in asking or responding questions (Firman and Rahman, 2020). Furthermore, students become more independent in solving problems because they are asked to be more active in searching information related to the course (Firman and Rahman, 2020; Herliandry et al., 2020). Although many support the online learning, some others criticize that online learning needs longer preparation time for the teaching and learning activities, considerably costly (Herliandry et al., 2020), and may result in anxiety due to slow feedbacks given to the students (Pangondian, Paulus, and Nugroho, 2019). Online learning also provides challenges for both students and teachers, one of which is due to the online learning infrastructure (Firman and Rahman, 2020). This online learning requires good internet access and gadgets (Bustomi, 2020), which are not always available (Dewi, 2020; Khasanah, Pramudibyanto, and Widuroyekti, 2020). Apart from the infrastructure, teachers/lecturers are also shocked as they have to master information and technology skills as online learning involves video conference and other learning media which force the teachers/lecturers to be more creative and adaptive in delivering the course materials (Bustomi, 2020; Djaja, 2016; Herliandry et al., 2020; Sole and Anggraeni, 2018). With the minimum supports from schools or educational institutions, teachers and students are borne by the costs to access the online learning (Abidin, Rumansyah, and Arizona, 2020; Firman and Rahman, 2020; Herliandry et al., 2020). Moreover, students also complain about their difficulties in understanding the course materials and completing the homework due to the communication barriers between the students and the teachers (Bustomi, 2020; Firman and Rahman, 2020; Juliane et al., 2017). To minimize those challenges, teachers may adopt the project-based or problem-based assignments which require the students to search information related to the assigned projects (Abidin, Rumansyah, and Arizona, 2020; Chasanah, Khoiri, and Nuroso, 2016). Although the learning is conducted through online platforms, the assessments must be able to measure students’ performances. Assessments also serves as instruments to check the student’s achievements, planned learning outcomes, feedbacks as well as the instructors’ monitoring instruments (Born, 2003). Online exams can be set to maximize and fasten the teachers’ scoring objectivity (Aripin, Silalahi, and Ulfa, 2020). It is expected that online exams possibly minimize the students’ cheating activities. However, without proper planning, those will encourage the students to do more cheating 15 A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 activities by creating notes, using gadgets to find the answers, or communicate with the others (Abdullahi and Mansor, 2015; Murdiansyah, Sudarman, and Nurkholis, 2017). The next section discusses the online exam designs to prevent from the students’ cheating activities by providing the individualized excel-based exams. METHODS In the final exam of the Intermediate Accounting course, there were seven questions consisting of four conceptual and three problem-based questions to answer. The conceptual questions were set the same for all students. However, each problem was designed individually for each student, both related to the accounting method and/or policy choices as well as the numbers involved in the problems. Hence, an excel spreadsheet was used to automatically generate methods and numbers based on the students’ identification number. Excel is an essential feature in accounting education (Beaman, Waldmann, and Krueger, 2005; Chandler and Marriott, 1994) that the accounting lecturers should have sufficient excel skills, especially in designing excel-based exams. In this higher education institution where the exams were conducted, a student’s ID has a 12-digit number, with some differences in the sixth and last four digits. Hence, the starting point to design an individualized exam was using those numbers with some excel functions to generate numbers. From those 12 digits, only five unique digits of each student were utilized instead of the last digit usually used to create a distinctive figure for each student. Students were first asked to fill 12 digits of their student’s IDs in the 12 cells. This ID was used to produce numbers and select the accounting method. To avoid incorrect input of two or more digits in a cell, data validation was set to constrain the number of only from 0 to 91. The cells were also shaded with a bright green color to attract the students’ attention. Instructions were clearly provided for the students to input 12 digits of their student’s IDs in the bright green-shaded cells, while the notification of numbers and methods for each question will be different based on each student’s ID (see Figure 1). Figure 1. Instructions to complete the cells 1 To do this, click on Excel Ribbon: Data – data validation - whole number – minimum 0 – maximum 9. 16 A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 Students were then classified into the odd or even group. This classification was to decide the accounting method used in solving the problems. Hence, the Excel’s MOD function was used with the last digit of student’s IDs as the number and 2 as the divisor2. There were two outcomes: 1 and 0, with 1 represented the odd group (Group A), and 0 represented the even group (Group B). This formula was then combined with the IF function to provide notifications for those students whether belonging to Group A or B (see Figure 1). This group needs to specify the students’ answer sheets. For example, students were asked to make journal entries either for the lessee (Group A) or lessor (Group B, see Figure 2). IF function was used to assign which method should be conducted by the students. Figure 2. Sample questions and formula Prior to assigning numbers, a base number was set and then the IF function was used to assign the unique numbers to each problem provided for the exam (see Figure 2). As previously explained, only five numbers are unique to each student (6th, 9th, 10th, 11th and 12th), and then the combination of 6th and 9th with 10th to 12th were used to set figures in the questions. For instance, continue the lease problem. The first value was set for the leased equipment’s book value. It was set as a combination of question codes and base numbers, added with the multiplication of student’s ID. The second value was set for the equipment’s fair value of IDR 200,000 higher than the equipment’s book value. Next, the cost of goods sold was set 80% of the book value. In this type of problem, the lecturer needs to be careful in determining the lease terms and estimated useful life as the results are in different leasing types; capital lease or operating lease. The next step, the lease terms (in cell O12) were then set by using IF function. The last digit of student’s ID (in cell L6) was used to create a different length of lease as follows: =IF(L6=1,3,IF(L6=2,4,IF(L6=3,5,IF(L6=4,5,IF(L6=5,5,IF(L6=6,8,IF(L6=7,9,IF(L6=8,10,IF(L6=9,1 1,12))))))))) ……….. (in cell O12) After determining the lease terms, the estimated useful life of asset was determined from cell O12: =IF(O12=3,9,IF(O12=4,10,IF(O12=5,8,IF(O12=6,7,IF(O12=7,8,IF(O12=8,8,IF(O12=9,11,IF(O12= 10,12,IF(O12=11,12,12))))))))) ……….. (in cell O13) 2 At the target cells type =MOD(L6,2) with L6 is the cell containing the last digit student’s ID. 17 A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 In determining the incremental borrowing rate, the second last digit used was: =IF(J6=1,7%,IF(J6=2,6%,IF(J6=3,5%,IF(J6=4,8%,IF(J6=5,9%,IF(J6=6,10%,IF(J6=7,11%,IF(J6=8, 4%,IF(J6=9,12%,13%))))))))) ……….. (in cell O14) Once the lease terms, life of equipment, and borrowing rate were set, the estimated unguaranteed residual value could be calculated by using the following formula: Residual value = {depreciation rate x (useful life – lease term)} + base number = {(book value / useful life) x (useful life – lease term)} + base number (1) In the excel sheet, the formula was: = ((O7/O13)*(O13-O12))+125000 ……….... (in cell O9) The last number was the annual rental payment. It was calculated with the following formula: Annual rental payment = (fair value – PV residual value) / lease term (2) In the excel sheet, the present value (PV) might be easily calculated that the annual rental payment (cell O11) became = (O8+PV(O14,O12,0,O9,1))/O123. Lastly, the sheet was protected, and the formulas were hidden before distributing the exam4. In addition to the creation of exam’s questions, the solutions to the problems were also designed in different Excel sheet. This scoring guide was purposively designed to make the exam scoring operate faster. The numbers in problems were connected with the solutions while the point for each correct answer was defined. Once student’s ID was input to the solution sheet, the correct answer changed. This method enables the scoring to operate faster, although the students submitted the answers in the form of photo. Exam Delivery The exams were conducted in three classes at the same time and at the same date. The individual exam question was available sequentially in the e-learning (Moodle-based) platform at one specific time. Students could only see the exam questions at the set time. As many students were from rural areas where internet connection is somehow inaccessible and/or limited, the individual exam question is also sequentially sent via WhatsApp Group at the same time with the question appeared through e-learning. WhatsApp has been used as a means of communication in medical schools (Dar et al., 2017; Willemse, 2015), engineering (Nitza and Roman, 2016), and education (Awada, 2016; Hamad, 2017; Mwakapina, Mhandeni, and Nyinondi, 2016; Susilo, 2014). This platform allows the students to have online discussions, while the teachers found it easier to deliver the online tutorials, learning materials, and assignments, including instructions (So, 2016; Susilo, 2014). Hence, it is not surprising that WhatsApp, including WhatsApp group, is very popular in Indonesia, although sometimes the participants send spams and hoaxes (Bouhnik and Deshen, 2014; Cetinkaya, 2017) while teachers should be prepared to spend time outside their normal working hours (Bouhnik and Deshen, 2014). Each question had different points; a more complex problem had a higher score and took more time to finish. Hence, the time allocation for each question was set differently. Students were given a limited time to answer each problem. This information was sent to the students a week before the exam to have better preparation. Instructions of how to answer the exam questions and how to submit their answers to be marked was sent before the exam date. Since the students’ typing speed could not be equalized, the students’ laptops or PCs had different technical specifications, and based on the agreement with the students; the answers were paper based; one sheet for one question. 3 The formula to calculate this present value is PV (interest rate, lease term period, annual payment of 0, guaranteed residual value, and 1 for beginning of the year). 4 Select all worksheets except for the green cells for the student’s ID, right click, choose protection tab and then select locked and hidden. Next, click Tools in the ribbon, protection, and protect sheet. Fill in the passwords to lock the worksheet. 18 A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 Once the students had completed one problem, they were asked to take a picture of each answer, scanned it, upload it either to the e-learning platform, or send it to the lecturers’ WhatsApp numbers. Students were given five minutes after the exam ended to upload their answer sheets. After this upload time ended, the next question was then opened for the students. These online exams were set to be invigilated. Hence, the students were asked to join the online classrooms via Webex5 with mode-on video that the lecturers were able to see what the students did during the exam to prevent from the students’ cheating activities. As previously explained, this excel-based exam is already individualized with each student receiving different methods and numbers. Hence, the online surveillance plus individualized-type questions are expected to reduce the cheating opportunities as the answers of each student are different from the others. The students’ perceptions and feedbacks regarding to this online, individualized excel-based exam are further discussed in the next section. RESULTS AND DISCUSSION To measure the effectiveness, Qualtrics online survey was conducted to the students participating in this online individualized excel-based final exam after the final scores were announced (n=80). Each questionnaire consisted of 16 questions. Students were questioned related to their genders, final exam scores, 14 questions on the easiness use of this online individualized excel-based final test, and their reflections (Alsadoon, 2017; Deutsch et al., 2012; Hillier, 2015; Özden, Ertürk, and Sanli, 2004), measured with the 5-point Likert scale. The survey was opened for a week, and 76 responses were collected. The students were further classified into high achievers (if the exam mark is more than or equal to 85) and nonhigh achievers (for those less than 85). For data screening, we checked the univariate outliers from the standardized value for each Likert scale item. We found three outlier cases, and these were then removed from the data for further analysis (Hair et al., 2014), resulting in a 91% response rate. After removing the outliers, a normality test was conducted using skewness and kurtosis, and showed that all the data was normally distributed. A principle component analysis (PCA) was then conducted on those 14 items with a direct oblimin rotation to classify the questionnaire into several factors and check the construct and discriminant validity. The Kaiser-Meyer-Olkin measure verified the sampling adequacy for the analysis, with KMO = 0.753, and all KMO values for individual items were more than the minimum threshold of 0.5 (Field, 2013). Bartlett’s test of sphericity with c2 (91) = 288.803 and p < 0.001, indicated that the correlations between items were sufficiently large for PCA. An initial analysis was conducted to obtain the eigenvalues for each data component. Five data components had the eigenvalues of more than one and in combination, explained by 69.56% of the variance. Table 1 shows the after-rotation loading factor. The items clustered in the same components suggested that component 1, 2, 3, 4 and 5 respectively represented the easiness, timeliness, self-efficacy, challenge, and integrity concern of the online exam. These factor scores were then used for further analysis. Table 1. Factor loading for each item Items Applicable to other courses Preferring the online to offline exam The system is easy to use Feeling Relax if future exam is conducted online Recommending online for future exams Online exams objectively reflect the students’ performance 5 Easiness .855 .820 .816 .767 .744 .518 Webex is an online video conferencing platform. 19 Time lines Selfefficacy Challenge Integrity A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 Not feeling panic when doing the exams Requiring less time finishing the online exams believing of having enough competence Not ashamed after finishing the exams Feeling Proud after finishing the exams Online exams are enjoyable challenges Reading on screen adding no difficulties during the exams Online exams reduce cheating opportunities Eigenvalues % of variance a .847 .696 .693 .649 .607 .887 .819 4.258 30.411 .869 1.654 11.814 .568 1.479 10.567 .562 1.321 9.437 .661 .995 1.026 7.331 N/A Based on those classifications, the descriptive statistical data were shown in Table 2. Although all mean scores were greater than 3, out of 5, the students relatively agreed that the online exam was technically easy to follow and appropriate to use for the future exams in other courses. In addition, students also felt that in the online exam environment, they believed that they had a higher competence and less panic. Hence, they felt that there was no feasible challenge when reading the questions on the screen instead of on paper. These positive experiences made the students preferred online to offline exam and agreed if the future exams will be conducted via online that they will feel relax. Students also considered that it was harder for them to do cheating activities during the personalized type exams were conducted. Hence, they believed that an online exam objectively reflected their performance. Table 2. Descriptive statistics Items Easiness Applicable for other courses Preferring online to offline exams The system is easy to use Feeling Relax if future exam is conducted online Recommending online for future exams Online exams objectively reflect the students’ performance Timeliness Not panic when doing the exams Need less time finishing the online exams Self-efficacy believing of having enough competence Not ashamed when finishing the exams Proud after finishing the exams Challenge Online exams are enjoyable challenges Reading on screen add no difficulties during the exams Integrity Online exams reduce cheating opportunities Min Max Mean Stdev 2 1 2 2 2 2 5 5 5 5 5 5 3.66 3.39 3.71 3.46 3.46 3.24 0.866 1.054 0.769 0.846 0.846 0.875 2 1 5 5 3.77 3.32 0.837 0.883 2 2 2 5 5 5 3.83 3.65 3.34 0.747 0.819 0.899 1 2 5 5 3.30 3.47 0.922 0.863 1 5 3.35 0.952 Independent sample t-tests were then conducted to check the differences between gender and high achiever students based on five factors. On average, female students expressed the decreasing cheating opportunities (M=0.159, SE=0.963) more than male students (M=-0.505, SE=0.975). This difference6 was significant with t(65) = -25.399 and p < 0.05; representing a medium-sized effect with r = 0.29. This finding implied that male students perceived that cheating opportunities during the online exams were higher than the female students did. This was probably caused by the female students’ learning efforts were 6 For robustness check, a non-parametric test was conducted and showed similar findings (Mann Whitney U = 246; p = 0.017). There was no significant different between the high and non-high achiever students. 20 A. W. Suryani/Journal of Accounting and Business Education, 5 (1), September 2020 bigger than male students’ (Dodeen, 2012), since male students perceived that cheating was more morally acceptable (Smyth and Davis, 2003). Although academic cheating activities happened more in offline classes than online environments (Watson, George, and Sottile, 2010), educators should take extra precautions when designing online tests, such as by designing subjective exams (essay questions) than objective measures (multiple choice questions). CONCLUSION This study aims at explaining the online exam methods in accounting subjects during the outbreak of Covid-19 pandemic. The exams are uniquely designed for each student to reduce cheating opportunities and relatively ease the scoring. This automatic scoring may improve the scoring process credibility scoring and minimize the calculation errors. From the students’ feedbacks, it is shown that online exam is not considered as a burden and believed reducing the cheating opportunities during the exam. This personalized exam may be further developed in class practices where the students may directly answer the questions presented in the excel sheets with auto scoring feature. 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https://openalex.org/W4288716732	https://hal.inrae.fr/hal-03794351/file/2022_Portier_microorganisms-10-01531-v2.pdf	English	null	Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species	Microorganisms	2,022	cc-by	8,258	Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species Perrine Portier, Géraldine Taghouti, Paul-Emile Bertrand, Martial Briand, Cécile Dutrieux, Audrey Lathus, Marion Fischer-Le Saux To cite this version: Perrine Portier, Géraldine Taghouti, Paul-Emile Bertrand, Martial Briand, Cécile Dutrieux, et al.. Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species. Microorganisms, 2022, 10 (8), pp.1531. 10.3390/microorganisms10081531. hal-03794351 To cite this version: Perrine Portier, Géraldine Taghouti, Paul-Emile Bertrand, Martial Briand, Cécile Dutrieux, et al.. Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species. Microorganisms, 2022, 10 (8), pp.1531. 10.3390/microorganisms10081531. hal-03794351 To cite this version: Perrine Portier, Géraldine Taghouti, Paul-Emile Bertrand, Martial Briand, Cécile Dutrieux, et al.. Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species. Microorganisms, 2022, 10 (8), pp.1531. 10.3390/microorganisms10081531. hal-03794351 Distributed under a Creative Commons Attribution 4.0 International License Keywords: Xylophilus; diversity; biological resources center; multi locus sequence analysis Citation: Portier, P.; Taghouti, G.; Bertrand, P.-E.; Briand, M.; Dutrieux, C.; Lathus, A.; Fischer-Le Saux, M. Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species. Microorganisms 2022, 10, 1531. https://doi.org/ 10.3390/microorganisms10081531 Academic Editor: Dawn L. Arnold Received: 14 June 2022 Accepted: 27 July 2022 Published: 28 July 2022 Citation: Portier, P.; Taghouti, G.; Bertrand, P.-E.; Briand, M.; Dutrieux, C.; Lathus, A.; Fischer-Le Saux, M. Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species. Microorganisms 2022, 10, 1531. https://doi.org/ 10.3390/microorganisms10081531 Academic Editor: Dawn L. Arnold Received: 14 June 2022 Accepted: 27 July 2022 Published: 28 July 2022 Perrine Portier * , Géraldine Taghouti, Paul-Emile Bertrand, Martial Briand, Cécile Dutrieux, Audrey Lathus and Marion Fischer-Le Saux Institut Agro, University Angers, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, F-49000 Angers, France; geraldine.taghouti@inrae.fr (G.T.); bertrand.paul.emile@gmail.com (P.-E.B.); martial.briand@inrae.fr (M.B.); cecile.dutrieux@inrae.fr (C.D.); audrey.lathus@inrae.fr (A.L.); marion.le-saux@inrae.fr (M.F.-L.S.) * Correspondence: perrine.portier@inrae.fr Abstract: Xylophilus ampelinus is the causal agent of blight and canker on grapevine. Only a few data are available on this species implying that the occurrence of this pathogen may be underestimated, and its actual ecological niche may not be understood. Moreover, its genetic diversity is not well known. To improve our knowledge of this species, an analysis of the complete genome sequences available in NCBI was performed. It appeared that several sequences are misidentiﬁed. The complete genome sequence of the type strain was obtained and primers designed in order to sequence gyrB and rpoD genes for the strains held in CIRM-CFBP. The genetic barcoding data were obtained for 93 strains, isolated over 35 years and from several geographical origins. The species revealed to be strongly homogenous, displaying nearly identical sequences for all strains. However, the oldest strains of this collection were isolated in 2001 therefore, a new isolation campaign and epidemiological surveys are necessary, along with the obtention of new complete genome sequences for this species. HAL Id: hal-03794351 https://hal.inrae.fr/hal-03794351v1 Submitted on 3 Oct 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License microorganisms Article Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species , Géraldine Taghouti, Paul-Emile Bertrand, Martial Briand, Cécile Dutrieux, Audrey Lathus her-Le Saux 1. Introduction https://doi.org/10.3390/microorganisms10081531 2 of 11 Microorganisms 2022, 10, 1531 This bacterium is distributed in several grapevine-growing areas, such as the Mediter- ranean basin (France, Greece Italy, Jordan, Moldova, Slovenia), South Africa, Russia and Japan. Reports of symptoms close to the diseases described as caused by X. ampelinus have been made from Argentina, Portugal, Switzerland, Tunisia, Turkey and former Yu- goslavia, but the presence of the bacterium had not been conﬁrmed (except for Slovenia). Formerly present in Spain, the disease is reported as no longer found since the 2010s. As the occurrence of the disease over the years can be erratic, the symptoms can be confused with other diseases and because of the absence of systematic surveys in many areas, there is uncertainty about its geographical distribution. X. ampelinus may be present in more grapevine-growing countries than is currently known [15]. Moreover, the distribution of the bacterium inside the plant can be heterogenous, adding to the difficulties for its detection [17]. p g g It may be possible that the actual ecological niche of the bacterium is not completely known. During the analysis of the American Gut Project, Perz et al. [18] remarked that the microbiota associated to autistic patients are enriched in Xylophilus ampelinus. In the MetaMetaDB [19], hits corresponding to Xylophilus ampelinus 16S (97% identity) appear in a variety of ecosystems (beetle: 22.46%, soil: 17.67%, rhizosphere: 13.01%, marine: 8.34%, freshwater: 6.98%, root: 6.88%, human lung: 5.79%, ant fungus garden: 5.72%, human skin: 5.08%, hydrocarbon: 4.53% bovine gut: 3.52%). The bacterium has also been recently isolated from the microbiota of blueberry [20], indicating that its actual occurrence in the environment is probably underestimated. p y Only little information is available through public databases; hence, the genetic di- versity of this bacterium is poorly known. In this regard, Komatsu et al. [21] established, using Eric-, Box- and Rep-PCR, that the population of the bacterium is homogenous even if they were able to discriminate three genetic types. In GenBank, only thirteen genomic data are available for Xylophilus. The complete genome sequence is available for three strains labeled as X. ampelinus (including the type strain CECT 7646T), two others are available for isolates labeled as Xylophilus sp. along with the type strain of ‘X. rhododendri’ (KACC 21265). Seven other sequences, corresponding to uncultured organisms retrieved from metagenomes, are labeled as Xylophilus sp. (https://www.ncbi.nlm.nih.gov/datasets/ genomes/?taxon=54066, (accessed on 26 July 2022)). 1. Introduction Xylophilus ampelinus is a Gram-negative betaproteobacterium [1,2] which causes blight and canker on grapevine (Vitis vinifera), its only known host. The disease was described in Greece in 1939 but its causal agent was only identiﬁed as the slow growing bacteria Xanthomonas ampelina in 1969 [3]. This bacterium was also shown to be responsible of different grapevine diseases such as ‘mal nero della vite’ in Italy [4], ‘maladie d’Oléron’ in France [5], ‘vlamsiekte’ in South Africa [6] and ‘necrosis bacteriana’ in Spain [7]. Severity of the disease appears to be dependent on cultivar and strain [8] and can lead to serious harvest losses [9,10]. A DNA and RNA study revealed that this bacterium is not related to Xanthomonas and was thus transferred in the Xylophilus genus as X. ampelinus [11]. This genus is, to date, composed of only two species X. ampelinus and “X. rhododendri”; the latter is not yet validated [12]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. In Europe, X. ampelinus was classiﬁed as a quarantine organism until 2019 (date of the revision of the list of quarantine organisms), but is still present on the A2 list of organ- isms established by the European Plant Protection Organization (https://www.eppo.int/, (accessed on 26 July 2022)), indicating that it is still considered as a potential threat for the European and Mediterranean agriculture. The control of the disease can be obtained by using preventive measures such as disinfection of pruning tools, detection and identiﬁca- tion of the bacterium to ensure the use of pathogen-free propagative and planting material. Hot water treatment of canes, at 52 ◦C for 45 min, was shown to eliminate X. ampelinus efﬁciently in grapevine cuttings, along with being efﬁcient toward other pathogens [13–15]. More recently, some extracts of the plant Limonium binervosum (G.E.Sm.) C.E.Salmon (rock sea-lavender), have shown some activity against X. ampelinus, and this could lead to new control strategies [16]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/microorganisms Microorganisms 2022, 10, 1531. 2.1. Bacterial Strains The 101 strains belonging to Xylophilus ampelinus held in CIRM-CFBP were isolated all from grapevine plants, over a period of 35 years in Greece, France, Spain and South Africa. The most recent strains present in the collection were isolated in 2001. The strains are preserved as freeze-dried or in sterile water with 40% glycerol at −80 ◦C or in liquid nitrogen at −196◦C. For routine cultivation, the strains are plated on YPGA (yeast extract 7 g.L−1; bacto peptone 7 g.L−1; glucose 7 g.L−1; agar 15 g.L−1) for 4 days at 25 ◦C. The type strain of Acidovorax anthurii CFBP 3232T was added in this study as an outgroup. Both species X. ampelinus and A. anthurii belong to the Comamonadaceae family. All strains information is listed in the Supplementary Materials, Table S1. All strains listed in Table S1 are preserved at CIRM-CFBP (https://cirm-cfbp.fr, (accessed on 26 July 2022)) and are available upon request for the international scientiﬁc community. 2.2. Genome Sequencing The complete genome sequence of the type strain CFBP 1192T was obtained as described by Merda et al. [25], using the Illumina technology and HiSeq 2000 (Genoscreen, Lille, France). Libraries of genomic DNA were performed using the Kit NextEra 141 XT (Illumina, San Diego, CA, USA). Paired-end reads of 2 × 100 bp were assembled in contigs using SOAPDENOVO 1.05 [26] and VELVET 1.2.02 [27]. Annotation was performed using Prokka [28]. 1. Introduction g y The French Collection for Plant-associated Bacteria (CIRM-CFBP; https://cirm-cfbp.fr, (accessed on 26 July 2022)) preserves bacterial resources strategic for plant health, mainly plant-pathogens. These resources serve as a tool available for worldwide researchers, to improve crop health and to better understand plant–bacteria interactions. CIRM-CFBP holds 101 strains of Xylophilus ampelinus, isolated from various locations over a long time period. In order to enhance the quality of the strains held in CIRM-CFBP, we decided to obtain the partial sequence of two housekeeping genes for all accessions of the collection. This technique allows to accurately identify the strains at the species level. Moreover, the data can also be used to build the phylogeny of the strains and to better understand the diversity of the considered taxa. This technique was successfully applied in different genera and the protocols (and associated references) used at CIRM-CFBP are available via the collection’s website (https://cirm-cfbp.fr/page/molecular_identiﬁcation, (accessed on 26 July 2022)). In order to apply this technique to Xylophilus ampelinus strains, we sequenced the complete genome of the type strain CFBP 1192T and designed primers for gyrB and rpoD genes. These two genes were chosen because they are used for the molecular identiﬁcation of Xanthomonas [22] and Pseudomonas [23,24] and revealed to be efﬁcient for species identiﬁcation and diversity analysis of these two genera. The sequences of these two genes were obtained for all the strains held in the collection. In order to complete our study of the diversity of this genus, we also analyzed the different whole genome sequences available in GenBank labeled as Xylophilus. 3 of 11 Microorganisms 2022, 10, 1531 2.3. Comparative Genomics The thirteen genomes labeled as Xylophilus available in GenBank on 1 June 2022 were retrieved. Six of these genomes correspond to isolates, the other seven sequences correspond to MAGs (Metagenome Assembled Genomes) with no associated cultured isolate. These genome features are listed in Table 1. All genomes were checked for quality using ChekM [29]. Table 1. Genomes available in GenBank on 1 June 2022 labeled as Xylophilus. Isolate/Genome Taxonomy Isolate/MAG Biotope Biosample Bioproject Assembly Total Length (bp) Assembly Level CECT 7646T Xylophilus ampelinus Isolate Plant, Vitis vinifera SAMN09074800 PRJNA463320 GCA_003217575.1 3731505 Scaffold CCH5-B3 Xylophilus ampelinus Isolate Bioﬁlm, hospital ward SAMN04299458 PRJNA299404 GCA_001556675.1 6019991 Contig BgEED09 Xylophilus ampelinus Isolate Human duodenum SAMEA5664384 PRJEB32184 GCA_901875635.1 6174221 Contig KACC 21265 Xylophilus rhododendri Isolate Plant, Rhododendron schlippenbachii SAMN13783577 PRJNA600143 GCA_009906855.1 5873400 Complete Genome ASV27 Xylophilus sp. Isolate Plant, Sarracenia purpurea SAMN17004937 PRJNA224116 GCA_016428875.1 4734944 Contig leaf220 Xylophilus sp. Isolate Plant, Arabidopsis thaliana SAMN04151686 PRJNA297956 GCA_001421705.1 4483623 Scaffold Gw_UH_bin_252 Xylophilus sp. MAG Wastewater treatment SAMN18119505 PRJNA524094 GCA_017989255.1 1400660 Scaffold Go_Prim_bin_55 Xylophilus sp. MAG Wastewater treatment SAMN18119707 PRJNA524094 GCA_017990095.1 2320559 Scaffold Gw_Prim_bin_50 Xylophilus sp. MAG Wastewater treatment SAMN18119294 PRJNA524094 GCA_018005875.1 1282324 Scaffold Gw_Inlet_bin_57 Xylophilus sp. MAG Wastewater treatment SAMN18119261 PRJNA524094 GCA_018006615.1 1897017 Scaffold SP210_2 Xylophilus sp. MAG Plant, rice SAMEA8944525 PRJEB45634 GCA_913776965.1 4051675 Contig SP51_3 Xylophilus sp. MAG Plant, rice SAMEA8944104 PRJEB45634 GCA_913777525.1 3038960 Contig cluster_DBSCAN _round5_1 Xylophilus sp. MAG Insect, Lagria villosa SAMN12995593 PRJNA531449 GCA_009914555.1 4706822 Contig Table 1. Genomes available in GenBank on 1 June 2022 labeled as Xylophilus. The genome sequence data retrieved from GenBank, along with the sequence of CFBP 1192T, were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinfor- matics platform (https://tygs.dsmz.de, (accessed on 26 July 2022)), for a whole genome- based taxonomic analysis [30,31]. The TYGS analysis permits accurate identiﬁcation, by determining the closest type strains present in the TYGS database, of the uploaded 4 of 11 Microorganisms 2022, 10, 1531 genomes. The Newick tree derived from this analysis was then edited using Mega 11 (https://www.megasoftware.net/, (accessed on 26 July2022)) [32]. genomes. The Newick tree derived from this analysis was then edited using Mega 11 (https://www.megasoftware.net/, (accessed on 26 July2022)) [32]. p g y The subsequent dDDH (digital DNA-DNA-hybridation) analysis was performed, still by the TYGS pipeline, between the uploaded genomes and a selection of the closest type strains’ genomes from the TYGS database. 2.3. Comparative Genomics g After TYGS analysis, ANIb calculation, using pyani [33] were performed with the 14 genomes along with the genome of the type strain of Xenophilus azovorans DSM 13620T, detected as closely related to the CCH5-B3 and BgEED09 genomes by TYGS analysis. 2.4. gyrB-rpoD Phylogeny Primers to amplify gyrB and rpoD genes were designed using the genome of the type strain CFBP 1192T (this study) and the sequence of strain CFBP 3232T (Acidovorax anthurii; GCA_003269065.1) using the online tool Primer Blast (https://www.ncbi.nlm.nih. gov/tools/primer-blast/, (accessed on 26 July 2022)), and software Ampliﬁx [34] (https: //inp.univ-amu.fr/en/amplifx-manage-test-and-design-your-primers-for-pcr, (accessed on 26 July 2022)) and Amplify4 (https://engels.genetics.wisc.edu/amplify/, (accessed on 26 July 2022)). For the 93 strains listed in Table S1, portions of the gyrB and rpoD genes were sequenced. PCR ampliﬁcation mix was as follows: Taq polymerase GoTaq (Promega) 5U, polymerase buffer 1X, MgCl2 1 mM, dNTP 100µM, boiled cells 10%. Primers and ampliﬁcation program are detailed in Table 2. Table 2. Primer sequences and PCR programs for partial gyrB and rpoD ampliﬁcation for diversity analysis of Xylophilus ampelinus strains. Table 2. Primer sequences and PCR programs for partial gyrB and rpoD ampliﬁcation for diversity analysis of Xylophilus ampelinus strains. Gene Primer Sequence 5’-3’ Expected Size (bp) Tm gyrB gyrB_XyF AGATGGACGACAAGCACGAG 841 60 gyrB_XyR TTGGTCTGGCTGCTGAACTT 60 30X 95 ◦C 95 ◦C 65 ◦C 72 ◦C 72 ◦C 15 ◦C 5′ 30′′ 30′′ 30′′ 5′ ∞ Gene Primer Sequence 5’-3’ Expected size (bp) Tm rpoD rpoD-XyF AAGGAACGCGCCTTGATGA 767 60 rpoD-XyR CCGTAGCCTTCCTTGTCGTAG 60 PCR Program 30X 95 ◦C 95 ◦C 58 ◦C 72 ◦C 72 ◦C 15 ◦C 5′ 30′′ 30′′ 30′′ 5′ ∞ Table 2. Primer sequences and PCR programs for partial gyrB and rpoD ampliﬁcation for diversity analysis of Xylophilus ampelinus strains. Gene Primer Sequence 5’-3’ Expected Size (bp) Tm gyrB gyrB_XyF AGATGGACGACAAGCACGAG 841 60 gyrB_XyR TTGGTCTGGCTGCTGAACTT 60 30X 95 ◦C 95 ◦C 65 ◦C 72 ◦C 72 ◦C 15 ◦C 5′ 30′′ 30′′ 30′′ 5′ ∞ Gene Primer Sequence 5’-3’ Expected size (bp) Tm rpoD rpoD-XyF AAGGAACGCGCCTTGATGA 767 60 rpoD-XyR CCGTAGCCTTCCTTGTCGTAG 60 PCR Program 30X 95 ◦C 95 ◦C 58 ◦C 72 ◦C 72 ◦C 15 ◦C 5′ 30′′ 30′′ 30′′ 5′ ∞ PCR products’ sequencing was performed by Genoscreen (Lille, France). The con- sensus sequences for each gene for each strain were extracted from forward and reverse sequence assemblies using Geneious Pro version 9.1.8 (www.geneious.com). The sequences were then aligned and trimmed using BioEdit version 5.0.6. A phylogenetic tree was con- structed with concatenated alignments of all genes with MEGA 7.0.26 using the neighbor- joining method with 1000 bootstrap replicates, and the evolutionary distances were com- puted by using the Kimura two-parameter method. 2.4. gyrB-rpoD Phylogeny Strains CECT 7646T and CFBP 1192T display ANIb and dDDH values at 100%, indicating that these two strains are equivalent (Table 4, Figure 1). The TYGS analysis (Figure 1) revealed that the genomes of the type strains of X. ampelinus (CECT 7646T, CFBP 1192T) and ‘X. rhododendri’ (KACC 21265T) correspond to their respective taxa. However, all the other genomes labeled as ‘Xylophilus’ do not correspond to taxa available in the TYGS database. The comparison of the dDDH and ANIb values conﬁrms these results. Strains CECT 7646T and CFBP 1192T display ANIb and dDDH values at 100%, indicating that these two strains are equivalent (Table 4, Figure 1). The genomes of strains CCH5-B3 and BgEED09 labeled as X. ampelinus, belong to a same species, but are in fact closer to Xenophilus strains. The comparison of these two genomes with the genomes of the type strain of Xenophilus azovorans added as reference (Table 4), showed that they probably belong to a not yet described species in this genus. On the other hand, strains ASV27 and leaf220 correspond to two undescribed species embedded inside the Xylophilus genus correspond to taxa available in the TYGS database. The comparison of the dDDH and ANIb values conﬁrms these results. Strains CECT 7646T and CFBP 1192T display ANIb and dDDH values at 100%, indicating that these two strains are equivalent (Table 4, Figure 1). The genomes of strains CCH5-B3 and BgEED09 labeled as X. ampelinus, belong to a same species, but are in fact closer to Xenophilus strains. The comparison of these two genomes with the genomes of the type strain of Xenophilus azovorans added as reference (Table 4), showed that they probably belong to a not yet described species in this genus. On the other hand, strains ASV27 and leaf220 correspond to two undescribed species g q g The genomes of strains CCH5-B3 and BgEED09 labeled as X. ampelinus, belong to a same species, but are in fact closer to Xenophilus strains. The comparison of these two genomes with the genomes of the type strain of Xenophilus azovorans added as reference (Table 4), showed that they probably belong to a not yet described species in this genus. ( ) y p y g y p g On the other hand, strains ASV27 and leaf220 correspond to two undescribed species embedded inside the Xylophilus genus. 2.4. gyrB-rpoD Phylogeny Even though the exact taxonomic position of these genomes may not be precise enough, it is sufﬁcient to conﬁrm that microbiotas can contain yet unknown members of Xylophilus, and that not all sequences assigned as Xylophilus are bona ﬁde Xylophilus. Finally, only two genome sequences belong to X. ampelinus, and both were obtained from the type strain (from two different collections). These data are far from enough to permit a comprehensive study of the diversity of this species. These results indicate two things. The ﬁrst is that the Xylophilus genus is far from well known, with unknown species detected in this genus, with unknown ecological niches and only a few data available. Secondly, that the taxonomic assignation of the publicly available sequences is not always accurate. Hence, this raises the question of the accuracy of the assignation of the sequences extracted from metagenomes and identiﬁed as Xylophilus. A more in-depth analysis is warranted to determine if they really correspond to Xylophilus or to other related genera. 3. Results and Discussion 3. Results and Discussion 3.1. Genome Comparison The genome features for strain CFBP 1192T and NCBI accession number are summa- rized in Table 3. rized in Table 3. Table 3. Features of the complete genome sequence of CFBP 1192T, type strain of Xylophilus ampelinus. Strain Size Scaffolds %GC N50 N50 BP Coverage CDS NCBI Accession CFBP 1192T 3,736,570 85 67.8 9 138.681 225 3307 JAMOFZ000000000 Table 3. Features of the complete genome sequence of CFBP 1192T, type strain of Xylophilus ampelinus. Table 3. Features of the complete genome sequence of CFBP 1192T, type strain of Xylophilus ampelinus. Strain Size Scaffolds %GC N50 N50 BP Coverage CDS NCBI Accession CFBP 1192T 3,736,570 85 67.8 9 138.681 225 3307 JAMOFZ000000000 The ChekM process revealed, unsurprisingly, that the genomes obtained from MAGs were of a lesser quality than the ones obtained from isolates (Table S3). However, all were uploaded for whole genome analysis by TYGS. The TYGS analysis (Figure 1) revealed that the genomes of the type strains of X. ampelinus (CECT 7646T, CFBP 1192T) and ‘X. rhododendri’ (KACC 21265T) correspond to their respective taxa. However, all the other genomes labeled as ‘Xylophilus’ do not correspond to taxa available in the TYGS database. The comparison of the dDDH and ANIb values conﬁrms these results. 2.4. gyrB-rpoD Phylogeny The sequences of gyrB and rpoD for type strains CFBP 1192T (X. ampelinus) and CFBP 3232T (A. anthurii) were retrieved from the complete genome sequences, the latter strain acting as an outgroup. The sequences were obtained for both genes for 93 strains. All the sequences used for the phylogenetic tree were deposited at NCBI, and the accession numbers are listed in Table S1. The sequence alignment is provided in Table S2. PCR products’ sequencing was performed by Genoscreen (Lille, France). The con- sensus sequences for each gene for each strain were extracted from forward and reverse sequence assemblies using Geneious Pro version 9.1.8 (www.geneious.com). The sequences were then aligned and trimmed using BioEdit version 5.0.6. A phylogenetic tree was con- structed with concatenated alignments of all genes with MEGA 7.0.26 using the neighbor- joining method with 1000 bootstrap replicates, and the evolutionary distances were com- puted by using the Kimura two-parameter method. The sequences of gyrB and rpoD for type strains CFBP 1192T (X. ampelinus) and CFBP 3232T (A. anthurii) were retrieved from the complete genome sequences, the latter strain acting as an outgroup. The sequences were obtained for both genes for 93 strains. All the sequences used for the phylogenetic tree were deposited at NCBI, and the accession numbers are listed in Table S1. The sequence alignment is provided in Table S2. PCR products’ sequencing was performed by Genoscreen (Lille, France). The con- sensus sequences for each gene for each strain were extracted from forward and reverse sequence assemblies using Geneious Pro version 9.1.8 (www.geneious.com). The sequences were then aligned and trimmed using BioEdit version 5.0.6. A phylogenetic tree was con- structed with concatenated alignments of all genes with MEGA 7.0.26 using the neighbor- joining method with 1000 bootstrap replicates, and the evolutionary distances were com- puted by using the Kimura two-parameter method. The sequences of gyrB and rpoD for type strains CFBP 1192T (X. ampelinus) and CFBP 3232T (A. anthurii) were retrieved from the complete genome sequences, the latter strain acting as an outgroup. The sequences were obtained for both genes for 93 strains. All the sequences used for the phylogenetic tree were deposited at NCBI, and the accession numbers are listed in Table S1. The sequence alignment is provided in Table S2. 5 of 11 Microorganisms 2022, 10, 1531 3. Results and Discussion 3.1. 2.4. gyrB-rpoD Phylogeny Genome Comparison The genome features for strain CFBP 1192T and NCBI accession number are summa- rized in Table 3. Table 3. Features of the complete genome sequence of CFBP 1192T, type strain of Xylophilus ampelinus. Strain Size Scaffolds %GC N50 N50 BP Coverage CDS NCBI Accession CFBP 1192T 3,736,570 85 67.8 9 138.681 225 3307 JAMOFZ000000000 The ChekM process revealed, unsurprisingly, that the genomes obtained from MAGs were of a lesser quality than the ones obtained from isolates (Table S3). However, all were uploaded for whole genome analysis by TYGS. The TYGS analysis (Figure 1) revealed that the genomes of the type strains of X. ampelinus (CECT 7646T, CFBP 1192T) and ‘X. rhododendri’ (KACC 21265T) correspond to their respective taxa. However, all the other genomes labeled as ‘Xylophilus’ do not correspond to taxa available in the TYGS database. The comparison of the dDDH and ANIb values conﬁrms these results. Strains CECT 7646T and CFBP 1192T display ANIb and dDDH values at 100%, indicating that these two strains are equivalent (Table 4, Figure 1). The genomes of strains CCH5-B3 and BgEED09 labeled as X. ampelinus, belong to a same species, but are in fact closer to Xenophilus strains. The comparison of these two genomes with the genomes of the type strain of Xenophilus azovorans added as reference (Table 4), showed that they probably belong to a not yet described species in this genus. On the other hand, strains ASV27 and leaf220 correspond to two undescribed species embedded inside the Xylophilus genus. The two MAGs SP210_2 and SP51_3 are closely related, belonging to a same species, well embedded in the Xylophilus genus, probably corresponding to a not yet described Xylophilus species. However, as these genomes were retrieved from MAGs, this assig- nation may not be accurate enough. The situation is equivalent for the genome clus- ter_DBSCAN_round5_1 which corresponds to another not yet described species located at the limit of the Xylophilus genus. Here also, the limited quality of the genome does not permit to ensure its precise taxonomic position. Finally, the cluster of MAGs retrieved from rice microbiota all belong to a not yet described species close to Macromonas bipunctata, but with the same reserves considering the quality of the genomic sequences. 2.4. gyrB-rpoD Phylogeny The two MAGs SP210_2 and SP51_3 are closely related, belonging to a same species, well embedded in the Xylophilus genus, probably corresponding to a not yet described Xylophilus species. However, as these genomes were retrieved from MAGs, this assig- nation may not be accurate enough. The situation is equivalent for the genome clus- ter_DBSCAN_round5_1 which corresponds to another not yet described species located at the limit of the Xylophilus genus. Here also, the limited quality of the genome does not permit to ensure its precise taxonomic position. Finally, the cluster of MAGs retrieved from rice microbiota all belong to a not yet described species close to Macromonas bipunctata, but with the same reserves considering the quality of the genomic sequences. Even though the exact taxonomic position of these genomes may not be precise enough, it is sufﬁcient to conﬁrm that microbiotas can contain yet unknown members of Xylophilus, and that not all sequences assigned as Xylophilus are bona ﬁde Xylophilus. q g Finally, only two genome sequences belong to X. ampelinus, and both were obtained from the type strain (from two different collections). These data are far from enough to permit a comprehensive study of the diversity of this species. These results indicate two things. The ﬁrst is that the Xylophilus genus is far from well known, with unknown species detected in this genus, with unknown ecological niches and only a few data available. Secondly, that the taxonomic assignation of the publicly available sequences is not always accurate. Hence, this raises the question of the accuracy of the assignation of the sequences extracted from metagenomes and identiﬁed as Xylophilus. A more in-depth analysis is warranted to determine if they really correspond to Xylophilus or to other related genera. 6 of 11 Microorganisms 2022, 10, 1531 Figure 1. Phylogenetic tree provided after TYGS analysis [30]. Tree inferred with FastME 2.1.6.1 [36] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers on branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 81.2%. The tree was rooted at the midpoint [37]. The Newick ﬁle was edited in MEGA11 [32]. The 14 blue dots correspond to the uploaded genomes. Figure 1. Phylogenetic tree provided after TYGS analysis [30]. Tree inferred with FastME 2.1.6.1 [36] from GBDP distances calculated from genome sequences. 2.4. gyrB-rpoD Phylogeny The branch lengths are scaled in terms of GBDP distance formula d5. The numbers on branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 81.2%. The tree was rooted at the midpoint [37]. The Newick ﬁle was edited in MEGA11 [32]. The 14 blue dots correspond to the uploaded genomes. Microorganisms 2022, 10, 1531 7 of 11 7 of 11 Table 4. ANIb (above diagonal) and dDDH (below diagonal) values, calculated respectively with pyani [33] and TYSG, formula d4 [35]. Highlighted in green, the values above the 95% (for ANIb) or 70% (for dDDH) thresholds for bacterial species delineation. The numbers featured on top, correspond to the genome number on the left. CFBP 1192T and CECT 7646T are both equivalent of the same type strain of the species held in two different collections. Genome Name Taxonomy (in Genbank) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1. CECT7646 X. ampelinus (Type strain) 1.00 1.00 0.93 0.82 0.81 0.81 0.80 0.79 0.78 0.78 0.78 0.77 0.77 0.76 0.76 2. Xya-CFBP1192 X. ampelinus (Type strain) 1.00 1.00 0.93 0.82 0.81 0.81 0.80 0.79 0.78 0.78 0.78 0.77 0.77 0.77 0.77 3. Leaf220 Xylophilus sp. 0.48 0.48 1.00 0.25 0.81 0.81 0.23 0.78 0.21 0.22 0.22 0.21 0.21 0.21 0.21 4. ASV27 Xylophilus sp. 0.25 0.25 0.82 1.00 0.80 0.81 0.23 0.79 0.79 0.79 0.79 0.77 0.77 0.77 0.77 5. SP210_2 Xylophilus sp. 0.24 0.24 0.24 0.24 1.00 0.98 0.22 0.78 0.21 0.22 0.22 0.20 0.20 0.21 0.20 6. SP51_3 Xylophilus sp. 0.24 0.24 0.24 0.24 0.86 1.00 0.23 0.78 0.22 0.22 0.22 0.21 0.20 0.22 0.21 7. KACC21265 X. rhododendri (Type strain) 0.23 0.23 0.79 0.80 0.79 0.79 1.00 0.78 0.77 0.78 0.78 0.76 0.75 0.76 0.76 8. cluster_DBSCAN_round5_1 Xylophilus sp. 0.22 0.22 0.22 0.22 0.22 0.22 0.21 1.00 0.22 0.22 0.22 0.21 0.20 0.21 0.21 9. BgEED09 Xylophilus ampelinus 0.22 0.22 0.78 0.22 0.77 0.77 0.21 0.78 1.00 1.00 0.84 0.76 0.76 0.76 0.76 10. CCH5-B3 Xylophilus ampelinus 0.22 0.22 0.78 0.22 0.78 0.78 0.22 0.78 0.99 1.00 0.83 0.76 0.76 0.76 0.76 11. JQKD01.1 Xenophilus azovorans DSM 13,620 (Type strain) 0.22 0.22 0.78 0.22 0.78 0.78 0.21 0.78 0.29 0.28 1.00 0.21 0.21 0.21 0.21 12. Gw_Inlet_bin_57 Xylophilus sp. 0.21 0.21 0.77 0.21 0.76 0.77 0.20 0.76 0.21 0.21 0.77 1.00 0.90 0.97 0.98 13. 3.2. Genetic Diversity of CIRM-CFBP Xylophilus Ampelinus Strains The gyrB and rpoD sequences were perfectly identical for 1382 base pairs (out of 1383) (Figure 2). The accession numbers for all gyrB and rpoD sequences are available in Table S1, the alignment of the sequences is available in Table S2, a version of the phylogenetic tree including the 93 X. ampelinus strains is available in Figure S1. A single 1 base-pair difference was observed in the rpoD sequence for 18 of the 92 strains, including the type strain of the species. A third gene (rpoB, results not shown) had been tested for a few strains leading also to perfectly identical sequences (thus, the analysis with this gene was not completed). These results are surprising considering that the strains have been isolated over a period of 35 years from different countries: Spain, Greece, France and South Africa. The number of analyzed genes is limited and may not reﬂect the actual diversity of the species. However, for other genera of plant-pathogenic bacteria, the analysis of only a few (1–3) housekeeping genes is enough to reveal the genetic diversity of the considered taxa. It is the case for Xanthomonas [38], Acidovorax [39] or Pectobacterium [40] for instance. A complete MultiLocus Sequence Analysis study of Curtobacterium ﬂaccumfaciens [41] used 6 loci, but each locus independently was enough to reveal the diversity of the species. The homogeneity of X. ampelinus is thus remarkable. Figure 2. Phylogenetic tree reconstructed from concatenated partial sequences of gyrB and rpoD housekeeping genes for 15 strains of Xylophilus ampelinus and the type strain of Acidovorax anthurii as outgroup. The phylogenetic tree was reconstructed with concatenated alignments of all genes with MEGA 7.0.26 using the neighbor-joining method with 1000 bootstrap replicates, and the evolutionary distances were computed by using the Kimura two-parameter method. Triangles indicate the two CFBP accession corresponding ot the type strain (accession duplicated in the CIRM-CFBP collection). The phylogenetic tree of the 93 Xylophilus ampelinus strains and all accession numbers of the sequences are available in Figure S1 and Table S1, respectively. In 2016, Komatsu et al. [21] described a limited genetic variability in X. ampelinus strains revealed by a combination of Box-, Eric- and Rep-PCR. The strains were divided in 4 groups, groups A and B comprising CFBP strains. The comparison of these results with the ones of the present study showed no correlations. The group A described by Komatsu et al. 2.4. gyrB-rpoD Phylogeny Go_Prim_bin_55 Xylophilus sp. 0.21 0.21 0.77 0.21 0.76 0.76 0.20 0.76 0.21 0.21 0.76 0.99 1.00 0.98 0.98 14. Gw_Prim_bin_50 Xylophilus sp. 0.21 0.21 0.77 0.21 0.76 0.77 0.21 0.76 0.21 0.21 0.77 0.90 0.91 1.00 0.98 15. Gw_UH_bin_252 Xylophilus sp. 0.20 0.20 0.77 0.20 0.77 0.77 0.20 0.77 0.21 0.21 0.77 0.90 0.89 0.89 1.00 Table 4. ANIb (above diagonal) and dDDH (below diagonal) values, calculated respectively with pyani [33] and TYSG, formula d4 [35]. Highlighted in green, the values above the 95% (for ANIb) or 70% (for dDDH) thresholds for bacterial species delineation. The numbers featured on top, correspond to the genome number on the left. CFBP 1192T and CECT 7646T are both equivalent of the same type strain of the species held in two different collections. Microorganisms 2022, 10, 1531 8 of 11 4. Conclusions The homogeneity of X. ampelinus species is a key fact for plant pathology, permitting to better choose how to design tools for detection and identiﬁcation of this species. However, more data on the diversity of the strains belonging to this species is necessary. Moreover, the analyzed collection does not extend further to strains isolated in 2001. Even though the analyzed strains are numerous, from diverse locations, and isolated at different times, these ﬁndings must be conﬁrmed by the analysis of more recent strains. Hence, new isolation campaign and epidemiological surveys are necessary. As highlighted by Broders et al. [42], the continuous isolation and reliable preservation of plant-pathogenic strains is beneﬁcial in the long term and can be of crucial help when epidemics arise. On the other hand, the identiﬁcation of the potential source of spread of a plant- pathogen such as X. ampelinus is of crucial importance for plant health. A better knowledge of the reservoirs of inoculum could indicate where and how the efforts should be concen- trated to limit the effects of the disease on crops. The analysis of the different genome sequences available in the public databases showed clearly that the ecological niche of the genus Xylophilus is largely unknown. Its actual ecological importance, beyond its pathogenicity on grapevine, is still to be described. The ongoing analysis of microbiota in various environments could help us to better understand this genus and its repartition, once the problem of the accuracy of the sequence assignation has been addressed. The better characterization of Xylophilus strains held in the collection can help with this task and we encourage scientists to characterize their strains and to make them available for the scientiﬁc community. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10081531/s1. Figure S1: phylogenetic tree con- structed from gyrB and rpoD concatenated sequences for the 93 strains of X. ampelinus held in CIRM-CFBP, Table S1: information on strains used in this study, Table S2: gyrB-rpoD sequence align- ment for all strains listed in Table S1, Table S3: Statistics derived from ChekM analysis [29] on the Xylophilus genomes retrieved from NCBI. Author Contributions: P.P.: conceptualization, writing—original draft, supervision and project administration; P.P. and M.F.-L.S.: funding acquisition, writing—review and editing; P.P., M.B., M.F.-L.S. and G.T.: formal analysis, writing—review and editing; G.T. and P.-E.B.: data acquisition, curation and methodology; C.D. and A.L.: preservation of resources. 3.2. Genetic Diversity of CIRM-CFBP Xylophilus Ampelinus Strains [21] clusters together strains belonging to both groups revealed by our study of gyrB and rpoD sequences. These different techniques do not analyze the diversity at the same level. Sequencing of housekeeping genes provide reliable information at the species/intra-speciﬁc level, while the Box-, Eric- and Rep-PCR are able to assess variations between individual strains. Thus, these two ﬁndings can be compatible. The analysis of a larger number of genomes of strains actually belonging to X. ampelinus is needed to bring a deﬁnitive answer on the actual diversity of this species. 9 of 11 9 of 11 Microorganisms 2022, 10, 1531 Our results suggest that the species is very homogenous considering the housekeeping genes, with a limited diversity existing between the different strains. Grall et al. [17], reported that sap and old wood are the main reservoirs for the bacterium. Hence, human activities such as pruning, grafting and plant cuttings’ transportation are highly susceptible to favorize the spread of the bacterium. If this bacterium is disseminated by human activities from plant to plant, this could explain the homogenic structure of the species. 4. Conclusions All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Projects Lycovitis (gyrB-rpoD sequencing) and TAXOMIC (Genome sequence of CFBP 1192T) both funded by INRAE. Institutional Review Board Statement: All CFBP strains are available upon request at CIRM-CFBP https://cirm-cfbp.fr; cfbp@inrae.fr. Data Availability Statement: The sequence data supporting this article can be found in GenBank under the accession numbers listed in Table 2 and Table S1. Acknowledgments: The authors would like to thank Emeline Théard for help in the sequencing of CFBP 1192T. Acknowledgments: The authors would like to thank Emeline Théard for help in the sequencing of CFBP 1192T. References g y , , ; p 10. Botha, W.J.; Serfontein, S.; Greyling, M.M.; Berger, D.K. Detection of Xylophilus ampelinus in grapevine cuttings using a nested polymerase chain reaction. Plant Pathol. 2001, 50, 515–526. [CrossRef] 11. Willems, A.; Gillis, M.; Kersters, K.; Van Den Broecke, L.; De Ley, J. Transfer of Xanthomonas ampelina Panagopoulos 1969 to a new genus, Xylophilus gen. nov., as Xylophilus ampelinus (Panagopoulos 1969) comb. nov. Int. J. Syst. Bacteriol. 1987, 37, 422–430. 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https://openalex.org/W3107996944	https://www.researchsquare.com/article/rs-36806/v1.pdf?c=1631860101000	English	null	The emotional and social burden of heart failure: integrating physicians’, patients’, and caregivers’ perspectives through narrative medicine	BMC cardiovascular disorders	2,020	cc-by	7,525	The Burden of Heart Failure: Physicians’, Patients’, and Caregivers’ Perspectives Using Narrative Medicine Marco Testa Cardiology Unit, Sant'Andrea Hospital, Rome Antonietta Cappuccio (  acappuccio@istud.it ) Fondazione ISTUD https://orcid.org/0000-0002-6259-8824 Maura Latella Novartis Farma SA Silvia Napolitano Fondazione ISTUD Massimo Milli Cardiology Unit, Santa Maria Nuova Firenze Hospital Massimo Volpe Universita degli Studi di Roma La Sapienza Facolta di Medicina e Psicologia Maria Giulia Marini Fondazione ISTUD Research article Keywords: heart failure, narrative medicine, doctor-patient relationship, quality of life, informal caregive Posted Date: July 15th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-36806/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License The Burden of Heart Failure: Physicians’, Patients’, and Caregivers’ Perspectives Using Narrative Medicine Marco Testa Cardiology Unit, Sant'Andrea Hospital, Rome Antonietta Cappuccio (  acappuccio@istud.it ) Fondazione ISTUD https://orcid.org/0000-0002-6259-8824 Maura Latella Novartis Farma SA Silvia Napolitano Fondazione ISTUD Massimo Milli Cardiology Unit, Santa Maria Nuova Firenze Hospital Massimo Volpe Universita degli Studi di Roma La Sapienza Facolta di Medicina e Psicologia Maria Giulia Marini Fondazione ISTUD Research article Keywords: heart failure, narrative medicine, doctor-patient relationship, quality of life, informal caregiver Posted Date: July 15th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-36806/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on December 12th, 2020. See the published version at https://doi.org/10.1186/s12872-020-01809-2. Key Points For Decision Makers: Patients and their informal caregivers have a strong need to be heard; narrative medicine provides this opportunity Patients and their informal caregivers have a strong need to be heard; narrative medicine provides this opportunity Two parallel lives were revealed: patients’ lives, which are affected by their HF-related physical limitations, and lives of informal caregivers, burdened emotionally by caring for a family member Two parallel lives were revealed: patients’ lives, which are affected by their HF-related physical limitations, and lives of informal caregivers, burdened emotionally by caring for a family member NM offers HF specialists the opportunity to better understand the patient experiences of HF, and an opportunity to actively recognize the role of the caregiver, and educate both NM offers HF specialists the opportunity to better understand the patient experiences of HF, and an opportunity to actively recognize the role of the caregiver, and educate both Abstract Background The TRUST (The Roadmap Using Story Telling) project used a Narrative Medicine (NM) framework to assess the impressions of people with heart failure (HF), their informal caregivers and HF specialists of the impact of heart failure (HF) on the daily life of patients and their carers. Methods Italian HF specialists participated on a voluntary basis, completing their own narratives, and inviting patients and their caregivers to write anonymously about their experiences, all on a dedicated online platform. The narratives were analyzed according to standard NM methodology. Results 82 narratives were collected from patients, 61 from caregivers, and 104 from HF specialists. Analysis of the three points of view revealed the extent of the burden of illness on the entire family, particularly that of the caregiver. The impact was mainly experienced as emotional and social limitations in patients’ and their caregivers’ daily lives. The analysis of all three points of view highlighted a strong difference between how HF is perceived by patients, caregivers, and HF specialists. Conclusions This NM project illustrates the complex issues of living with HF and gave new insight into integrating three different perspectives into the HF pathway of care. 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 on December 12th, 2020. See the published version at https://doi.org/10.1186/s12872-020-01809-2. Page 1/22 Page 1/22 1 Background Heart failure (HF) is a progressive chronic disease that needs long-term management. It affects 1–2% of the European population and about 10% of people between the ages of 75 and 80 years [1]. The incidence of HF is currently increasing, mainly due to innovative therapies and improved survival from myocardial infarction [2]. Standard treatment for HF requires the patient to take several different drugs daily, which is not only difficult to adhere to but further burdens their daily lives, negatively affecting patient and caregiver quality of life [3, 4]. Moreover, most people with HF have comorbidities, including hypertension (58.4%), atrial fibrillation (25.3%), chronic kidney disease (51.4%), and dyslipidemia (44.6%) [5], which add to the burden on the patient and caregiver/family. Page 2/22 Page 2/22 Patient symptoms of HF have been found to be associated with strain in their caregivers [6]. Indeed, the risk of depression and anxiety, as well as financial loss, increases over time in families as a direct consequence of providing care for a person with deterioriating health; this is especially true for female informal caregivers [7]. Risk of hospital readmission rates for people with HF are correlated with lack of social support [8], suggesting the important role of the family environment in the care of people with HF. The need for research into clinical- and person-oriented outcomes of both the person with HF and their caregiver(s) has been recognized [6]. Narrative Medicine (NM) is a methodology based on the analysis of narratives of illness experiences [9]. NM promotes the integration of the disease-centered approach, focused on the clinical aspects, with the illness-centered approach, focusing on personal coping with the pathology, and sickness-centered approach, focusing on social perception of a specific condition[10]. NM is considered a very informative tool, since the integration of all the points of view involved in the pathway of care help reveal common issues as well as possible interventions or solutions about living with a specific disease [9, 11–13]. The main tools of this methodology are parallel charts and illness plots, dedicated to healthcare professionals and patients-caregivers, respectively [14, 15]. Recent studies have demonstrated advantages of applying the parallel chart in exploring a healthcare professional’s point of view about the pathway of care for chronic conditions (i.e., chronic obstructive pulmonary disease [COPD]) and doctor-patient relationships [16, 17]. 2.1 Participants This was a cross-sectional project conducted at 21 HF clinics across Italy. Beginning May 2018, 25 HF specialists working at these clinics were invited to take part in a voluntary training session on NM where the methods and aims of the project were described. The 21 specialists who decided to participate then invited people with HF and their informal caregivers to participate, providing them with information materials about the TRUST project. 1 Background Similarly, patient and caregiver illness plots were recently shown to be a source of information on personal coping with the disease, and on how patients and their families rearrange their lives after a diagnosis in studies in other chronic diseases [18–20]. The main aim of the TRUST (The Roadmap Using Story Telling) project was to investigate the impressions of people with HF, their informal caregivers and HF specialists of the impact of HF on the daily life of patients and their carers, by using NM methodology. 2.2 Narrative tools A board composed of two Italian HF specialists (MV and MT) and one patient reviewed the NM tools and patient informed consent forms developed by the “ISTUD Foundation” (Fondazione ISTUD, Milan, Italy) [21], and adapted them for use in this project. The NM tools used were a semi-structured parallel chart (for physicians) and two different versions of illness plots (for patients and caregivers). In these tools the Page 3/22 Page 3/22 prompts were composed of brief sentences with the aim of easing response writing (Appendix 1), and were specifically designed to overcome writer’s block [22]. All narratives were written in Italian. 2.3 Data collection From June to November 2018, physicians completed a parallel chart for each enrolled patient. There were no restrictions in terms of patients’ disease severity or other clinical parameters; the only inclusion criterion was to write about a person with a confirmed diagnosis of HF whom they had seen at least twice. Patients and caregivers accessed a dedicated online platform to provide their narrations, allowing them to independently and anonymously describe their experiences. The platform is designed to facilitate research in the healthcare sector and includes safeguards to guarantee anonymity for survey participants by not registering sensitive data, such as country of residence or IP address. It is fully compliant with the European General Data Protection Regulations. Only the authors had access to the survey responses, and authors were blinded to the participants’ identities. From June to November 2018, physicians completed a parallel c no restrictions in terms of patients’ disease severity or other clinic criterion was to write about a person with a confirmed diagnosis twice. Patients and caregivers accessed a dedicated online platfo them to independently and anonymously describe their experienc research in the healthcare sector and includes safeguards to gua by not registering sensitive data, such as country of residence or European General Data Protection Regulations. Only the authors authors were blinded to the participants’ identities. 2.4 Narrative analysis All collected narratives were analyzed according to the Grounded Theory [23] by three independent researchers (AC, SN, MGM), with the aid of the qualitative data analytics software NVivo10 (QSR International ). Additional analyzes were carried out according to Kleinman’s theory [10], Plutchik’s theory [24] and Frank’s classification [25]. 2.5 Ethical considerations Since physicians, patients and caregivers completed their contributions anonymously, the patient described by physicians in the parallel charts could be different from the patient that participated in the project, and there was no possibility for physicians or researchers to identify any potential relationship between them. Informed consent was obtained online from all participants when they first accessed the online platform, prior to writing their narratives. The project was carried out in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the Santa Maria Nuova Hospital (Florence, Italy). 3.1 Sociodemographic characteristics A total of 82 narratives were collected from patients, 61 from caregivers, and 104 from HF specialists, for a total of 247 narratives (Appendix 2). Most of the people with HF were male (75%) with a mean age of 68 years; most caregivers were patients’ wives (47%) or daughters (35%), with a mean age of 60 and 46 years, respectively. Most patients were pensioners (71%) and 37% of caregivers were employed. The sociodemographic characteristics of participants, both patients and caregivers, were similar to those of the general Italian population, except for education attainment level, which was higher than the Italian Page 4/22 Page 4/22 average [26]. The HF specialist narratives described patients with a similar sociodemographic profile to that of participating patients (Appendix 2). 3.2 Management and awareness of the disease HF diagnosis frequently occurred before 60 years of age, and the mean disease duration was 10 years for patients and as described by caregivers, and was 8 years according to physicians (Table 1). Physicians reported other cardiovascular comorbidities affecting 63% of their patients. Families had to cover 37 km on average to reach the cardiology center, with follow-up every 6 months or more frequently (86%). At home, disease management included the administration of a mean total of eight different drugs per day across seven different times. Most patients reported having New York Heart Association (NYHA) class II or III HF (class II: 27% of patients, class III: 26%). However, physicians described the people with HF in their care as suffering from more severe HF (class II: 44%, class III: 34%). When patients and caregivers were asked to report the patient’s ejection fraction (EF), 16% and 17%, respectively, did not know the answer (Table 1). Page 5/22 Table 1 Disease management and clinical characteristics of patients with HF reported by patients, HF specialists, and caregivers Patients (N = 82) Patients described by physicians (N = 104) Patients described by caregivers (N = 61) Age (mean ± SD), years 57 ± 3 57 ± 3 – Disease duration (mean ± SD), years 10 ± 2 8 ± 1 10 ± 3 Recurrence of specialist visits, n (%) (n = 73) – (n = 52) ≥ 1 in 6 months 28 (38) – 25 (48) 1 per 6 months 35 (48) – 23 (44) 1 per year 8 (11) – 2 (4) < 1 per year 3 (4) – 1 (2) Just in emergency cases – – 1 (2) NYHA class, n (%) (n = 73) (n = 92) (n = 53) I 18 (25) 4 (4) 17 (32) II 27 (37) 44 (48) 16 (30) III 26 (36) 34 (37) 18 (34) IV 2 (2) 10 (11) 2 (4) Ejection fraction, n (%) (n = 73) (n = 102) (n = 52) > 40% (HF-pEF) 18 (25) 17 (17) 14 (27) < 40% (HF-rEF) 39 (53) 85 (83) 21 (40) I don’t know 16 (22) – 17 (33) Table 1 HF heart failure, NYHA New York Heart Association, pEF preserved ejection fraction, rEF, reduced EF; SD standard deviation 3.3 Analysis of the narratives 3.3.1 Frequent words Analysis of the 100 most frequently used words in the narratives showed differences in the points of view between the three types of participant. The most frequently used words by patients evoked previous life conditions and expressed fatigue, tiredness, and difficulty carrying out activities that were previously Page 6/22 Page 6/22 considered normal, like walking, working, and climbing stairs (e.g., «Before my illness I was always active, I’d walk at least two hours a day and I had a balanced diet. The only unhealthy thing I did was smoking»). considered normal, like walking, working, and climbing stairs (e.g., «Before my illness I was always active, I’d walk at least two hours a day and I had a balanced diet. The only unhealthy thing I did was smoking»). The word “fear” was used more commonly by the caregivers than the patients, revealing concern for the quality of life and life expectancy of the patients they cared for. Also present were words relating to the medical domain (e.g., “physicians”, “therapy”, “follow-up visit”). The physicians’ narratives highlighted the improvements obtained with treatments, but the caregivers were represented as secondary, background figures, as helpers in the event that the patients should fail to comply wth the treatments. In most of their narratives the physicians showed trust at the time of diagnosis but felt an urge to reassure their patients (e.g., «none of them should blame themselves, but they all had to undertake to follow the doctor’s instructions from the time of diagnosis onwards»). The physicians proved to be aware of the importance of knowing how to actively listen to their patients and in depth, not only in the clinical domain but also in the domains of emotions and planning everyday life. 3.3.2 Burden of the disease on the patient and caregiver All the narratives were rich in detail about the level of the patients’ quality of life. The consequences of the disease were so burdensome that only 26% of patients and 16% of caregivers stated that they had returned to their usual life (e.g., «I’ve had to reduce my working hours and ask my children and family for help. Today we spend much of our time at home. We don’t go anywhere») [Table 2]. 3.3.4 Perception of the disease Patients were asked to define their HF by using a metaphor (Fig. 2). The metaphors were analyzed and grouped into four main classes: (a) malignant nature metaphors, relating to something frightful or unpredictable (e.g., "volcano eruption"), (b) limitation metaphors, in which the disease is perceived as disabling (e.g., "a very fast car without fuel"), (c) fight metaphors, where the disease is seen as an enemy (e.g., a "trench war"), (d) threat metaphors, in which danger is the main feature (e.g., "the sword of Damocles"). The patients mostly expressed limitation metaphors (72% of patients), the caregivers evil nature metaphors (52% of caregivers) [e.g., "slowness" of life], as did the physicians (60%) [e.g., "earthquake", "panther"]. The physicians also frequently used fight metaphors (13%) [e.g., "trench war"]. 3.3.3 The emotional impact of the disease The emotional impact of the disease on participants’ lives and physicians’ care practice was also investigated through the application of the Plutchik’s classification2 to the narratives [24]. Physicians’ prevalent emotions at the diagnosis were predominantly trust and optimism (61%), and this positivity was still present at the time of writing the narrative (71%) (Fig. 1). On the other hand, the most frequent patient emotions at diagnosis were fear (53%) and sadness (15%), and this emotional impact on ill individuals was also confirmed by both caregivers’ and physicians’ perceptions. Although at the time of writing their narratives, patients’ emotions were characterized by optimism and trust (31% and 23% of patients, respectively), a significant proportion still felt fear (34%) [‘Today’ Fig. 1], suggesting that they continue to feel afraid about the disease. Similarly, more than 80% of caregivers described having felt fear and anguish at the moment of diagnosis, and these emotions remained in a significant proportion at the time of writing narratives (38%) [Fig. 1]. Furthermore, the future was described as frightening by 21% of patients and 15% of caregivers, and 17% and 23%, respectively, declared they did not want to think about the future. Indeed, 41% of caregivers’ narratives contained references to the fear of the sudden death of their loved ones. 3.3.1 Frequent words Page 7/22 Table 2 Table 2 Patients’, physicians’ and caregivers’ perception of the impact of HF on daily activities Patients Informal Caregivers Patients, as described by physicians Patients, as described by caregivers Impact on work, n (%) (n = 33) (n = 21) No changes 6 (18) 5 (24) – – Feeling disadvantaged at work 3 (9) 1 (5) – – Limiting activities at work 14 (42) 9 (43) – – Work interrupted 10 (30) 6 (29) – – Spare-time activities before the diagnosis of HF, n (%) (n = 70) (n = 33) (n = 79) Social Life (i.e. Dinner with friends, theatre, etc.) 32 (46) 19 (58) 39 (49) – Sport 17 (24) 5 (15) 10 (13) – Work and little spare time 10 (14) – 13 (16) – Taking care of the family 7 (10) 8 (24) 13 (16) – Gardening 4 (6) 1 (3) 4 (5) – Impact on daily activities today, n (%) (n = 69) (n = 32) (n = 74) (n = 44) Social Life (i.e. friends, theatre, etc.) 18 (26) 5 (16) 34 (46) 5 (11) Light physical activities (i.e. bike, walk, etc.) 14 (20) – 14 (19) Reading and watching TV 7 (10) – 1 (1) Taking care of the family 5 (7) 7 (22) 4 (5) Art (i.e. painting, music, etc.) 3 (5) – 5 (7) Limited activities 15 (22) – 10 (14) 18 (41) Impossible to restore activities 6 (9) 9 (28) 6 (8) 16 (36) Activities not restored due to fear 1 (1) 11 (34) – 5 (11) HF heart failure Table 2 Patients’, physicians’ and caregivers’ perception of the impact of HF on da Between the three types of participant in this project it was mainly the female caregivers who denounced their caregiving burden, and whose duration of caregiving exceeded 8 hours per day in 55% of the Page 8/22 narratives. A total of 34% of the informal caregivers’ narratives considered a return to past activities impossible as it would imply leaving the patients on their own (Table 2). Based on Kleinman’s1 classification of narratives [10], the most representative style of writing among participants was ‘illness’ (96% for patients, 100% for caregivers, 96% for physicians). Based on Kleinman’s1 classification of narratives [10], the most representative style of writing among participants was ‘illness’ (96% for patients, 100% for caregivers, 96% for physicians). 3.3.7 Perception of treatment Therapies were described as effective and were often considered to have contributed to positive relationships between patients and caregivers. Generally, both patients and caregivers were satisfied with patient treatment (more than 80% of each considered them effective or very effective), while surgery, cited by 20% and 6% respectively, was considered the most critical treatment in terms of both risk and effect on outcomes. On the other hand, HF specialists perceived the treatment plan for patients as complex and burdensome in 21% of the cases, more often than the patients and their caregivers did so themselves. 3.3.5 Disease awareness A high proportion of both patients (69%) and caregivers (84%) stated that they had not initially recognized the first symptoms of the disease, and this underestimation was also reported by 44% of physicians. No initial symptoms were described by participants since the disease occurred suddenly during daily activities (according to 39% of patients and 66% of caregivers), although 25% of patients reported having noticed unusual fatigue before the diagnosis (Fig. 3). In fact, 55% of physicians reported first meeting the person with HF in an emergency situation. Furthermore, several elements in both patients’ and caregivers’ narratives showed a lack of awareness about the disease (85% of patients and 74% of caregivers) exemplified by their misuse of clinical terms and lack of knowledge about what exactly HF is (Fig. 3). 3.3.6 Doctor-patient relationship Relationships in the pathway of care were clustered in three main ways: ‘easy’ relations, when described as comfortable and trustworthy, ‘difficult’, when described as unsatisfactory, and ‘evolved’, when initially difficult but with a positive evolution. Physicians established good relationships with patients and their families (Fig. 4). Furthermore, 7% of physician-patient relationships (from the perspective of the physician) that were difficult initially improved over time (Fig. 4). The highest proportion of difficult relationships early after diagnosis reported by patients were for those between patient and caregiver when the caregiver was a family member (48%). These caregivers were often described by the patient as being more afraid than necessary, and as annoying to the patient, who often desired more autonomy. 3.3.8 Participation in NM The patients, physicians and informal caregivers reported their general appreciation for being able to write about their experience, and sharing it was perceived as a liberation and an opportunity to reflect (e.g., «I was pleased I was able to describe our experience in the hope that it may be useful; indeed, I wanted to thank you for giving me this opportunity»). Twelve percent of the narratives stated that it was difficult to share the experience (e.g., «remembering the single moments arouses a feeling of emotional suffering for a situation that is still unsolved and that presents an objective uncertainty for the future of all our family»). 1Kleinman's theory [10] distinguishes between illness, disease and sickness. Disease means the disease in the biomedical sense, illness indicates the subjective experience of the disease and sickness means disease as percieved by a social group or population (‘social recognition’). 2Plutchik defines 8 basic emotions: joy, trust, fear, surprise, sadness, anticipation, anger, and disgust. Plutchik applies a framework to illustrate the various ways in which the 8 basic emotions relate to one another, including which ones are opposites and which ones can easily turn into another one [24]. 4 Discussion Page 10/22 Page 10/22 Page 10/22 The TRUST project aimed to explore living with HF in a multiperspective way through the integration of patients’, informal caregivers’, and HF specialists’ points of view. Firstly, the large number of narratives collected for this project can be considered an excellent result, highlighting the need of patients and their caregivers alike to be listened to. Indeed, participating in the project was rated as a positive experience by about 90% of patients and caregivers, and an even higher percentage of physicians perceived the task of writing as a way to reflect on their work. The TRUST project aimed to explore living with HF in a multiperspective way through the integration of patients’, informal caregivers’, and HF specialists’ points of view. Firstly, the large number of narratives collected for this project can be considered an excellent result, highlighting the need of patients and their caregivers alike to be listened to. Indeed, participating in the project was rated as a positive experience by about 90% of patients and caregivers, and an even higher percentage of physicians perceived the task of writing as a way to reflect on their work. Our project results show that HF strongly limits the life of both the patient and their caregiver. The narratives revealed two parallel lives: the life of the patients, which is physically limited, and that of the informal caregivers, which is affected by the need to look after a family member. A substantial proportion of patients adopted hobbies like playing cards or reading in place of strenuous activities such as sport. A strong emotional impact of HF emerged in terms of anxiety and fear of sudden death for both patients and caregivers. This anguish led to additional limitations to activities, and caregivers reported being not only responsible for co-ordinating the patient’s complex therapy plan and medical visits, but also having to be constantly in close proximity to the patient due to their deep fear of the patient experiencing sudden disease worsening. Furthermore, most caregivers were women, partners, and daughters, and their narratives exemplified the sacrifices they made. This strong emotional impact of HF on the patient and their family is in agreement with a previously published study, in which caregiving demands have been related to depression in the caregiver [27]. Both had to change their daily lives to cope with this new condition. 4 Discussion Participants’ narratives included many elements of confusion, and lack of knowledge of HF emerged from their narratives, reflected by their deep feelings of fear and anguish, and those of their caregivers. Moreover, even when the diagnosis was recent, almost 30% of the affected people and their families didn’t know their EF, suggesting that they probably didn’t understand the severity of their condition. Indeed, families’ poor health literacy and knowledge of HF have been recognized in previous studies [30, 31], especially in terms of understanding specific terminology. Nevertheless, participating patients and caregivers in our project had educational attainment levels higher than the Italian standard, so they had the necessary means to understand the disease course of HF. Interestingly, scarce knowledge of HF was shown even in the case of participants involved in the ‘HF Awareness Day’ initiative launched by the European Society of Cardiology HF Association [31], suggesting that their desire to be involved in such initiatives was not correlated with higher acceptance or awareness of the disease. What could be inferred from their participation in the HF Awareness Day initiative was their greater desire for effective care, and ultimately complete healing. The metaphor analysis revealed a large difference between patients and physicians in how they defined the disease (essentially an ‘internal’ understanding or knowledge of disease). While doctors expressed awareness of the inevitable progression of HF, using ‘malignant nature’ metaphors, patients were mainly focused on the ‘limitations’ they experienced because of the disease (Fig. 2). Furthermore, the high level of fear and anguish felt by patients and the caring attitude of physicians could have contributed to the lack of communication from doctors of the severe disease prognosis. This requires further study. A limitation of this project is that all data were gathered by self-report. The large number of narratives collected and the integration of different points of view may help reduce to some extent the possible bias of using a qualitative methodology. Another possible source of bias was the high level of educational attainment among participating patients and caregivers. 3Using the concept ‘‘narrative type’’ Frank [25] described three types of illness narratives: Restitution, Chaos and Quest. The restitution narrative evolved through three stages, beginning with health, followed by sickness, and then by looking forward to a return to health in the future. 4 Discussion Almost all project participants wrote about the patient’s HF as an ‘illness’, according to our analysis using Kleinman’s theory [10]. These results are in contrast with those seen with other chronic diseases, for example, COPD [19], in which patients report ‘sickness narratives’. Therefore, although some people with HF had engaged in harmful behaviors (i.e., smoking, alcohol consumption, over-eating) that may have contributed to HF development, they did not feel judged for the onset of HF. All the relationships were described with positivity and patients often expressed gratefulness to their doctors and for effective therapies, as confirmed by the high percentage of ‘restitution’ style of writing, according to Frank’s classification3[25]. Our results are in contrast with a recent Swedish study, which showed that caregivers felt unrecognized for their role in HF management [28], which suggests that caregivers may have difficulty in establishing a positive relationship with the physician. HF care is particularly challenging, not only for the elderly age of occurrence and frequent comorbidities, but also for the high number of different drugs per day patients have to consume. However, it is interesting to notice that the complexity of the treatment plan was considered more burdensome by the physicians than by either the patient or their caregiver. A recent study in patients with COPD demonstrated a link between the physician’s style of narrative writing about their relationship with their patient and the quality of care of their patients [29]. In that study, participating physicians wrote ‘illness-centered’ narratives. Our results were possibly biased towards a positive physician-patient relationship because the HF specialists participating in the TRUST project were already attentive to their relationship with patients. Page 11/22 Both caregivers and HF patients frequently avoided seeking the help of a HF specialist or other healthcare professionals until the patient’s condition had dramatically worsened, even if they had recognized unusual fatigue. Participants’ narratives included many elements of confusion, and lack of knowledge of HF emerged from their narratives, reflected by their deep feelings of fear and anguish, and those of their caregivers. Both caregivers and HF patients frequently avoided seeking the help of a HF specialist or other healthcare professionals until the patient’s condition had dramatically worsened, even if they had recognized unusual fatigue. 5 Conclusions Our NM project enabled us to describe the profile of those who live with HF and those who take care of people with HF in Italy. Through the integration of the three points of view, the burden of illness on the entire family emerged from understanding the key role of the caregiver in the daily management of the complex care of HF. The impact described in the narratives was mainly focused on the emotional and social limitations of both patients’ and caregivers’ daily lives, impeding their work activities and impacting on their hobbies and friendships. The strong presence of fear and anguish in patient and Page 12/22 caregiver narratives were probably a consequence of their general lack of knowledge and understanding of HF. Recent evidence showed that low levels of literacy and limited disease awareness are influenced by age-related factors and communication of information [32]. caregiver narratives were probably a consequence of their general lack of knowledge and understanding of HF. Recent evidence showed that low levels of literacy and limited disease awareness are influenced by age-related factors and communication of information [32]. An area deserving multidisciplinary attention is that of the informal caregiver; the stress of caregiving affects not only the caregiver’s wellbeing but that of the whole family, which may be defined as a "second victim" of HF. To improve the caregiver’s condition, it would be useful to strengthen the therapeutic alliance of the physician-patient-caregiver triad, by acting in several directions: 1. (a) Providing individual psychological support such as brief or extensive counseling, organized in such a way as not to require an excessive time investment or constitute an additional burden on the subject’s psychophysical resources. 2. (b) Promoting caregiver interaction within support groups, in order to stimulate and facilitate elaborative/transformative processes allowing the acquisition of new strategies in the daily management of the patient and preventing social isolation. 3. (c) Providing adequate information to the patient and caregiver at the time of communicating the diagnosis, so as to allow adequate understanding of the disease and make the necessary changes to their behavior. An Italian study has demonstrated the effectiveness of including NM training in the education pathway of cardiology specialists in terms of obtaining relevant healthcare information that their patients may not otherwise disclose and optimizing visits with patients [33]. 5 Conclusions The application of NM could therefore be considered an effective tool for integrating the different perspectives on life with HF, and to strengthenthe triad of care and the therapeutic alliance. Abbreviations TRUST - The Roadmap Using Story Telling TRUST - The Roadmap Using Story Telling NM - Narrative Medicine HF - Heart Failure EF - Ejection Fraction NM - Narrative Medicine HF - Heart Failure EF - Ejection Fraction Funding The work was unconditionally supported by Novartis Farma Italia, since Fondazione ISTUD is a not-for- profit organization. The publication of results was not contingent on the sponsor's approval. Authors’ contributions AC, ML, and MGM were involved in the conceptualization of the TRUST project. AC, SN and MGM contributed to data analysis, and MT, MV, MM, ML, and MGM contributed to project investigation. MT, MV, MM, and the TRUST Group enrolled people with HF. AC and MGM were involved in methodology; AC and SN were involved in project administration; AC, SN and MGM were involved in the analysis of the narratives; and MT, MV, MM, and the TRUST Group were involved in data validation. AC and SN contributed to writing; and all authors contributed to report visualisation and read and approved the final draft for submission. Availability of data and material All data relevant to the project are included in the present manuscript. Original narratives are available in Italian upon request to the researchers at the following email address: areasanita@istud.it. Consent for publication Not applicable. Conflicts of interest/Competing interests A Cappuccio, S Napolitano, and MG Marini report grants from Novartis Farma Italy, during the conduct of the project. G Maiocchi and M Latella are employees in the medical department of Novartis Farma Italy. M Volpe, M Milli, and M Testa have no conflict of interest to declare. Ethics approval and consent to participate The project was carried out in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the Santa Maria Nuova Hospital (Florence, Italy). Written informed consent was obtained online from all participants when they first accessed the online platform, prior to writing their narratives. Page 13/22 Page 13/22 Acknowledgments The authors wish to thank Novartis Farma Italia for its unconditional contribution to this project and the researchers of the Healthcare Area of ISTUD Foundation for their useful role throughout this project. The authors would also like to thank all the people with HF, their caregivers and physicians who took part in this project, Dr. Giuseppe Maiocchi for his assistance in the planning and management of the project, Mr. Gianni Forlani for his assistance with adapting the materials, Antonino Giorgi and Martina Roverselli who provided input on the manuscript, and Tracy Harrison of Springer Healthcare Communications who edited Page 14/22 and styled the manuscript prior to submission. This editorial assistance was funded by Novartis Farma Italia. TRUST Group: Belloli Daniela, Cacciatore Francesco, Candela Pietro, Carigi Samuela, Casale Giuseppe, Clemenza Francesco, Cosentino Eugenio, Donadeo Vittorio, Floresta Agata, Granata Nicoletta, Graziano Gabriella, Marini Marco, Paino Anna Maria, Palvarini Michela, Paolini Carla, Sarzani Riccardo, Tramontana Luca, Versace Antonio Giovanni, and Villani Alessandra. References 1. 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Living with Chronic Spontaneous Urticaria in Italy: A Narrative Medicine Project to Improve the Pathway of Patient Care. Acta Derm Venereol. 2017;97(1):81-5. doi:10.2340/00015555-2478. 18. References De Vincentis G, Monari F, Baldari S, Salgarello M, Frantellizzi V, Salvi E et al. Narrative medicine in metastatic prostate cancer reveals ways to improve patient awareness & quality of care. Future Oncol. 2018;14(27):2821-32. doi:10.2217/fon-2018-0318. 18. De Vincentis G, Monari F, Baldari S, Salgarello M, Frantellizzi V, Salvi E et al. Narrative medicine in metastatic prostate cancer reveals ways to improve patient awareness & quality of care. Future Oncol. 2018;14(27):2821-32. doi:10.2217/fon-2018-0318. 19. Gatti V, Banfi P, Centanni S, D'Antonio S, Giustini S, Piraino A et al. Enlightening chronic obstructive pulmonary disease through patients' and caregivers' narratives. Int J Chron Obstruct Pulmon Dis. 2018;13:3095-105. doi:10.2147/COPD.S172214. 20. 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Istat - National Statistical Institute, Rome. 2019. http://dati.istat.it/Index.aspx?DataSetCode=DCIS_INDDEMOG1. 27. Grigorovich A, Lee A, Ross H, Woodend AK, Forde S, Cameron JI. A longitudinal view of factors that influence the emotional well-being of family caregivers to individuals with heart failure. Aging Ment Page 16/22 Page 16/22 Health. 2017;21(8):844-50. doi:10.1080/13607863.2016.1168361. 28. Gusdal AK, Josefsson K, Adolfsson ET, Martin L. Informal Caregivers' Experiences and Needs When Caring for a Relative With Heart Failure: An Interview Study. J Cardiovasc Nurs. 2016;31(4):E1-8. doi:10.1097/JCN.0000000000000210. 29. Cappuccio A, Sanduzzi Zamparelli A, Verga M, Nardini S, Policreti A, Porpiglia PA et al. Narrative medicine educational project to improve the care of patients with chronic obstructive pulmonary disease. ERJ Open Res. 2018;4(2). doi:10.1183/23120541.00155-2017. 30. Della Pelle C, Orsatti V, Cipollone F, Cicolini G. Health literacy among caregivers of patients with heart failure: A multicentre cross-sectional survey. J Clin Nurs. 2018;27(3-4):859-65. doi:10.1111/jocn.14137. 31. Stork S, Kavoliuniene A, Vinereanu D, Ludwig R, Seferovic P, Dickstein K et al. What does the lay public know about heart failure? Findings from the Heart Failure Awareness Day Initiative. Eur J Heart Fail. 2016;18(1):66-70. doi:10.1002/ejhf.425. 32. Selan S, Siennicki-Lantz A, Berglund J, Fagerstrom C. Self-awareness of heart failure in the oldest old-an observational study of participants, >/= 80 years old, with an objectively verified heart failure. BMC Geriatr. 2016;16:23. doi:10.1186/s12877-016-0195-4. 32. Selan S, Siennicki-Lantz A, Berglund J, Fagerstrom C. Self-awareness of heart failure in the oldest old-an observational study of participants, >/= 80 years old, with an objectively verified heart failure. BMC Geriatr. 2016;16:23. doi:10.1186/s12877-016-0195-4. 33. Volpe M. Tales in Cardiology: A revival for narrative medicine is taking place in Rome. Eur Heart J. 2019;40(10):800-2. doi:doi.org/10.1093/eurheartj/ehz064. 33. Volpe M. Tales in Cardiology: A revival for narrative medicine is taking place in Rome. Eur Heart J. 2019;40(10):800-2. doi:doi.org/10.1093/eurheartj/ehz064. Figures Page 17/22 Figure 1 The emotional impact of HF reported by patients, informal caregivers, and HF specialists: a comparison of the emotions felt at diagnosis (via recall) versus those felt at the time of providing their narrative. Data are reported as proportion of patients/caregivers/HF specialists. HF heart failure Figure 1 The emotional impact of HF reported by patients, informal caregivers, and HF specialists: a comparison of the emotions felt at diagnosis (via recall) versus those felt at the time of providing their narrative. Data are reported as proportion of patients/caregivers/HF specialists. HF heart failure Page 18/22 Page 18/22 Figure 2 Language analysis of participants’ metaphors to describe HF. Data are reported as proportion of patients/caregivers/HF specialists. HF heart failure Figure 2 Language analysis of participants’ metaphors to describe HF Data are reported as proportion of Figure 2 Language analysis of participants’ metaphors to describe HF. Data are reported as proportion of patients/caregivers/HF specialists. HF heart failure Language analysis of participants’ metaphors to describe HF. Data are reported as proportion of patients/caregivers/HF specialists. HF heart failure Page 19/22 Page 19/22 Figure 3 Awareness of the disease symptoms as described in patients’ and caregivers’ narratives. Figure 3 Figure 4 Positive relationships involving patients, caregivers, and HF specialists from the patients’, caregivers’, and physicians’ perspectives, at the beginning of the narrative (light blue) and the incremental increase due to evolved relationships at the end of the narrative (blue) Figure 4 Figure 4 Figure 3 Awareness of the disease symptoms as described in patients’ and caregivers’ narratives. Awareness of the disease symptoms as described in patients’ and caregivers’ narratives. Page 20/22 Page 21/22 Figure 4 Positive relationships involving patients, caregivers, and HF specialists from the patients’, caregivers’, and physicians’ perspectives, at the beginning of the narrative (light blue) and the incremental increase due to evolved relationships at the end of the narrative (blue) Supplementary Files Supplementary Files Page 21/22 This is a list of supplementary files associated with this preprint. Click to download. trustcoreq.pdf trustcoreq.pdf Marcoetal.Appendix.docx Marcoetal.Appendix.docx Page 22/22
https://openalex.org/W4389409424	http://www.thieme-connect.de/products/ejournals/pdf/10.1055/a-2223-5458.pdf	English	null	Altmetric Analysis of Artificial Intelligence Articles in Plastic Surgery	Archives of plastic surgery	2,023	cc-by	1,241	Altmetric Analysis of Artiﬁcial Intelligence Articles in Plastic Surgery gdanovich, BS1 Pearl Shah, BS, MBA1 Parth A. Patel, MD1 Tommy Bui, BS1 Brennan Bogdanovich, BS1 Pearl Shah, BS, MBA1 Parth A. Patel, MD1 Tomm Carter J. Boyd, MBA, MD2 Address for correspondence Carter J. Boyd, MBA, MD, Hansjörg Wyss Department of Plastic Surgery, New York University Langone Health, 305 East 47th Street, Suite 1A, New York, NY 10017 (e-mail: Carterjosephboyd@gmail.com). Address for correspondence Carter J. Boyd, MBA, MD, Hansjörg Wyss Department of Plastic Surgery, New York University Langone Health, 305 East 47th Street, Suite 1A, New York, NY 10017 (e-mail: Carterjosephboyd@gmail.com). 1Medical College of Georgia, Augusta University, Augusta, Georgia 2Hansjörg Wyss Department of Plastic Surgery, NYU Langone Health, New York, New York Arch Plast Surg 2024;51:262–264. Arch Plast Surg 2024;51:262–264. Arch Plast Surg 2024;51:262–264. Artiﬁcial intelligence (AI) is becoming increasingly relevant and integrated into the medical space. Current adoption in AI is distinct from prior attempts, as computer processing power, larger data storage libraries, and current AI workforce talent outweigh previous capabilities. These advances have enabled AI-based systems to ﬂourish within health care. In a report produced by Accenture, it is estimated that AI, by 2026, has the potential to save the health care industry over $150 billion annually.1 Plastic surgery speciﬁcally could leverage AI to optimize patient care. lation coefﬁcient, Mann–Whitney U test, Kruskal–Wallis test, and Fisher’s exact test, where appropriate. p < 0.05 was considered statistically signiﬁcant. The mean AAS of the 50 most disseminated articles online was 11.3 19.2, primarily driven by mentions on Twitter (12.2 16.5). No correlation was identiﬁed between AAS and citation count (r ¼ 0.13; p ¼ 0.38). No articles were published prior to 2014, with 68% published between 2020 and 2022 (►Supplementary Table S1, available in the online version only). Forty-two percent of articles were open access, a similar proportion relative to the 50 most cited articles (44%; p > 0.99). Plastic and Reconstructive Surgery was the most common journal of publication for the 50 most dis- seminated articles online (26%) and articles published in this journal accrued greater AAS relative to other journals (p ¼ 0.04). A majority of articles (64%) were multi-institu- tional collaborations and 34% were multinational collabo- rations (►Supplementary Table S2, available in the online version only). The most common subspecialty of social interest was general/burn (28%; ►Supplementary Table S3, available in the online version only). Accepted Manuscript online: 2023-12-06 Article published online: 2024-01-29 Accepted Manuscript online: 2023-12-06 Article published online: 2024-01-29 Communication 262 © 2024. 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 Publishers, Inc., 333 Seventh Avenue, 18th Floor, New York, NY 10001, USA DOI https://doi.org/ 10.1055/a-2223-5458. eISSN 2234-6171. Altmetric Analysis of Artiﬁcial Intelligence Articles in Plastic Surgery First authors were predominantly male (84%) and from the United States (54%). Given this rapid expansion of AI in the literature, it is necessary to identify the most salient articles in the ﬁeld. Traditionally, bibliometric analyses have enabled identiﬁca- tion of seminal articles through quantiﬁcation of citation count. However, citation count, while useful, fails to account for social dissemination. We provide an alternative perspec- tive from traditional bibliographic analysis by using Altmet- ric Attention Scores (AAS) to determine the online social inﬂuence of AI-related plastic surgery articles. Intended to be complementary to traditional, citation-based metrics, AAS reﬂects the digital attention a research article is garnering across multiple sources, including, but not limited toTwitter, news outlets, Facebook, Google þ , LinkedIn, Reddit, etc.2 As noted by Elmore in 2019, AAS presents with certain limitations, including but not limited to an absence in the ability to analyze the quality of an article and difﬁculty with ﬁeld normalization. Additionally, the Altmetric algorithm uses sites like Facebook, Reddit, and Twitter, but does not use TikTok or Instagram. It is also important to note that AAS is not related to the scientiﬁc importance of an article but rather the social inﬂuence. However, analyzing the social inﬂuence acts as an important complementary measure to traditional, citation-based metrics.2 The Web of Science database was searched with a combi- nation of Boolean operators and descriptive terms to identify articles relevant to AI and plastic surgery. Articles were manually examined to ensure relevance to the present analysis. In total, 285 articles were identiﬁed from the database search and 266 were eligible for screening after removing duplicates. After eliminating irrelevant articles, 141 articles remained. AAS, which measures the social dissemination of an article, was determined using Altmetric Explorer. Articles were ranked by citation count and AAS, and their characteristics were analyzed using the Pearson corre- The collaborative effort of plastic surgeons in AI-related research was a notable ﬁnding of our analysis. Most of the DOI https://doi.org/ 10.1055/a-2223-5458. eISSN 2234-6171. received May 11, 2023 accepted after revision November 30, 2023 accepted manuscript online Decembe 6, 2023 article published online January 29, 2024 Altmetric Analysis of Artificial Intelligence Articles Bogdanovich et al. 263 Writing original draft: B.B., P.S., P.A.P., and C.B. Writing review edit: B.B., P.S., P.A.P., and C.B. plastic surgery articles analyzed were multi-institutional and greater than one-third were multinational. Conﬂict of Interest None declared. Conﬂict of Interest None declared. Funding None. There is limited literature available on this topic. Our analy- sis of plastic surgery-related AI papers revealed 266 articles within the Web of Science database, with 141 articles ultimate- ly analyzed. Conversely, in a recent paper that analyzed AAS of AI in the ophthalmology literature, the authors identiﬁed 2,927 total articles.5 Although plastic surgery is a ﬁeld that prides itself on innovation, approximately one-tenth the number of articles regarding AI in plastic surgery was initially identiﬁed relative to ophthalmology. We hypothesize that the lack of available literature is due to the novelty of the subject and its potentially unclear role within the ﬁeld of plastic surgery. As such, lest plastic surgeons fall behind, it is paramount for the ﬁeld to discover novel means of integrating and leveraging AI within the specialty, either in clinical or business operations. Altmetric Analysis of Artiﬁcial Intelligence Articles in Plastic Surgery Moreover, while female authorship percentages in plastic surgery have been increasing according to Silvestre et al,3 only 16% of articles published in the plastic surgery/AI space cross- section were female. Interestingly, 22% of employees in the AI workspace are female, closely mirroring our ﬁndings and pointing to a potential coexisting male bias in the ﬁeld of AI.4 Ethical Approval No IRB approval required for this study. Archives of Plastic Surgery Vol. 51 No. 2/2024 © 2024. The Author(s). References 1 CollierM Artiﬁcialintelligenceinhealthcare. Accenture, Accessedon 10 January, 2023, at: https://www.accenture.com/au-en/insights/ health/artiﬁcial-intelligence-healthcare 1 CollierM Artiﬁcialintelligenceinhealthcare. Accenture, Accessedon 10 January, 2023, at: https://www.accenture.com/au-en/insights/ health/artiﬁcial-intelligence-healthcare 1 CollierM Artiﬁcialintelligenceinhealthcare. Accenture, Accessedon 10 January, 2023, at: https://www.accenture.com/au-en/insights/ health/artiﬁcial-intelligence-healthcare 2 Elmore SA. The Altmetric Attention Score: what does it mean and why should i care? Toxicol Pathol 2018;46(03):252–255 3 Silvestre J, Wu LC, Lin IC, Serletti JM. Gender authorship trends of plastic surgery research in the United States. Plast Reconstr Surg 2016;138(01):136e–142e 4 World Economic Forum The Global Gender Gap Report 2018 Insight Report. 2018. Accessed on 13 January, 2023, at: https:// www3.weforum.org/docs/WEF_GGGR_2018.pdf Authors’ Contributions Conceptualization: All authors. Methodology: All authors. 5 Bui T, Patel PA, Boyd CJ. Altmetric Analysis of the Online Attention Directed to Artiﬁcial Intelligence Literature in Ophthalmology. Asia Pac J Ophthalmol (Phila) 2023;12(06):625–626
https://openalex.org/W3115551718	https://zenodo.org/record/4392501/files/123-128.pdf	English	null	Decision Analytic Pricing with Constant Price Elasticities	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	2,021	Abstract This article introduces a new pricing model which uses decision analysis and isoelastic demand functions. Using the methodologies discussed here will enable companies to choose prices that will maximize the profit of a given product based on the state of the economy. An exponential utility function is used to incorporate the risk attitude of the company. The use of the model is demonstrated through a case study on 2018 Chevrolet Malibus. The proper use of the model will “take the guesswork out” of the pricing decision and help companies make pricing decisions using the best available data. Keywords: Pricing, Decision Analysis, Elasticity, Utility. International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 http://ijbe.ielas.org/index.php/ijbe/index ISSN (online) 2545-4137 Decision Analytic Pricing with Constant Price Elasticities Kenneth Ko Graziadio Business School Pepperdine University 6100 Center Drive, Fourth Floor Los Angeles, CA 90045 United States of America E-mail: kenneth.ko@pepperdine.edu Kenneth Ko Graziadio Business School Pepperdine University 6100 Center Drive, Fourth Floor Los Angeles, CA 90045 United States of America E-mail: kenneth.ko@pepperdine.edu Accepted: 2020-12-14 Published online: 2020-12-24 Accepted: 2020-12-14 Published online: 2020-12-24 Accepted: 2020-12-14 Published online: 2020-12-24 Received: 2020-11-13 Published online: 2020-12-24 8 https://www.bdc.ca/en/articles-tools/marketing-sales-export/marketing/pages/pricing-5-common-strategies.aspx 1. Introduction Pricing is an important strategic decision for every company for every product that it sells. Through pricing, a company can maximize its profit for a given product. Of course, it is not easy to determine what the optimal price should be. Certainly, there are many different approaches to pricing including: cost-plus pricing, competitive pricing, value-based pricing, price skimming, and penetration pricing8 123 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 http://ijbe.ielas.org/index.php/ijbe/index ISSN (online) 2545-4137 Decision analysis is a proven methodology used to make decisions through taking into account alternatives, values (e.g., maximizing profit), and uncertainty (states of nature). Decision analysis has proved quite effective in helping companies to make business decision regarding pricing (Khouja and Robbins, 2005; Cobb, 2009). In order to use decision analysis to make pricing decisions, knowledge the demand function of the product is required. Isoelastic demand functions, which have constant price elasticities, can be used effectively. Furthermore, pricing decisions with the use of constant price elasticities has been examined (McAfee and Veldee, 2008; Helmes and Schlosser, 2013). The focus of this paper is to introduce a model that uses decision analysis in conjunction with isoelastic demand functions to make pricing decisions. The model is applied to 2018 Chevrolet Malibus to illustrate its use. 2. Model The basis of decision analysis is to choose the best alternative which takes into account uncertainty to maximize a given objective. When it comes to pricing decisions, the clear decision is to pick the best price that will maximize profit. For modeling purposes, there are three prices: low, medium, and high. Many different uncertainties could be considered, but the focus will be on the state of the economy as this is perhaps the biggest single factor in determining the overall demand of a product. Simply put, when the economy is strong, people generally have more disposable income and will purchase more, which increases the demand for products. Conversely, when the economy is weak, people generally have less disposable income and will purchase less, which decreases the demand for products. For the purposes of our model, three possibilities for the economy will be considered: a declining economy, a flat economy, and a growing economy. Some assumptions about how specifically the economy will affect product demand are needed. The optimal decision will be the price that maximizes profit taking all of the information into account. A decision tree can be used to not only model the entire situation but also determine the optimal decision. Information about the product, including price information, cost information, and the price elasticity is needed. As mentioned earlier, isoelastic demand functions are used, and they are fully determined once the elasticity is found. Using the above approach will enable companies to make optimal pricing decisions based on the available information. In order to choose the optimal price, not only the decision which maximizes the expected value of profit but also the decision which maximizes expected utility should be considered. In order to determine utilities, the exponential utility function is used as 124 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 http://ijbe.ielas.org/index.php/ijbe/index ISSN (online) 2545-4137 it has been shown to accurately model risk averse utility functions: U(x) = 1 – e-x/R where R is the risk tolerance. Thankfully, there is a good way of determining the risk tolerances of corporations (Howard, 1988): 1. 6.4% of total sales 2. 1.24 times net income 3. 15.7% of equity. The risk tolerance of a corporation can be determined through taking an average of these three measures. 3. Case Study: 2018 Chevrolet Malibu The use of a model will be illustrated which incorporates decision analysis and price elasticities through a case study involving the Chevrolet Malibu. In 2018, 144,542 Chevrolet Malibus were sold in the United States. Using this as the baseline, a decision analysis model was developed. Please see the expected value decision tree in Figure 1. Figure 1. Expected Value Decision Tree Figure 1. Expected Value Decision Tree 125 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 http://ijbe.ielas.org/index.php/ijbe/index ISSN (online) 2545-4137 As can be seen from Figure 1, the decision for Chevrolet is to charge a low, medium, or high price. The base model 2018 Chevrolet Malibu L had an MSRP of $22,555. The highest model 2018 Chevrolet Malibu Premier had an MSRP of $31,850. For the medium price, the average of these two prices is used, which is $27,202.50 or approximately $27,200. This price is used as the medium price. For the high price and low price, 1% above and 1% below the medium price are used, respectively. So, this means that the low price is $26,928, and the high price is $27,472. From the 2018 General Motors annual report, the net sales and revenue from automobiles is $133,045,000,000 and that cost and expenses for automobiles and other cost of sales is $120,656,000,000. So, this leads to a profit margin per automobile of 10.27%, and to an average cost of $24,670 per 2018 Chevrolet Malibu. An isoelastic demand function is used: Q=AP-e where Q is the quantity, A is a constant, P is the price and e is the price elasticity of demand. The price elasticity for Chevrolet automobiles was found to be 4.0 (Gwartney and Stroup 1997). So, given that 144,542 Chevrolet Malibus were sold at the $27,202.50 price point, it is easy to find that A=7.911022. So, the demand function for Chevrolet Malibus is Q=(7.911022)P-4. Furthermore, economic conditions are taken into account. A flat economy is assigned a probability of 50%. A growing economy id given a probability of 25%, and the sales are estimated to be 10% more than in the average economy. A declining economy is given a probability of 25%, and the sales are estimated to be 10% less than in the average economy. 3. Case Study: 2018 Chevrolet Malibu Given all of the above information, all the data is incorporated into the decision tree in Figure 1. From Figure 1, Chevrolet’s base case profit for the Malibu is estimated to be approximately $366,000,000 in 2018. However, our analysis shows that given that the price elasticity demand is 4, that Chevrolet could have generated an optimal profit of $389,000,000 had they charged a 1% higher price. Next General Motors’ risk attitude needs to be taken into account in order to maximize its expected utility for this decision. Please see Table 1 for the calculation of its Risk Tolerance. Table 1. General Motors Risk Tolerance Table 1. General Motors Risk Tolerance Financial Measure Multiplier Amount Risk Tolerance Total Sales 0.064 $147,049,000,000 $9,411,136,000 Net Income 1.24 $8,075,000,000 $10,013,000,000 Equity 0.157 $42,777,000,000 $6,715,989,000 Average $8,713,375,000 126 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 http://ijbe.ielas.org/index.php/ijbe/index ISSN (online) 2545-4137 General Motors’ utility function is now obtained because the average Risk Tolerance as calculated in Table 1 of $8,713,375,000 can be inserted it into the exponential utility function described above. Taking into account the decisions, uncertainties, and utilities, the expected utility decision tree in Figure 2 can be used to determine General Motors optimal decision. As it turns out, the optimal decision given General Motors Risk Attitude is still to choose the highest price as this gives the highest expected utility – 0.043678 – and certainty equivalent (the certain amount of money that would have the same utility for General Motors given its risk attitude) - $389,160,001. $389,160,001. Figure 2. Expected Utility Decision Tree Doing sensitivity analysis, the price elasticity of demand would have had to gone up to 10.27 before charging the average price of $27,200 would be optimal. (At a price elasticity of 11.32, the low price becomes optimal.) Please see Table 2. Given that an elasticity of 10.27 is well above the actual elasticity of 4.0, it is obvious that Chevrolet should have charged a higher price for the Chevrolet Malibu in 2018. So, this model can be used to not only determine the optimal decision but also do sensitivity analysis. Figure 2. Expected Utility Decision Tree Figure 2. 3. Case Study: 2018 Chevrolet Malibu Expected Utility Decision Tree Doing sensitivity analysis, the price elasticity of demand would have had to gone up to 10.27 before charging the average price of $27,200 would be optimal. (At a price elasticity of 11.32, the low price becomes optimal.) Please see Table 2. Given that an elasticity of 10.27 is well above the actual elasticity of 4.0, it is obvious that Chevrolet should have charged a higher price for the Chevrolet Malibu in 2018. So, this model can be used to not only determine the optimal decision but also do sensitivity analysis. 127 International Journal of Business and Economics Vol. 5, No. 2, 2020, pp. 123-128 http://ijbe.ielas.org/index.php/ijbe/index ISSN (online) 2545-4137 Table 2. Price Elasticity Sensitivity Analysis Price Elasticity Pricing Decision e <= 10.26 $27,472 – high price 10.27 <= e <= 11.31 $27,200 – medium price e >= 11.32 $26,928 – low price Table 2. Price Elasticity Sensitivity Analysis 4. Conclusion All companies would like to make the best decisions possible. In this paper, the focus was on using decision analysis to make optimal pricing decisions. Isoelastic demand functions model well the demand for products. So, incorporating decision analysis with the use of isoelastic demand functions provides a rigorous analytic approach which forms the basis of good pricing decision making. In this model, an isoelastic demand function is input into an exponential utility function. The risk tolerance can be computed easily using some key financial measures. The use of the model was shown through a case study involving 2018 Chevrolet Malibus. The model demonstrated that General Motors would have generated an additional $23,507,110 ($389,160,001– $365,652,891) of profit on a certainty equivalent basis for the 2018 Chevrolet Malibu if it would have increased the price by 1%. Furthermore, sensitivity analysis revealed that this optimal price is robust. More generally, the effective use of the modeling techniques described above will enable companies to choose the optimal price that will maximize profit for a given product. References Cobb, B., (2009). Influence Diagrams for Capacity Planning and Pricing under Uncertainty. Journal of Management Accounting Research, 21, 75-97. https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.613.5140&rep=rep1&ty pe=pdf Helmes, K.L., and Schlosser R. (2013). Dynamic Advertising and Pricing with Constant Demand Elasticites.Journal of Economics Dynamics & Control, 37(12), 2814-2832. Gwartney, J.D., and Stroup R. L. (1997). Economics: Private and Public Choice, eighth edition, Cengage Learning. Howard, R. A. (1988). Decision Analysis: Practice and Promise.Management Science, 34(6), 679-695. //pdfs.semanticscholar.org/7921/7d44239aacdb4b3a8460679a858404699 https://pdfs.semanticscholar.org/7921/7d44239aacdb4b3a8460679a85840469901d.pdf Khouja, M., and Robbins S. S.(2005). Optimal Pricing and Quantity of Products with Two Offerings. European Journal of Operations Research, 163(2), 530-544. g McAfee, R.P., and Velde,V. t. (2008). Dynamic pricing with Constant Demand Elasticity.Production & Operations Management, 17(4), 432-438. 128
https://openalex.org/W4313229771	https://ejournal.yasin-alsys.org/index.php/arzusin/article/download/107/85	Indonesian	null	Meningkatkan Prestasi Belajar Peserta Didik melalui Manajemen Sarana dan Prasarana di Sekolah	Arzusin	2,021	cc-by	2,413	MENINGKATKAN PRESTASI BELAJAR PESERTA DIDIK MELALUI MANAJEMEN SARANA DAN PRASARANA DI SEKOLAH Rudi Herianto1, Fitriyani Sanuhung2, Muhammad Farid Wajdi3 Universitas Ahmad Dahlan yogyakarta fitriyani1900031128@webmail.uad.ac.id A R Z U S I N Jurnal Manajemen dan Pendidikan Dasar https://doi.org/10.58578/arzusin.v1i1.107 A R Z U S I N Jurnal Manajemen dan Pendidikan Dasar e-ISSN : 2809-4093 p-ISSN : 2809-4484 Terindeks: Crossref, Dimensions, Scilit, Garuda, Google Scholar, Moraref, Base, OneSearch, etc. A R Z U S I N Jurnal Manajemen dan Pendidikan Dasar e-ISSN : 2809-4093 p-ISSN : 2809-4484 Terindeks: Crossref, Dimensions, Scilit, Garuda, Google Scholar, Moraref, Base, OneSearch, etc. Abstract This research was conducted to determine the effect of facilities and infrastructure in improving student achievement and how the invisible relationship between the management of facilities and infrastructure with student achievement. Facilities and infrastructure are one of the supporting factors so that students' academic and non-academic achievements in schools can increase, through good management of facilities and infrastructure that can meet the needs of students, teachers, staff, and school employees. Student achievement can increase if the management of facilities and infrastructure can be carried out effectively and efficiently. Facilities and infrastructure are not only carried out by maintaining them but facilities must be managed through planning, procurement, management, maintenance. Keywords : Management of Facilities and Infrastructure, Student Achievement Abstrak : Penelitian ini dilakukan untuk mengetahui pengaruh sarana dan prasarana dalam peningkatan prestasi siswa serta bagaimana hubungan tidak terlihat antara manajemen sarana dan prasarana dengan prestasi siswa . Sarana dan prasana merupakan Salah satu faktor yang menjadi penunjang agar prestasi akademik maupun non akademik siswa disekolah bisa meningkat, melalui pengelolaan sarana dan prasarana yang baik dapat memenuhi kebutuhan peserta didik, guru, staf, dan karyawan disekolah. Prestasi peserta didik dapat meningkat jika pengelolaan sarana dan prasana dapat dilakukan dengan efektif dan efisien. Sarana dan prasarana tidak hanya dilakukan dengan memeliharanya tetapi sarana harus di kelola dengan melalui perencanaan, pengadaan, pengelolaan, pemeliharaan. Kata Kunci : Manajemen Sarana dan Prasarana, Prestasi Siswa Volume 1, Nomor 1, Desember 2021; 56-63 https://ejournal.yasin-alsys.org/index.php/arzusin Jurnal Arzusin is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi Volume 1, Nomor 1, Desember 2021 PENDAHULUAN Pendidikan adalah salah satu investasi yang baik serta mempunyai peran yang strategis sebagai wadah untuk menciptakan kualitas SDM. Semakin jelas pendidikan suatu bangsa maka perkembangan dan kemajuan bangsa tersebut semakin terlihat. Pendidikan juga menjadi sentral untuk mengambangkan SDM masyarakat. Peningkatan mutu, relevansi, serta efisiensi pendidikan harus mampu dijamin oleh sistem pendidikan dan majamen pendidikan.(Manurung et al., 2020). Pendidikan yaitu satu faktor yang sangat penting bagi masyarakat. Setiap orang berhak duduk di bangku pendidikan dengan kualitas yang baik. Dengan pendidikan seseorang bisa melihat dan mengetahui apa yang belum pernah diketahuinya. Pendidikan juga bisa menjadi pandangan sosial dilingkungan masyarakat. Generasi muda merupakan masa depan untukbangsa ini, dan masa depan anak muda ada pada pendidikan bangsa ini. Pelaksanaan pendidikan dilakukan oleh beberapa lembaga diantaranya adalah lembaga pendidikan formal, informail, dan non formal. Lembaga pendidikan merupakan tempat dilaksanakannya proses pendidikan berlangsung seperti sekolah, madrasah, bimpel, dan lain sebagainya. (Ike Malaya Sinta, 2019) Melaksanakan pendidikan tentunya tidak mudah, ada proses yang harus dilaksanakan serta ada aturan yang harus di taati. Proses pendidikan dilaksanakan untuk mewujudkan tujuan pendidikan tersebut dengan efektif. Agar mewujudkan tujuan dari pendidikan tersebut dapat dilakukan dengan memperhatikan hal-hal yang mendukung dan mendorong tujuan pendidikan tersebut dapat berhasil. Salah satu faktor yang mendukung berhasilnya tujuan pendidikan yaitu pelaksanaan pembelajaran. Norma-norma dan nilai-nilai budaya ada pada proses pembelajaran oleh sebab itu proses pembelajaran sangat penting sebagai pewarisan nilai budaya. Oleh karena itu sangat baik apabila pada kegiatan belajar mengajar membangun suasana yang efektif dan efisien sehingga para siswa bisa menerima pelajaran dengan baik dan tidak merasa jenuh. (Nurmaidah, 2018) Proses pembelajaran berlangsung dengan baik dan efektif karena faktor baiknya fasilitas di lembaga pendidikan. Pendidikan yang baik juga di latar belakangi oleh pengelolaan sarana prasarana yang baik juga (Pahlevi et al., 2016). Efektif atau tidaknya proses pembelajaran dapat di lihat dari manajemen sarana prasarana suatu Volume 1, Nomor 1, Desember 2021 57 57 Volume 1, Nomor 1, Desember 2021 Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi sekolah. Sehingga pemanfaatan dan pengelolaan sarana dan prasarana harus dilakukan dengan efektif serta efisien untuk mencapai arah dan tujuan dari pendidikan. (Ike Malaya Sinta, 2019) Manajemen sarana prasarana mengatur seluruh fasilitas pembelajaran dimulai dari perencanaan, pengadaan sampai pada tahap pengelolaan. Tujuan dari manajemen sarana prasarana yaitu untuk melakukan yang terbaik pada kegiatan pengelolaan tersebut agar fasilitas yang ada dapat digunakan dengan efektif (Herawati et al., 2020). METODE Artikel ini ditulis dengan menggunakan metode kualitatif serta pendekatan deskriptif. Penulis menggunakan data berupa jurnal, buku serta internet (menggunakan data dari tangan kedua). Sebagaimana diungkapkan oleh sugiyono jika penelitian yang menggunakan objek secara alamiah untuk meneliti objek. Peran kunci ada pada peneliti. Data dikumpulkan dengan melakukan analisis data dengan sifat induktif serta hasil dari penelitian yang berfokus pada makna, dan triangulasi, .(Anjassari et al., n.d.) PENDAHULUAN Proses kerjasama yang dilakukan oleh sekelompok orang supaya mengatur serta menggunakan fasilitas pendidikan dengan baik dan efektif disebut juga Manajemen sarana dan prasarana.(Nurmaidah, 2018) Prestasi siswa merupakan hasil yang diproleh oleh peserta didik selama melakukan pembelajaran. Prestasi siswa bisa dilihat dari berbagai bidang. Pada umumnya prestasi siswa terbagi atas dua bagian yaitu prestasi secara akademik dan prestasi non akademik. Peningkatan prestasi siswa dapat dilaksanakan melalui beberapa faktor diantaranya motivasi diri, dukungan orang tua, manajemen pendidikan seperti manajemen sarana dan prasarana. Peserta didik bisa menjadikan prestasi yang diperolehnya sebagai motivasi untuk mendapatkan prestasi-prestasi lainnya. Manajemen sarana dan prasaran yang baik sangat menunjang prestasi belajar siswa. Jika fasiltas suatu lembaga pendidikan sangat memadai sehingga pembelajaran di dalam kelas dapat berlangsung dengan efektif serta pesera didik mampu mendapat materi pelajaran dengan baik pula, sebab itu prestasi siswa juga dapat dilihat dari fasilitas yang dimiliki oleh lembaga pendidikan. Namun masih banyak sekolah dan lembaga pendidikan lain yang fasilitasnya belum memadai bahkan masih banyak sekolah-sekolah yang ada di indonesia yang manajemen sarana dan prasarana masih kurang. Ini merupakan hal yang harus dilihat dan dapat diselesaikan oleh pemerintah. Hubungan tidak terlihat yang ada dalam manajemen sarana prasarana dan prestasi siswa menjadi tujuan dari penulisan artikel ini. Penulis menulis artikel ini untuk membahas faktor yang menjadi penunjang dalam meningkatkan prestasi siswa yaitu Manajemen sarana prasarana. ARZUSIN : Jurnal Manajemen dan Pendidikan Dasar 58 58 ARZUSIN : Jurnal Manajemen dan Pendidikan Dasar ARZUSIN : Jurnal Manajemen dan Pendidikan Dasar Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi Volume 1, Nomor 1, Desember 2021 A. Pengertian Manajemen, Sarana dan Prasarana A. Pengertian Manajemen, Sarana dan Prasarana Proses yang dilakukan oleh sekelompok orang guna mengolah sumebr daya yang ada dengan baik dan efektif supaya mewujudkan tujuan yang sudah ditentukan tersebut Manajemen.(Nurmaidah, 2018) Sarana yaitu fasilitas yang digunakan secara langsung pada proses pembelajaran, seperti meja, bangku, gedung sekolah dan lainnya. Sedangkan prasana merupakan peralatan atau perangkat yang digunakan dalam proses pendidikan secara tidak langsung seperti taman bunga, lapangan, pagar dan lainnya.(Supiana et al., 2019) Manajemen sarana dan prasarana adalah proses yang dilakuakn guna memanfaatkan sumberdaya pada lembaga pendidikan dengan efektif dan efisisen sehingga dapat bermanfaat untuk warga sekolah. Dalam melakukan manajemen pendidikan terdapat beberapa fase yang harus dilakukan yaitu, perencanaan, pengadaan, dan pengelolaan(Ananda & Banurea, 2017) a. Perencanaan sarana dan prasarana a. Perencanaan sarana dan prasarana Langkah yang dilaksanakan untuk menetapkan kebutuhan sarana prasarana sesuai dengan kondisi sekolah merupakan perencanaan sarana prasarana. Dalam melakukan perencanaan ini sekolah biasanya akan melakukan rapat untuk menetapkan sarana prasarana yang akan dibutuhkan. Perencanaan sarana dan prasarana yang dilakukan disekolah biasanya mencakup beberapa prosedur diantaranya: Langkah yang dilaksanakan untuk menetapkan kebutuhan sarana prasarana sesuai dengan kondisi sekolah merupakan perencanaan sarana prasarana. Dalam melakukan perencanaan ini sekolah biasanya akan melakukan rapat untuk menetapkan sarana prasarana yang akan dibutuhkan. Perencanaan sarana dan prasarana yang dilakukan disekolah biasanya mencakup beberapa prosedur diantaranya: 1. Guru, staf, kepala sekolah, serta karyawan mengadakan rapat 59 Volume 1, Nomor 1, Desember 2021 59 Volume 1, Nomor 1, Desember 2021 Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi 2. Membuat susunan kebutuhan sarana prasarana pada proses pendidikan 3. Mengevaluasi fasilitas yang paling diperlukan berdasarkan dana yang tersedia 4. Melakukan penetapan untuk pengadaan sarana prasarana (Parid & Alif, 2020) b. Pengadaan sarana dan prasarana b. Pengadaan sarana dan prasarana Pengadaan sarana prasarana adalah langkah yang dilakukan agar tersedia fasilitas sesuai yang diperlukan sekolah. Pengadaan sarana dan prasarana dapat dilaksanakan melalui beberapa cara yaitu dengan membeli produk yang dibutuhkan, membuat produk itu sendiri, serta menyewa sarana dan prasarana dari tempat lain. c. Penyimpanan sarana dan prasarana pendidikan Penyimpanan sarana prasana pendidikan bisa dilakukan melalui beberapa kegiatan yaitu penerimaan barang, penyimpanan barang, serta menyalurkan barang tersebut. Penyimpanan dilakukan untuk menjaga keamanan barang- barang tersebut d. Penyaluran sarana dan prasarana Penyaluran sarana pendidikan dilakukan dengan membagi atau menyalurkan barang berdasarkan atas keperluan guru dan siswa pada kegiatan pendidikan. Penyaluran sarana dan prasarana ini dilakukan atas persetujuan dari kepala sekolah. e. Pemeliharaan sarana dan prasarana Melakukan pemeliharaan sarana prasarana harus setiap saat, semua warga sekolah dari kepala sekolah sampai ke para siswa wajib memlihara fasilitas yang diberikan. Pemeliharaan sarana prasarana memiliki peran yang sangat penting pada pengelolaan sarana prasarana. f. Rehabilitasi sarana dan prasarana Melakukan rehabilitas sarana prasarana artinya memperbaiki sarana prasarana yang rusak dengan cara ditambal atau membeli alat suku cadang yang rusak sehingga barang tersebut dapat digunakan kembali. (Megasari, 2014) ARZUSIN : Jurnal Manajemen dan Pendidikan Dasar 60 Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi Pengelolaan sarana prasarana berperan paling penting pada kegiatan pendidikan, karena sarana prasarana adalah salah satu faktor yang menunjang keberhasilan serta kesuksesan aktifitas dalam pendidikan (Hartoni, Amirudin, 2018) Sarana dan prasarana yang dikelola dengan baik merupakan kunci untuk mengetahui fasilitas tersebut memberikan manfaat atau tidak. Sehingga itu pengelolaan saran dan prasarana harus dilakukan dengan baik dan efektif agar fungsi dan manfaat barang tersebut dapat tersalurkan. (Anjassari et al., n.d.) B. Manajemen sarana Prasarana dan Prestasi Siswa Pengelolaan sarana dan prasarana menjadi suatu penunjang dari berhasilnya aktifitas pada pendidikan. Terpenuhinya Sarana dan prasarana tentu akan memberikan dampak positif bagi pendidikan. Pengaruh sarana dan prasana bukan hanya dilihat dari kualitas sekolah tetapi dapat kita lihat dari pencapaian siswa pada bidang akademik ataupun non akademik (Hartoni, Amirudin, 2018). Terpenuhinya sarana prasarana memudahkan siswa dalam dalam melakukan kegiatan-kegiatan yang berhubungan dengan proses pendidikan baik itu proses belajar mengajar maupun melakukan kegiatan lain seperti ekstrakurikuler dan kokirukuler. (Sari & Budhi, 2017). Jika fasilitas disekolah dapat digunakan dan dioptimalkan secara baik maka akan mendukung peningkatan prestasi siswa. Sarana dan prasarana sangat dibutuhkan pada jalannya pendidikan baik itu yang langsung ataupun tidak langsung. Pada kegiatan pendidikan fasilitas harus bisa memenuhi standart untuk menciptakan suasana efektif pada kegiatan pendidikan. Sarana prasarana yang baik serta pengoptimalan yang baik sangat membantu dalam keberhasilan peningkatan presetasi siswa.(Huda, 2018) Meningkatkan prestasi siswa melalui manajemen sarana prasarana dapat dilakukan dengan memenuhi kebutuhan sarana dan prasarana peserta didik, guru serta staf dan karyawan di sekolahsesuai dengan kebutuhan mereka. Meyalurkan sarana prasarana juga harus sesuai dengan kebutuhan warga sekolah karena jika penyaluran sarana prasarana kurang dari kebutuhan pihak yang membuthkan akan mengalami kesulitan. Namun jika penyaluran dilakukan secara berlebihan sarana prasarana tersebut tidak dapat dimanfaatkan dengan baik (Firmansyah et al., 2018). Rehabilitasi Volume 1, Nomor 1, Desember 2021 61 61 Volume 1, Nomor 1, Desember 2021 Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi sarana prasarana juga sangat penting dalam pengelolaan sarana dan prasarana. Jika rehabilitasi dilakukan secara efektif dana untuk sarana prasarana akan berkurang sehingga dana tersebut dapat digunakan untuk hal yang lain (Kurniawati & Sayuti, 2013). Pengelolaan sarana prasarana yang baik akan menunjang peningkatan prestasi siswa. Dengan terpenuhinya sarana dan prasarana di dalam kelas, misalnya buku paket, papan tulis, alat tulis, bangku, meja dan lainnya, menjadikan peserta didik lebih optimal saat mendapat materi yang diajarkan atau disampaikan oleh guru. Sedangkan terpenuhinya sarana dan prasarana diluar kelas seperti lapangan, alat olahraga dan lainnya, mampu menunjang dan meningkatkan prestasi siswa dibidang akademik. Dengan optimalnya sarana dan prasarana tersebut peserta didik mampu melakukan apa yang ingin dilakukannya baik itu dibidang akademik dan non akademik. KESIMPULAN Proses yang dilakukan oleh sekelompok orang untuk mendayagunakan fasilitas pendidikan agar mewujudkan tujuan yang telah ditetapkan adalah manajemen sarana prasarana. Manajemen sarana prasana pendidikan bisa diartikan sebagai proses dalam mengelola sarana untuk mencapai tujuan pendidikan baik itu secara langsung ataupun tidak langsung. Salah satu penunjang dalam peningkatan prestasi belajar peserta didik baik itu prestasi akademik maupun non akademik adalah sarana prasarana yang baik. Pengelolaan sarana dan prasana yang baik baik itu didalam kelas maupun diluar kelas membantu siswa untuk mewujudkan apa yang ingin dilakukannya disekolah. ARZUSIN : Jurnal Manajemen dan Pendidikan Dasar DAFTAR PUSTAKA Ananda, R., & Banurea, O. K. (2017). Manajemen Sarana dan Prasarana (S. Saleh (ed.); Pertama). Anjassari, R., Sukmawati, & Suib, M. (n.d.). Pengelolaan sarana dan prasarana dalam meningkatkan prestasi non-akademik di SD IT. Firmansyah, T., Supriyanto, A., & Timan, A. (2018). Efektivitas Pemanfaatan Sarana Dan Prasarana Dalam Meningkatkan Mutu Layanan Di Sma Laboratorium. Jurnal Manajemen Dan Supervisi Pendidikan, 2(3), 179–184. https://doi.org/10.17977/um025v2i32018p179 62 ARZUSIN : Jurnal Manajemen dan Pendidikan Dasar Rudi Herianto, Fitriyani Sanuhung, Muhammad Farid Wajdi Hartoni, Amirudin, S. (2018). Impelementasi Manajemen Sarana Dan Prasarana di Sekolah Menengah Kejuruan. Al-Idarah: Jurnal Kependidikan Islam, VIII(1), 179– 185. http://ejournal.radenintan.ac.id/index.php/idaroh/article/view/3088 Herawati, S., Arafat, Y., & Puspita, Y. (2020). Manajemen Pemanfaatan Sarana Dan Prasarana Pembelajaran. Attractive : Innovative Education Journal, 2(3), 21. https://doi.org/10.51278/aj.v2i3.68 Huda, M. N. (2018). OPTIMALISASI SARANA DAN PRASARANA DALAM MENINGKATKAN PRESTASI BELAJAR SISWA. Ta’dibi : Jurnal Manajemen Pendidikan Islam, VI, 30. Ike Malaya Sinta. (2019). MANAJEMEN SARANA DAN PRASARANA. Jurnal Islami Education Manajemen, 4, 79. https://doi.org/10.1575/Isema.v312.5645 Kurniawati, P. I., & Sayuti, S. A. (2013). Manajemen Sarana Dan Prasarana Di Smk N 1 Kasihan Bantul. Jurnal Akuntabilitas Manajemen Pendidikan, 1(1), 98–108. https://doi.org/10.21831/amp.v1i1.2331 Manurung, R., Harahap, E., Tahrun, T., & Suharyadi, A. (2020). Manajemen Sarana Prasarana di Sekolah Dasar Negeri 1 Kota Prabumulih. Jurnal Manajemen Pendidikan: Jurnal Ilmiah Administrasi, Manajemen Dan Kepemimpinan Pendidikan, 2(2), 168–177. https://doi.org/10.21831/jump.v2i2.33747 Megasari, R. (2014). Peningkatan Pengelolaan Sarana dan Prasarana Pendidikan Untuk Meningkatkan Kualitas Pembelajaran Di SMPN 5 Bukittinggi. Jurnal Administrasi Pendidikan, 2, 638–831. Nurmaidah. (2018). Manajemen Sarana dan Prasarana. Jurnal Al-Afkar, VI, 30. Pahlevi, R., Imron, A., & Kusumaningrum, D. . (2016). Manajemen Sarana dan Prasarana untuk Meningkatkan Mutu Pembelajaran. Jurnal Manajemen Pendidikan, 25(1), 88–94. Parid, M., & Alif, A. L. S. (2020). Pengelolaan Sarana dan Prasarana Pendidikan. Tafhim Al-’Ilmi, 11(2), 266–275. https://doi.org/10.37459/tafhim.v11i2.3755 Sari, A. R., & Budhi, W. (2017). Hubungan Antara Karakter siswa, Kedisiplinan siswa, dan Kelengkapan sarana prasarana sekolah dengan prestasi belajar fisika. Jurnal Ilmiah Pendidikan Fisika-COMPTON, 4. Supiana, S., Hermawan, A. H., & Hilmy, M. F. (2019). Manajemen Sarana Dan Prasarana Diniyah Takmiliyah. Jurnal Isema : Islamic Educational Management, 3(2), 23–41. https://doi.org/10.15575/isema.v3i2.5007 63 Volume 1, Nomor 1, Desember 2021
https://openalex.org/W2985775307	https://www.ecocycles.net/ojs/index.php/ecocycles/article/download/144/133	English	null	Mapping the ‘presency’ of women in cities	Ecocycles	2,019	cc-by	3,716	ECOCYCLES ISSN 2416-2140 ECOCYCLES ISSN 2416-2140 Open access scientific journal of the European Ecocycles Society Ecocycles, Vol. 5, No. 2, pp. 1-5 (2019) DOI: 10.19040/ecocycles.v5i2.144 Ecocycles, Vol. 5, No. 2, pp. 1-5 (2019) DOI: 10.19040/ecocycles.v5i2.144 Mapping the ‘Presency’ of Women in Cities May East UNITAR Fellow, Chief Executive of Gaia Education, Edinburgh, Scotland, UK UNITAR Fellow, Chief Executive of Gaia Education, Edinburgh, Scotland, UK E-mail address: may.east@gaiaeducation.org Abstract – This paper explores how innovative ways of mapping both the presence and the agency of contemporary women in cities may support the emergence of emancipatory placemaking perspectives and previously unrecorded narratives. It starts by proposing ‘presency’ as a new concept, merging the meaning of presence, as a mindful way of paying attention to life; and agency, as critical awareness of the context and capacity to act. It examines the pace of urbanisation of the world and the revisited role of women in their mediation of space and making of place including efforts to forge a new framework of regenerative urban development. It proposes different mapping approaches to capture a mosaic of regenerative practices led by women addressing how cities of present and future can be green and inclusive. It concludes by suggesting that the act of mapping spatially and ‘from within’ the way women experience and act in the city may unleash women’s emancipatory place-making skills, moving cities systems up to higher orders of integrated expression. Keywords – women; mapping; placemaking; regenerative development; agency Keywords – women; mapping; placemaking; regenerative development; agency Accepted: October 7, 2019 Prospects and Restraints All movement occurs while it is being inhibited (Maturana and Bunnell, 1999). The Agenda 2030 positions women and girls as diverse and innovative agents of change and gender equality is central to the achievement of all SDGs. However, investigating SDG 11 Sustainable Cities and Communities at the target level (Targets 11.2 and 11.7), the language is revealing. Women are characterised as amongst the vulnerable members of society requiring protection alongside children, older persons and persons with disabilities. Almost 40 years after the Convention on the Elimination of all Forms of Discrimination against Women, the international community continues to associate women with those in need of protection, instead of proposing a framework that inspires and propels women to help to shape the regeneration of contemporary cities. The accelerated pace of what Davis called ‘the urbanization of the human population’ (1965) provided the backdrop for the emergence of global cities (Sassem, 2005). Reflecting on the pace of urbanisation, Fuller (1978) thought that ‘unsettlement’ would be a more accurate description of the mobile character of modern life. While Jacobs (1968) argues for a piecemeal growth as a process of creating a vibrant built environment, Campbell (2018) proposes ‘making massive small change’ as a conceptual antidote to the ‘bigness’ approach which has informed recent urban evolution. Given that women live in so many diverse urban settings, there can be no single approach or strategy to address the apparent disconnect between SDG 11- Sustainable Cities and SDG 5- Gender Equality. However, in innovative ways, women are already meeting the challenge, working to co- create a society that uses energy and resources with greater efficiency (SDG 12), distributes wealth equitably (SDG 10), and makes quality of life, rather than open-ended economic growth (SDG 8), the focus of future thinking. Urbanisation is often associated with greater independence and opportunity for women- but also with hostility and constraints on employment, mobility and leadership that reflect deep gender-based inequalities (Tacoli, 2013). According to the Agenda 2030 (UN, 2015) and its 17 Sustainable Development Goals (SDGs) adopted by UN members states in 2015, new trends in urbanisation are likely to unfold over the coming years. The revisited role of women in their ‘mediation of space and making of place’ is crucial to the implementation of the 2030 Agenda including efforts to forge a new framework of regenerative urban development. Accepted: October 7, 2019 For Freire (1970), a word contains both © 2019 The Author(s). Ecocycles © European Ecocycles Society, ISSN 2416-2140 Volume 5, Issue 2 (2019) reflection and action. The sacrifice of action and emphasis on reflection leads to rhetorical verbalism, while poor reflection in detriment to action generates shallow activism. Therefore, the term ‘presency’ proposes a balance between inner and outer, thought and praxis, critical attention and action emerging from an experienced context. by claiming men cannot represent women’s interest adequately. In this context, emerging women’s collectives reflect on how to ensure that gender equality is at the heart of the planning and governance of sustainable cities and human settlements. How are sidewalks built, public transport networked, alleyways illuminated, neighbourhood funds distributed, should be informed by the ‘presency’ of women. This investigation aims to map a mosaic of regenerative practices led by women as agents of the re-enchantment of cities addressing how cities of present and future can be green, inclusive, liveable and poetic (UNSW, 2015). Prospects and Restraints For instance, like the material feminists of 1930’s who argued that women rather than men must control the infrastructure, water pipes, communication lines and fuel lines and use them as their base for economic power (Hayden, 1981) today active participation of women as consumers and workforce in the renewables industry is considered essential for the global energy transformation (IRENA, 2018). Recent research from IRENA (2019) revealed that women represent 32% of the full- time employees of the renewable energy industry substantially higher than the 22% average in the global oil and gas industry. This reinforces the fact that women are in the best position to define how new social accords and spatial infrastructures can shape the quality of their experience as residents and protagonists of urban environments. The case for cities supporting the sustainability agenda is incredibly compelling. Cities generate their own wealth, shape local and often national policies and are spearheading a thrilling new vision of governance for the implementation of the SDGs. Pragmatic in approach, close to real people and their problems (Barber, 2014), cities also contain the seeds of their own regeneration (Jacobs, 1961). Barber argues that in this century, it will be the city, not the state, which becomes the nexus of economic and political power (2014). However, cities have been planned, developed and built primarily by men for men (Greed, 1994) embedding inequality as a recurrent socio-economic pattern within the urban space. The Context In 2007, for the first time in history, the global urban population exceeded the global rural population (UN DESA, 2014). The world population has remained predominantly urban ever since. Cities occupy just 3% of the planet surface (Columbia University Earth Institute, 2005) but account for 60 - 80% of global energy consumption, 75% of carbon emissions (UNEP, 2012) and more than 75% of the world’s natural resources (Girardet, 2008). Continuing population growth and urbanisation is projected to add 2.5 billion people to the world’s urban population by 2050, with nearly 90 per cent of the increase concentrated in Asia and Africa (UNDESA, 2014). Accepted: October 7, 2019 Received: September 18, 2019 ‘The moving about that the city multiplies and concentrates makes the city itself an immense social experience of lacking a place - an experience that is, to be sure, broken up into countless tiny deportations (displacements and walks), compensated for by the relationships and intersections of these exoduses that intertwine and create an urban fabric, and placed under the sign of what ought to be, ultimately.’ This paper explores how innovative ways of mapping both the presence and the agency of contemporary women in cities may support the emergence of emancipatory placemaking perspectives and previously unrecorded narratives (University of Reading, 2019). It examines both the prospects and the impediments to mapping women’s presence and agency in cities from policy to community development (Colau, 2017 cited in Johnston-Zimmerman), from social capital (Campbell, 2018) to impact investment, from participatory budgeting to civic ecology (Krasny and Tidball, 2015). It also highlights the catalyst role of mapping as a tool to explore both the rhizomatic (Deleuze and Guattari, 1972) and the sociological (Simmel, 1903) nature of cities from a women’s perspective. - De Certeau, 1984 - De Certeau, 1984 Maps are often considered accurate visual representations of the world untainted by social factors (Harley, 1989) and providing a mirror effect to particular sections of the earth (Rorty, 1979). However, many see ‘the process of selection, omission, isolation, distance and codification of mapping’ (Corner, 1999 p.215) repeatedly reinforcing the legal statutes and representing specific political territorial imperatives (Harley, 1989). In contrast to the ‘scientific’ cartography approach, maps can be used as a liberating instrument (Corner, 1999), generating new thinking and holding the power of changing urban reality (Petrescu, 2018). Despite what cartographers may claim (Erskine, 2018), maps have a strategic, constitutive and inventive role (Corner, 1990) as they articulate diverse intentional arguments about the world (Harley, 1989). This paper is the first attempt to describe ‘presency’ as a new concept. It blends the words presence and agency to create a new concept which combines both meanings. It adopts the Heideggerian perspective of presence experienced in the now and or in the timeless aspect of the eternal now (1927). It also considers the Buddhist view of presence as a mindful way of paying attention to life, moment by moment (Levman, 2017). It describes agency as critical awareness of the context and capacity to act. Agency in Mapping Sassen (2016) claims that urban planning is not gender neutral. While there has been some research on how urban systems fail to respond to women’s needs, only recently cities have been addressing the ‘urban-planning gender gap’. This paper investigates the concept of the gendered nature of regenerative planning embedded in the proliferation of movements by and for women. It also describes agency as critical awareness of the context and capacity to act. A research can be emancipatory or supportive of dysfunctional power relations (Silva et al., 2015). The word emancipation is derived from the Latin e- ‘out’, manus- ‘hand’ and capere- ‘to take’, meaning freeing of an individual from the strong hand or the legal authority to make her or his own way in the world (Etymological Dictionary, 2019). This paper suggests that the act of mapping spatially and ‘from within’ the way women experience and interact with the city may unleash women’s emancipatory place-making skills, moving cities systems up to higher orders of integrated expression. In Darjeeling at the foothills of the Himalayas, for instance, a group of women are advancing the SDGs, prioritising #SDG6 Clean Water and Sanitation by harvesting rooftop rainwater and promoting responsible consumption (#SDG12) amongst its growing population (East, 2018). Taking into consideration the influx of tourism, and the lack of integrated planning policies and conservation measures, the group also prioritised #SDG11 - Sustainable Cities and Communities - as a potential catalyst for the changes that need to occur in the context of rapid unplanned urbanisation and its impact on infrastructure, mobility, waste management, noise and air pollution. This hypothesis invites further investigation in four key domains as illustrated in the table below. Table 1- Mapping Women’s ‘Presency’ in the City by Author Mapping Women’s Presency in the City From Within Experience of the City Spatially Socio-urban Practices Presence Mediation of Space Safe/Unsafe Reinforcing/undermining Sense of Self Belonging/Outsider Reinforcing/undermining Sense of Identity Agency Making of Place Gazing/Avoiding Gaze Walking/Strolling How political or invisible is the personal? Empowered/Disempowered Beneficiary/Protagonist Who rules? Who designs, plans? In the Brazilian city of Santana de Parnaiba, it is women, many of them urban planners, administrators and housing officials, who are leading the process of Voluntary Local Review on the SDGs implementation (East, 2018, Nichols, 2019). Presence in the City A core insight of regenerative development is the idea that we can shift from dominance to intimacy with the entity of place (Reed, 2007). This depends on knowing the ‘place’ on the Jacobs (1961) makes the case that ‘big city plans never stirred women’s blood’. De Beauvoir (1949) reinforces the argument 2 © 2019 The Author(s). Ecocycles © European Ecocycles Society, ISSN 2416-2140 Volume 5, Issue 2 (2019) level of relationship and experience. Referring to the way men experience the city, Baudelaire wrote ‘the crowd is his domain, just as the air is the bird’s, and water that of the fish. His passion and his profession are to merge with the crowd’ (1893). More than a century later, women continue to navigate cities in a profoundly different way from men, often seeking protection from risks of violence and striving to be respected and safe (Elkin, 2016). relational self. Spatially, how urban development supports or challenges ‘the fluid complexities of women’s identities’ (McCann and Kim, 2016, pp 20) and belonging to cities, towns and neighbourhoods could be considered. Questions to be researched at the level of agency includes the emancipatory decision of women to stand-up or blend in with the crowd (Elkin, 2016) while taking goal-oriented walks or slow strolls exploring city life (Gehl, 2010). Other parameters at the heart of their urban experience include how political or invisible is the personal (De Beauvoir, 1949) or how much they want to attract, initiate or escape gaze. For Awan (2016) at an intimate level, there is a disconnect between digital technology infrastructures of the so-called smart cities and women from low-income neighbourhoods. For instance, continuous and widespread Violence Against Women (VAW) in urban India highlights the challenge of delivering SDGs 11 and SDG 5 together. Officials from UN Women agency argue no city can be smart and sustainable if half of its population is not safe and lives in fear of violence. At the spatial domain mapping parameters could take in consideration ways in which women use the built environment as beneficiary or protagonist and what are their expectations in respect to planning process (Sassen, 2016) and place-making. This is about to change. With the rise of technology, smart phones and apps, women may today swiftly gather, produce and navigate city data. Presence in the City A myriad of apps such as Safe and the City, BSafe, Shake2Safety, Safitipin Nite, have been used by women from Delhi to Sydney, Kampala to Madrid, helping women to navigate back home safely. Similar GPS apps can serve as emancipatory tools for women to map ‘from within’ the socio-political cultural space (Petrescu), and spatially their essential contribution to place-making. Agency in Mapping Discussing how urban systems fail to respond to women’s needs, the female Secretary of Housing recently argued that while male planners see dislocation as natural and a way to concentrate commercial activities, women in the same role would plan services to be offered at walking distance of neighbourhoods. For men, the issue is mobility while for women, the issue is proximity. Mapping Women’s Presency in the City Table 1- Mapping Women’s ‘Presency’ in the City by Author Given that women live in so many different social, cultural economic and political circumstances, there can be no best global strategy for changes (McCann and Kim, 2016). However, it is expected that the mapping of women’s Parameters for mapping the presence of women at the level of experience may include the level of safety she experiences from within, reinforcing or undermining her sense of 3 © 2019 The Author(s). Ecocycles © European Ecocycles Society, ISSN 2416-2140 Volume 5, Issue 2 (2019) ‘presency’ would bear impact on the way women experience and act in cities. De Beauvoir, S.,1949. The Second Sex. 2nd Edition. The Modern Library, New York, 1968. De Certeau, M., 1984. The Practice of Everyday Life. University of California Press. October 2019]. Freire, P., 1970. Pedagogy of the Oppressed. Continuum International Publishing Group, Inc.; New York. Discussions and Conclusions For UN-Habitat, urban centres are where the battle for sustainable development will be won or lost. The potential triumph could only be achieved with a new integrated vision placing women as protagonists of an urban environment that reflects their needs and aspirations. Working class women, women of colour, mothers, academicians, professionals, the revolutionary and the conservative, the mature and the young, wearing burkas, saris, black jeans or business attire. Mapping how they walk, experience and act in the city may accelerate what the first female Mayor of Barcelona Colau (2016) stated: Deleuze, G. and Guattari, F., 1972. Anti-Oedipus: Capitalism and Schizophrenia. Paris: Les Editions de Minuit. East, M., 2018. Do-it-yourself ideas take root in the fertile minds of India’s hill peoples. Available at: East, M., 2018. Do-it-yourself ideas take root in the fertile minds of India’s hill peoples. Available at: https://www.scotsman.com/news/opinion/may-east-do-it- yourself-ideas-take-root-in-the-fertile-minds-of-india-s-hill- peoples-1-4766535 [Accessed 01 August 2019]. East, M., 2018. Setting goals for a sustainable future is the starting place for global changes. Available at: https://www.scotsman.com/news/opinion/may-east-setting- goals-for-a-sustainable-future-is-the-starting-place-for- global-changes-1-4819115 [Accessed 01 August 2019]. ‘For every woman, regardless of the privilege there is an opportunity for those individuals who have traditionally been let down as ‘second-class citizens’ to become the main characters. This is about achieving what we have never had the opportunity to impact before: the design and management of our cities, by and for our fellow women’. Elkin, L., 2016. Flâneuse Women Walk the City, Chatto & Windus. London. Elkin, L., 2016. Flâneuse Women Walk the City, Chatto & Windus. London. Taking in consideration that the value and meaning of women’s lives must be defined from their own point of view (McCann and Kim, 2016), this paper suggests mapping exercises as a way to investigate how women experience the city from within and spatially. Future investigations may consider ethnographic walks utilising apps combined with conversations carefully conducted so that they are not usurped by the presence of technology (Campbell, 2018). Beyond illustration and tracing, the visual aspect of mapping may function as a form of research, a platform for agency, a practice of presence. Erskine, J., 2018. Politics and Cartography: The Power of Deception through Distortion. Carnegie Council for Ethics in International Affairs. Available at: https://www.carnegiecouncil.org/publications/ethics_online/ politics-and-cartography-the-power-of-deception-through- distortion. [Accessed 05 July 2019]. https://www.carnegiecouncil.org/publications/ethics_online/ politics-and-cartography-the-power-of-deception-through- distortion. [Accessed 05 July 2019]. Etymological Dictionary. Available at: https://www.etymonline.com/word/emancipate [Accessed 06 October 2019]. References Awan, N., 2016. Diasporic Agencies: Mapping the City Otherwise. Routledge. Awan, N., 2016. Diasporic Agencies: Mapping the City Otherwise. Routledge. Fueller, B., 1978. Accommodating Human Unsettlement. Town Planning Review. Volume 49, n 1, pp. 51-60. Barber, B. R., 2014. If Majors Ruled the World. Yale University Press. Barber, B. R., 2014. If Majors Ruled the World. Yale University Press. Gehl, J., 2010. Cities for People. Island Press Gehl, J., 2010. Cities for People. Island Press Giddens, A., and Sutton, P. W., 2017. Essential Concepts in Sociology. Second Edition. Cambridge: Polity Press. Baudelaire, C.,1893. The Painter of Modern Life. Phaidom Press. Baudelaire, C.,1893. The Painter of Modern Life. Phaidom Press. Girardet, H., 2008. Cities People Planet: Urban Development and Climate Change. Second Edition. Wiley. Campbell, K., 2018. Making Massive Small Change. Chelsea Green Publishing. Greed, C., 1994. Women and Planning: Creating Gendered Realities. Taylor & Francis. Columbia University Earth Institute, 2005. The Growing Urbanization of the World Columbia University Earth Institute, 2005. The Growing Urbanization of the World http://www.earth.columbia.edu/news/2005/story03-07- 05.html [Accessed 25/6/2018] Harley, J. B., 1989. Deconstructing the Map. Cartographica, 26, pp. 1-20. Corner, J., 1999. The Agency of Mapping: Speculation, Critique and Invention in Cosgrove, D. E., Mappings. London, Reaktion, 2001, pp. 213-233. Hayden, D., 1981. The Grand Domestic Revolution. The MIT Press. Heidegger, M., 1927. Being and Time. Harper Perennial Modern Thought. Davis, K., 1965. The Urbanization of the Human Population. Scientific American. 4 © 2019 The Author(s). Ecocycles © European Ecocycles Society, ISSN 2416-2140 Volume 5, Issue 2 (2019) Jacobs, J., 1961. The Death and Life of Great American Cities. New York: Random House. Regenesis Group, 2017. The Regenerative Practitioner. Systemic Frameworks. Regenesis Group, Inc. Rorty. R., 1979. Philosophy and the Mirror of Nature. Princeton University Press. Johnston-Zimmerman. K., 2017. Urban Planning Has a Sexism Problem. Next City. Available at: https://nextcity.org/features/view/urban-planning-sexism- problem. [Accessed 01 August 2019]. Sassem, S., 2016. Built Gendering. Harvard Design Magazine. N.41 / Family Planning. Sassem, S., 2005. The Global City: Introducing a Concept. Brown Journal of World Affairs. Volume XI, Issue 2. Krasny, M. E., and Tidball, K. G., 2015. Civic Ecology Adaptation and Transformation from the Ground Up. MIT Press. Silva, E. A., Healey, P., Harris, N., Van den Broeck, P., 2015. The Routledge Handbook of Planning Research Methods. Routledge DOI: 10.7551/mitpress/9780262028653.001.0001 IRENA, 2018. Active Participation of Women Essential to the Global Energy Transformation. Available at: https://www.irena.org/newsroom/articles/2009/Apr/Active- participation-of-women-essential-to-the-global-energy- transformation. [Accessed 02 August 2019]. © 2019 by the author(s). 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 Simmel, G., 1903. The Metropolis and Mental Life. New York: Free Press, https://www.irena.org/newsroom/articles/2009/Apr/Active- participation-of-women-essential-to-the-global-energy- transformation. [Accessed 02 August 2019]. Tacoli, C., 2013. The benefits and constraints of urbanization for gender equality. Environment & Urbanization Brief- 27. IIED’s Human Settlements Group. Levman, B., 2017. Putting smrti back into sati (Putting remembrance back into mindfulness). Journal of the Oxford Centre for Buddhist Studies. 2017 (13) pp. 121-149. UNSW, 2015. Re-enchanting the city- designing the human habitat. University of South Wales, Sydney. Available at: https://www.futurelearn.com/courses/re-enchanting-the-city. [Accessed 02 June 2019]. Maturana, H. R. and Bunnell, P., 1999. The Biology of Business. Part II. The SOL Journal. McCann, C. R. and Kim, S. K., 2016. Feminist Theory Reader. Routledge. University of Reading, 2019. The Missing Map Symposium. United Nations, Department of Economic and Social Affairs, Population Division, 2014. World Urbanization Prospects: The 2014 Revision, Highlights (ST/ESA/SER.A/352). Nichols, M., 2019. Cities to step up at U.N. to push climate fight, sustainable development. Reuters. Available at: Nichols, M., 2019. Cities to step up at U.N. to push climate fight, sustainable development. Reuters. Available at: https://www.reuters.com/article/us-climate-change-un-cities- exclusive/exclusive-cities-to-step-up-at-u-n-to-push-climate- fight-sustainable-development-idUSKBN1W127O [Accessed 02 June 2019]. https://www.reuters.com/article/us-climate-change-un-cities- exclusive/exclusive-cities-to-step-up-at-u-n-to-push-climate- fight-sustainable-development-idUSKBN1W127O [Accessed 02 June 2019]. UNEP, 2012. 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https://openalex.org/W2211314610	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0141013&type=printable	English	null	Enhanced Recyclable Magnetized Palm Shell Waste-Based Powdered Activated Carbon for the Removal of Ibuprofen: Insights for Kinetics and Mechanisms	PloS one	2,015	cc-by	8,698	RESEARCH ARTICLE Enhanced Recyclable Magnetized Palm Shell Waste-Based Powdered Activated Carbon for the Removal of Ibuprofen: Insights for Kinetics and Mechanisms Kien Tiek Wong1, Yeomin Yoon2, Min Jang1,3* 1 Department of Civil Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur, Malaysia, 2 Department of Civil and Environmental Engineering, University of South Carolina, Columbia, United States of America, 3 Nanotechnology and Catalysis Research Centre (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia * minjang@um.edu.my; heejaejang@gmail.com OPEN ACCESS Citation: Wong KT, Yoon Y, Jang M (2015) Enhanced Recyclable Magnetized Palm Shell Waste- Based Powdered Activated Carbon for the Removal of Ibuprofen: Insights for Kinetics and Mechanisms. PLoS ONE 10(10): e0141013. doi:10.1371/journal. pone.0141013 Editor: Yogendra Kumar Mishra, Institute for Materials Science, GERMANY Received: July 14, 2015 Accepted: October 2, 2015 Published: October 23, 2015 Copyright: © 2015 Wong 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. Editor: Yogendra Kumar Mishra, Institute for Materials Science, GERMANY Received: July 14, 2015 Accepted: October 2, 2015 Published: October 23, 2015 Editor: Yogendra Kumar Mishra, Institute for Materials Science, GERMANY Received: July 14, 2015 Accepted: October 2, 2015 Published: October 23, 2015 Editor: Yogendra Kumar Mishra, Institute for Materials Science, GERMANY Received: July 14, 2015 Accepted: October 2, 2015 Published: October 23, 2015 Copyright: © 2015 Wong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Wong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Abstract A novel preparation method of magnetized palm shell waste-based powdered activated car- bon (MPPAC, avg. size 112 μm) was developed. The prepared MPPAC was assessed by several physicochemical analyses, and batch tests were performed for ibuprofen (IBP) removal. Field emission scanning electron microscopy (FESEM) and N2 gas isotherms revealed that magnetite and maghemite were homogeneous and deposited mostly on the surface of PPAC without a significant clogging effect on the micropores. Isotherm results showed that 3.8% Fe (w/w) impregnated PPAC [MPPAC-Fe(3.8%)] had about 2.2-fold higher maximum sorption capacity (157.3 mg g-1) and a 2.5-fold higher sorption density (0.23 mg m-2) than pristine PPAC. Both Fourier-transform infrared spectroscopy (FTIR) and isotherm data indicated that the high sorption capacity and density of IBP by MPPAC was primarily attributable to donor-acceptor complexes with the C = O group and dispersive π-π interactions with the carbon surface. Based on kinetic and repeated adsorption tests, pore diffusion was the rate-limiting step, and MPPAC-Fe(3.8%) had about 1.9~2.8- and 9.1~15.8-fold higher rate constants than MPPAC-Fe(8.6%) and palm shell-waste granular activated carbon (PGAC, avg. size 621 μm), respectively. MPPAC showed almost eight fold greater re-adsorption capacity than PPAC due to a thermal catalytic effect of magnetite/ maghemite. * minjang@um.edu.my; heejaejang@gmail.com Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water degree of chemical property diversity, conventional treatment processes, such as biological deg- radation, chlorine oxidation, coagulation, flocculation, and sedimentation, are often ineffective in removing pharmaceuticals [5]. Thus, the occurrence of pharmaceuticals in water resources reported in surveys conducted in several countries [6, 7] is stimulating the need for water treat- ment processes that are highly effective, relatively easy to operate, and energy efficient [8, 9]. Numerous studies have been performed to remove pharmaceuticals using membrane filtra- tion (nanofiltration and reverse osmosis), photocatalysis [10], sonolysis [11], oxidation using ozone [12, 13], and electrochemical degradation [14]. Nevertheless, drawbacks, such as toxic transformation products, incomplete removal, very high capital and operation costs, and sophisticated maintenance and skilled personnel requirements have become hurdles to the implementation of these technologies. Additionally, oxidative and biological processes often transform substances from one form to another that may not result in detoxification [15]. Among water treatment technologies, adsorption is used frequently because of the ease of oper- ation, simplicity of design, potential for media regeneration, effectiveness in contaminant attenuation, and absence of transformation products. Soil minerals, activated carbons (ACs), mesoporous silicas, zeolites, and other materials have been studied for their characteristics and adsorption mechanisms [16–19]. Of the adsorbents, AC is used most widely for the removal of organic constituents in water. Furthermore, the production of inexpensive AC from natural materials, such as municipal solid waste and agricultural by-products, has received much atten- tion because it can lead to economical water treatments [18, 20, 21]. In this research, we developed a novel synthesis route to prepare magnetized palm shell- waste based powdered activated carbon (MPPAC), based on a cheap and abundant agriculture carbonaceous waste in many tropical countries. Theoretically, MPPAC could have a lower sorption capacity than non-impregnated PPAC because of significant pore blocking by the nano-magnetite impregnation. To reduce micropore blockage, we attempted to coat magnetic materials primarily on the surface of PPAC. In particular, the preparation procedure developed can produce large quantities of MPPAC in an energy-efficient and cost-effective manner. To our knowledge, this is the first reported study to develop a preparation route of magnetized activated carbon for the application of water treatment. As a model pharmaceutical pollutant, ibuprofen (IBP) was chosen because it is one of the most widely consumed medicines worldwide, and has been found in many water sources, as well as in waste water [22, 23]. The physical and chemical characteristics of IBP are listed in Table A in S1 File. The main objectives of this investigation were to (i) prepare MPPAC coated with various amounts of Fe, (ii) characterize the MPPACs using various spectroscopic analysis, (iii) assess the sorption capacities and rates of IBP in batch and repeated operations, (iv) determine the thermodynamic parameters and adsorption mechanism, and finally (v) to assess the regenera- tion and re-adsorption capability of MPPACs by low thermal treatment. Introduction Funding: This work was supported by the Malaysian Government Ministry of Higher Education through the High Impact Research Grant (D000062-16001). With more than 65 million chemicals and formulations currently available commercially, the aquatic environment is often contaminated with a wide variety of organic constituents [1]. Of particular concern are pharmaceuticals; they are continually introduced to the environment through wastewater outfalls and have been found in drinking water [2–4]. Due to the high Competing Interests: The authors have declared that no competing interests exist. 1 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Physicochemical characterization The prepared media were analyzed by X-ray diffraction (XRD), field emission scanning elec- tron microscopy and energy dispersive X-ray spectroscopy (FESEM/EDS), N2 gas isotherms, Fourier-transform infrared (FTIR), and pHpzc. An explanation of these analyses is provided in S1 File. Materials and Methods Commercial PPAC (mesh size 100 × 200, avg. 112 μm) as a solid (pH 9.8) activated by KOH was purchased from Bravo Green Sdn. Bhd., Malaysia. Ferrous sulfate heptahydrate (FeS- O47H2O), sodium hydroxide (NaOH), potassium nitrate (KNO3), methanol (HPLC grade), and sodium chloride (NaCl) were purchased from R&M Chemical. For the adsorption, IBP (99%) was obtained from Sigma-Aldrich. 2 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Determination of Fe content in MPPACs The amount of iron loaded into the PPAC was determined by an aqua regia method, in which a strong acid mixture was prepared by the combination of 10 mL of nitric acid with 30 mL of hydrochloric acid. MPPAC (0.1 g) was then added to 10 mL of this mixture, and the suspension sonicated for 30 min. Then, 5 mL of the suspension were diluted with distilled water to 50 mL. The suspension was filtered (0.45 μm pore size) and analyzed using inductively coupled plasma mass spectrometry (ICP-MS). Aqua regia extraction results showed 3.8, 7.8, or 8.6% Fe loading on PPAC with 27, 54, or 72 g FeSO47H2O addition, respectively. Thus, these media are referred to as MPPAC-Fe(3.8%), -Fe(7.8%), and -Fe(8.6%), respectively. Preparation of MPPACs Different amounts of FeSO47H2O (27 g, 54 g, or 72 g) were dissolved in 200 mL of distilled water, and 50 g PPAC were added. This suspension was heated at 353 K for 2 h. Then, 50 mL of alkaline solution were prepared using 2.25 g of potassium nitrate (KNO3) and 15 g of NaOH. This alkaline solution was added drop-wise into the PPAC suspension under constant stirring. The suspension was then sonicated for 1 h at 353 K. The applied power and frequency of the ultrasound irradiation were 500 W and 50–60 Hz, respectively. The suspension was kept overnight for aging, before washing and drying. The precipitate was washed thoroughly with distilled water to remove chemical residue. Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water parameters were calculated for the adsorption of IBP on MPPAC-Fe(3.8%) at a constant adsorption uptake equal to 50 mg g-1 and at pH 7. Repeated adsorption tests Two sets of repeated adsorption tests were conducted. In the first trial, MPPAC-Fe(3.8%), MPPAC-Fe(8.6%), and palm shell-waste-based granular activated carbon (PGAC, 20 × 40 mesh) were also used to compare the adsorption speeds. Four cycles of experiments were con- ducted at pH 7 with 5 mg L-1 IBP and 0.1 M ionic strength. In the second trial, 13 cycles of repeated adsorption were conducted using MPPAC-Fe(3.8%) with the same conditions described in the first trial, but with a shortened operational time. MPPAC media were sepa- rated from the suspension using a permanent magnet once adsorption was complete, whereas PGAC was separated using a membrane filter (Whatman no. 1, 0.45 μm pore size). Regeneration and re-adsorption Adsorption was conducted by adding 0.01 g of medium [PPAC, MPPAC-Fe(3.8%), or MPPAC-Fe(8.6%)] into 30 mg g-1 IBP (100 mL) for 24 h. Then, the recovered medium was washed to remove excess IBP and was dried in the oven at 353 K. Six repeated thermal regener- ations of the saturated medium were conducted using a muffle furnace (Dae Heung Science, DF-2) for 3 h at 623 K. PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Batch tests of kinetics and isotherms All kinetic tests were conducted at pH 7 and room temperature at various ionic strengths. Dif- ferent MPPACs (8 mg) were used to treat 20 mg L-1 IBP solution (100 mL) with ionic strength of 0, 0.1 and 0.5 M. IBP samples were collected at different time intervals over 6 h, and concen- trations were subsequently determined using a UV spectrophotometer (Spectroquant Phoro 100, Merck) at 220 nm. The experimental kinetics data were fitted using a pseudo-second order kinetic model (see SI). Adsorption isotherms of IBP for the various MPPACs were inves- tigated using different masses of MPPAC (8–400 mg), temperatures (298, 308, or 318 K), and pH (4 and 7) at 0.1 M of NaCl. For isotherms, the IBP solution applied and the operational conditions of the suspension were the same as for the kinetics experiments. More information about the Langmuir and Freundlich isotherm models are noted in the SI. The isosteric Gibbs free energy, ΔG° of adsorption of IBP onto MPPAC-Fe(3.8%) was calculated using the results of the isotherm and the following equation: LnðkdÞ ¼ Ln Ca Ce ¼ DG RT ð1Þ ð1Þ where Kd is the equilibrium constant, Ca is the amount of IBP adsorbed at equilibrium (mg L-1), and Ce is the concentration of IBP remaining in the solution at equilibrium (mg L-1), T is the solution temperature (K), and R is the ideal gas constant. The isosteric enthalpy ΔH° and entropy ΔS° of adsorption were also calculated from the slope and the intercept of the plot of ln(Kd) versus 1/T, respectively, using the equation: ΔG° = ΔH° - TΔS°. Thermodynamic PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 3 / 18 Characterization The FESEM micrographs and EDS data for PPAC and MPPACs coated with 3.6% and 8.6% Fe are shown in Fig 1A–1I. A rough surface texture of PPAC is seen in Fig 1A. After iron impreg- nation [Fig 1D and 1G], the smooth surface of PPAC were occupied, and the spongy texture were observed. EDS results, shown in Fig 1C, 1F and 1I, indicate that PPAC did not contain any Fe, while MPPAC-Fe(3.6%) and -Fe(8.6%) had 19.1 and 66.5 wt%, respectively. In particu- lar, the homogeneous dispersion of Fe in MPPAC-Fe(8.6%) observed from elemental mapping indicated that US irradiation was a reliable technique for coating of Fe on the surface of PPAC (Fig 1J). Fig 2A shows XRD patterns of PPAC and MPPAC-Fe(8.6%). The XRD patterns of PPAC indicated a typical amorphous carbon shape and showed broad asymmetric peaks, correspond- ing from 25° to ~45° [24]. Additionally, the pattern showed one peak at ~30°, corresponding to the (4 0 0) reflexes of graphite (JCPDS File, No. 1–640). The XRD analysis of MPPAC-Fe (8.6%) showed that the medium contained two different phases of iron oxide: magnetite at 35.42° (3 1 1) and maghemite at 43.47° (4 0 0), 53.88° (4 2 2), and 57.16° (5 1 1) (JCPDS file, No. 19–0629). The intensity of the graphite peak was reduced sharply in MPPAC-Fe(8.6%) due to the incorporation of magnetite and maghemite. The N2 adsorption-desorption isotherms of all media [Fig 2B] indicated a type I isotherm character, based on the IUPAC classification. This result indicated that all materials had rela- tively high numbers of micropores. Specifically, as shown for PPAC, the marked knee at the low relative pressure and a very low slope in the multilayer (0.8 < P/P0 < 1.0) specified a low external surface and the absence of significant mesoporosity. According to the IUPAC classifi- cation, all media possessed a H4-type hysteresis loop, displaying a slit-shaped pore characteris- tic where both adsorption and desorption branches are parallel [25]. As the amount of impregnated Fe increased, however, the graph displayed type IV character isotherms at a high relative pressure (0.8 < P/P0 < 1.0), indicating the formation of meso- and macro-pore 4 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 1. Characterization FESEM and EDS images for (a-c) PPAC, (d-f) MPPAC-Fe(3.8%), (g-i) MPPAC-Fe(8.6%), and elemental mapping of (j) Fe, (k) O and (l) C for MPPAC-Fe(8.6%). doi:10.1371/journal.pone.0141013.g001 Fig 1. FESEM and EDS images for (a-c) PPAC, (d-f) MPPAC-Fe(3.8%), (g-i) MPPAC-Fe(8.6%), and elemental mapping of (j) Fe, (k) O and (l) C for MPPAC-Fe(8.6%). doi:10.1371/journal.pone.0141013.g001 structures. The Halsey equation with FAAS correction was used to describe differential pore volume according to pore size [20–2000 Å; Fig 2C]. Although all media had a similar primary peak at 39 Å, they had markedly different trends in pore volume at > 40 Å. MPPAC-Fe(3.8%) had a higher pore volume at < 100 Å than the other media, while higher Fe content media had a higher pore volume at > 100 Å. Based on this result, it can be concluded that more-polymer- ized structures could create higher inter-particle spaces between magnetite and maghemite as the Fe content increased, leading to higher pore volume at > 100 Å. Detailed pore characteristics of all materials are summarized in Table 1. Among the media, PPAC had the highest total (776.5 m2 g-1), BET (754.8 m2 g-1), and micropore surface area (603.7 m2 g-1). The pore properties, such as total, BET, and micropore surface area and volume, decreased linearly as the Fe amounts incorporated increased, while total and BJH pore volume increased with amount of Fe incorporated (Fig A in S1 File). The magnetization of media impregnated with different percentages of Fe was measured at 300 K [Fig 3A]. All media had non-linear and reversible behaviors with magnetic hysteresis 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 2. (a) XRD pattern for PPAC and MPPAC−Fe(8.6%), (b) N2 gas adsorption–desorption isotherms and (c) differential pore volume according to pore width calculated by Halsey equation with FAAS correction. doi:10.1371/journal.pone.0141013.g002 PACFe(8.6%), (b) N2 gas adsorption–desorption isotherms and (c) differential pore volume according to on with FAAS correction. Fig 2. (a) XRD pattern for PPAC and MPPACFe(8.6%), (b) N2 gas adsorption–desorption isotherms and pore width calculated by Halsey equation with FAAS correction. Fig 2. (a) XRD pattern for PPAC and MPPACFe(8.6%), (b) N2 gas adsorption–desorption isotherms and (c) differential pore volume according to pore width calculated by Halsey equation with FAAS correction. doi:10.1371/journal.pone.0141013.g002 doi:10.1371/journal.pone.0141013.g002 loops. doi:10.1371/journal.pone.0141013.g002 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Table 1. Pore characteristics of PPAC and MPPACs. Sample PPAC MPPAC-Fe(3.8%) MPPAC-Fe(7.8%) MPPAC-Fe(8.6%) Total surface areaa (m2 g-1) 776.5 684.9 585.0 568.1 BET surface area (m2 g-1) 754.8 666.5 571.5 555.5 Micropore surface area (m2 g-1) 603.7 (77.7%b) 503.2 (73.5%) 446.3 (76.3%) 408.2 (71.9%) BJH surface areac (m2 g-1) 88.9 (11.4%) 111.2 (16.2%) 96.2 (16.4%) 109.1 (19.2%) Total pore volume (cm3 g-1) 0.39 0.40 0.40 0.43 Micropore volumed (cm3 g-1) 0.28 0.23 0.20 0.19 BJH pore volumee (cm3 g-1) 0.084 (21.5%) 0.150 (37.5%) 0.183 (45.8%) 0.229 (53.3%) pHPZC 9.68 NM f NM 9.11 a Single point surface area at p/p° = 0.201775532. b Portions of speciﬁc surface area [(speciﬁc surface area/total surface area)×100%]. c,d BJH desorption cumulative volume of pores between 17.000 Å and 3,000.000 Å width. e Single point adsorption at p/p° = 0.995. f not measured. Table 1. Pore characteristics of PPAC and MPPACs. a Single point surface area at p/p° = 0.201775532. doi:10.1371/journal.pone.0141013.t001 magnetic field. Fig 3B shows an illustration of the separation phenomena of MPPAC-Fe(3.8%) with a nickel-plated neodymium magnet (1.25 T). The complete separation took ~30 s. FTIR analysis was conducted to detect the presence of various organic groups on the surface of PPAC, MPPAC-Fe(3.8%), and -Fe(8.6%) [Fig 3C]. Two wide bands of the hydroxyl (OH-) group at 3238 and 3309 cm-1 were seen for both MPPAC-Fe(3.8%) and -Fe(8.6%), while they were hardly seen for PPAC. Instead, PPAC had two peaks at 2979 and 2877 cm-1, indicating the presence of CH stretching [26]. Additionally, a peak at 1544 cm-1 in the IR spectrum of PPAC indicated C = C of an aromatic ring [27]. When Fe was impregnated, the C = C aromatic signal disappeared, but carbonyl (C = O) and phenolic groups were found at 1627/1639 and 1415/1448 cm-1 for 3.8/8.6% Fe-impregnated media [28]. Also, the 3.8% Fe-impregnated medium had a smaller C = O signal than 8.6%. Based on these results, it can be concluded that the Fe impregnation using ultrasound resulted in oxidation of the C = C group, forming phe- nolic and C = O groups, although the degree of oxidation differed. Sharp peaks at 928 and 887 cm-1 were found for 3.8 and 8.6% Fe-impregnated media, while PPAC did not show those peaks. These peaks correspond to the intrinsic stretching vibration of Fe at a tetrahedral site. Furthermore, MPPAC-Fe(8.6%) had a peak at 790 cm-1, assigned to an octahedral Fe-stretch- ing vibration [28]. Characterization As an important magnetic parameter, the saturation magnetization (Ms) of MPPAC-Fe (3.8%), -Fe(7.8%), and -Fe(8.6%) were 2.3, 6.2, and 9.8 emu g-1, respectively. Thus, the mag- netic saturation increased as the amount of Fe coated on the PPAC increased. This magnetic property can facilitate ready separation of the media from the suspension by an external 6 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Kinetics with various ionic strength and pH (Fig 4A–4C) shows the kinetics of IBP uptake by PPAC or MPPACs in different ionic strengths at pH 7.0. All media showed rapid sorption rates initially, during the first 100 min, and pseudo-equilibrium was reached in 200 min. The rapid initial adsorption may present a more accessible surface as well as high availability of active sites, whereas the observed plateau corre- sponds to a slower rate of adsorption due to the accumulation of IBP on the active sites. At 0 M ionic strength, PPAC had the highest initial sorption rate for 50 min, while MPPAC-Fe(3.8%) had a higher sorption speed and capacity than other media at 0.1 M ionic strength. In all cases, the sorption capacities and rates of MPPAC-Fe(7.8%) and -Fe(8.6%) were similar, but lower than that of MPPAC-Fe(3.8%). Through a pseudo-second order kinetic model, the experimental data showed determina- tion coefficients (R2) higher than 0.99, indicating reliable fitting. The data obtained for each medium, in terms of values of qeq, k2, and v0, are compared in Table B in S1 File. The PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 7 / 18 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 3. (a) Magnetization versus applied magnetic field for all MPPAC materials and (b) shows settling time of the MPPAC from suspension by external magnet, (c) FTIR spectrum for PPAC, MPPAC–Fe (3.8%) and–Fe(8.6%). doi:10.1371/journal.pone.0141013.g003 Fig 3. (a) Magnetization versus applied magnetic field for all MPPAC materials and (b) shows settling time of the MPPAC from suspension by external magnet, (c) FTIR spectrum for PPAC, MPPAC–Fe (3.8%) and–Fe(8.6%). doi:10.1371/journal.pone.0141013.g003 Fig 3. (a) Magnetization versus applied magnetic field for all MPPAC materials and (b) shows settling time of the MPPAC from suspension by external magnet, (c) FTIR spectrum for PPAC, MPPAC–Fe (3.8%) and–Fe(8.6%). doi:10.1371/journal.pone.0141013.g003 equilibrated adsorption capacities increased for all media as the ionic strength increased from 0 to 0.5 M. This can be determined by the salting-out effect. That is, as the ionic strength increases, the solubility of IBP decreases so that the sorption onto the medium can be enhanced [29]. Overall, MPPAC-Fe(3.8%) showed qeq of 80.7–94.1 mg g-1, which was comparable to PPAC (76.3-94.7 mg g-1). Based on the total surface area, MPPAC-Fe(3.8%) had a higher sorp- tion density (0.118–0.137 mg m-2) than PPAC (0.098–0.122 mg m-2). equilibrated adsorption capacities increased for all media as the ionic strength increased from 0 to 0.5 M. Kinetics with various ionic strength and pH This can be determined by the salting-out effect. That is, as the ionic strength increases, the solubility of IBP decreases so that the sorption onto the medium can be enhanced [29]. Overall, MPPAC-Fe(3.8%) showed qeq of 80.7–94.1 mg g-1, which was comparable to PPAC (76.3-94.7 mg g-1). Based on the total surface area, MPPAC-Fe(3.8%) had a higher sorp- tion density (0.118–0.137 mg m-2) than PPAC (0.098–0.122 mg m-2). 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 4. (a-c) Adsorption kinetics of IBP by various media at pH 7.0 at different ionic strength (0 ~ 0.5 M) (fit lines obtained from Pseudo-second order kinetic model), kinetics of IBP uptake by media at pH 4 (d) and 7 (e) at 0.1 M of ionic strength. doi:10 1371/journal pone 0141013 g004 Fig 4. (a-c) Adsorption kinetics of IBP by various media at pH 7.0 at different ionic strength (0 ~ 0.5 M) (fit lines obtained from Pseudo-second order kinetic model), kinetics of IBP uptake by media at pH 4 (d) and 7 (e) at 0.1 M of ionic strength. doi:10.1371/journal.pone.0141013.g004 (a-c) Adsorption kinetics of IBP by various media at pH 7.0 at different ionic strength (0 ~ 0.5 M) (fit lines obtained from Pseudo-second order c model), kinetics of IBP uptake by media at pH 4 (d) and 7 (e) at 0.1 M of ionic strength. doi:10.1371/journal.pone.0141013.g004 The solution pH is a significant parameter in the sorption process, because it determines the charge of both adsorbent and adsorbate, and thereby governs the sorbent-sorbate electrostatic interaction [30]. Accordingly, pH plays a dominant role in determining the sorption capacities for all types of medium [16]. With a pKa value of 4.91, when pH values are higher than pKa, the IBP molecules convert to anionic species, and almost all will be deprotonated at pH 7. Based on the calculated log D value (Table A in S1 File) of IBP at pH 4 and 7, this indicates that with a higher value of log D, the hydrophobicity of the molecules increases. Thus, the hydro- phobicity of IBP is higher at a pH lower than the pKa. The pHPZC value (9.11) of MPPAC-Fe(3.8%) was lower than that (9.68) for PPAC (Fig B in S1 File.). PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Isotherm Adsorption isotherm data were fitted to the Langmuir and Freundlich models (Fig 5) and were used to evaluate the Qmax of PPAC and MPPACs for IBP uptake. The equilibrium data and other calculated values are presented in Table 2. Based on the values of the determination coef- ficients (R2), the Freundlich model better fitted the data for PPAC and MPPAC-Fe(3.8%), whereas the Langmuir model was best MPPAC-Fe(7.8%) and -Fe(8.6%). As a function of adsorption strength, the values of 1/n for PPAC and MPPACs were 0.12-0.31, which were < 1, indicating that the adsorption is favorable under the conditions studied. MPPAC-Fe(3.8%) had the highest Qmax (157.3 mg g-1), which is about 2.2-fold higher than PPAC (72.7 mg g-1). Based on the total surface area, MPPAC-Fe(3.8%) had a sorption density 0.23 mg m-2, which is 2.5-fold higher than PPAC (0.094 mg m-2). g g ( g ) To verify the removal mechanism of IBP by PPAC and MPPACs, a sole magnetite/maghe- mite was synthesized with the same procedure as for the MPPACs, and its adsorption capacity was tested at 5 mg L-1 IBP. Adsorption tests showed that magnetite/maghemite had no sorp- tion capacity. Thus, dispersive π-π interactions between the aromatic ring of IBP and hexago- nal graphite of the activated carbon can be proposed to be the major removal mechanism of IBP by PPAC or MPPACs [34]. As shown with FTIR, the chemical properties of the pore sur- face for MPPACs differed from that of PPAC. The high sorption density of IBP for MPPAC-Fe (3.8%) may be linked to the C = O group created by the oxidation of the carbon surface during magnetite/maghemite impregnation. Guedidi et al. suggested that IBP was attracted mainly to the C = O surface group, where the carbonyl group serves as an electron donor, while the aro- matic ring of IBP as the acceptor, leads to the formation of donor-acceptor complexes. How- ever, as shown by the fact that MPPAC-Fe(3.8%) exhibited lower levels of C = O groups and a higher sorption capacity than MPPAC-Fe(8.6%), a slightly oxidized carbon surface may pro- mote IBP removal because pore structures could be blocked when higher amounts of the oxy- genated group are formed during the oxidation process [32]. As additional evidence, Kyzas et al. Isotherm reported that the adsorption capacity of pharmaceutical compounds for 4 h-oxidized AC was 117 mg g-1, while 5 h of oxidation showed a decrease in adsorption capacity (91 mg g-1), although the total oxygen functional group increased twofold [35]. Fig 6 shows the overall syn- thesis route of MPPACs and a plausible IBP removal mechanism. Kinetics with various ionic strength and pH Although Fe-impregnated AC had a lower pHPZC, both media showed a predomi- nantly basic nature caused by delocalization of π electrons on the carbon plane [31]. However, as shown in the FTIR analysis, acidic oxygen functional groups formed on MPPACs, decreas- ing the pHPZC value due to their acidic properties [25, 32]. The equilibrated adsorption capacities of IBP by PPAC, MPPAC-Fe(3.8%), and MPPAC-Fe (8.6%) were 111.7, 113.9, and 87.6 mg g-1 at pH 4, respectively, which were 1.23-1.35-fold higher than those at pH 7 [(Fig 4D and 4E)]. Similar to previous results, MPPAC-Fe(3.8%) had better adsorption capacities than the other media under both pH conditions. Moreover, the 9 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water kinetic parameters, K2 (4.88–5.98×10−3 g mg-1 min-1) and v0 (2.72–4.08 mg g-1 min-1), were higher at pH 7 than at pH 4. Thus, under neutral conditions, a faster sorption rate was observed. In terms of kinetics, a neutral pH was ‘better’ than pH 4 due to the electrostatic inter- actions between anionic IBP and the positively charged surface of the media at pH lower than pHPZC [33]. At pH 4, IBP exists mostly as a non-ionic species (89 mole %), restricting the favorable electrostatic interaction. Nevertheless, pH 4 showed a higher adsorption capacity than pH 7. As the main removal mechanism, the aromatic ring of IBP was positioned at the oxygen molecule of the C = O group as a donor-acceptor complex, as well as dispersive π-π interactions with the carbon surface. However, as a negatively charged species, IBP at pH 7 (55 mole %), it is difficult to have a molecular position due to the electrostatic interaction between the carboxylic group and the positively charged groups on the carbon surface. doi:10.1371/journal.pone.0141013.g005 Temperature effect and thermodynamic parameters Temperature dependency for IBP uptake by MPPAC-Fe(3.8%) was studied using isotherm tests at pH 7. All isotherm data were fitted to the Langmuir and Freundlich models. As a result, both isotherm models provided the good fits (R2 = 0.98–0.99). Thermodynamic parameters were also calculated using the isotherm data. At various temperatures, the Qmax values obtained 10 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 5. (a-d) Adsorption isotherms of IBP uptake by media at pH 7.0 and 0.1 M ionic strength, Langmuir (long dashed line) and Freundlich models (solid line). Fig 5. (a-d) Adsorption isotherms of IBP uptake by media at pH 7.0 and 0.1 M ionic strength, Langmuir (long dashed line) and Freundlich models (solid line). Fig 5. (a-d) Adsorption isotherms of IBP uptake by media at pH 7.0 and 0.1 M ionic strength, Langmuir (long (solid line). doi:10.1371/journal.pone.0141013.g005 with the Langmuir model were similar, in the range of 127.5–131 mg g-1 (Fig C in S1 File). The Langmuir and Freundlich constants, KL and KF, were also comparable, although adsorption affinity to IBP increased slightly at higher temperatures. This was also confirmed by the increase in ‘n,’ indicating higher adsorption affinity. g g y Based on the Kd values calculated at 50 mg g-1 sorption capacity, the estimated ΔG° were -1.69, -3.67, and -7.99 kJ moL-1 at 298, 308, and 318 K, respectively. Generally, the low range of Table 2. Fitting parameters to the Freundlich and Langmuir models. Model Parameters PPAC MPPAC-Fe(3.8%) MPPAC-Fe(7.8%) MPPAC-Fe(8.6%) Langmuir KL (g mg-1 min-1) 21.8 0.74 0.52 0.28 Qmax (mg g-1) 72.7 157.3 100.6 114.7 R2 0.989 0.947 0.993 0.837 Freundlich KF (g mg-1 min-1) 56.3 75.1 52.1 42.3 1/n 0.12 0.31 0.21 0.3 R2 0.997 0.978 0.977 0.78 doi:10.1371/journal.pone.0141013.t002 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 11 / 18 Table 2. Fitting parameters to the Freundlich and Langmuir models. 11 / 18 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 6. Synthesis route for MPPAC and possible IBP adsorption mechanism on activated carbon. Fig 6. Synthesis route for MPPAC and possible IBP adsorption mechanism on activated carbon. doi:10.1371/journal.pone.0141013.g006 doi:10.1371/journal.pone.0141013.g006 doi:10.1371/journal.pone.0141013.g006 ΔG° (0 to -20 kJ mol-1) can be defined as physi-sorption, while the high range (-80 to -400 kJ mol-1) can be interpreted as chemi-sorption [36]. Thus, IBP adsorption by MPPAC-Fe(3.8%) was a spontaneous physi-sorption process. Temperature effect and thermodynamic parameters The calculated ΔH° and ΔS° values were 6.35 kJ mol-1 and 21.6 J K-1 mol-1, respectively. Due to the positive value of ΔH°, the adsorption of IBP by MPPAC-Fe(3.8%) can be defined as an endothermic process. Although the positive value of ΔS° is indicative of the increase in disorder at the solid-solution interface due to the desorption of water, the ΔS° value for MPPAC-Fe(3.8%) was lower than other reference values (Table 3). This indicates that adsorption of IBP is less competitive with water molecules. As a result, the parabolic diffusion had the highest R2 for all cases, suggesting that the adsorption of IBP was diffusion-controlled (Table D in S1 File) [39, 40]. The depletion time was also obtained by fitting data with the pseudo-sec- ond order kinetic model (Fig 7E). As K2 and v0 decreased, the depletion time of IBP increased, to 36-67 min in the 10th-13th cycles. had the highest removal speed for IBP [Fig 7A]. The kinetic results demonstrated that IBP was removed completely by MPPAC-Fe(3.8%) within 6 min, except for the first cycle, which required 10 min for completion. In the case of MPPAC-Fe(8.6%), all IBP was removed completely within 30 min during the second to fourth cycles, but about 0.16 mg L-1 IBP remained at 30 min for the first cycle. This can be explained by the establishment of Na+ layer on the media’s surface during the first cycle. PGAC did not remove IBP completely, and its removal efficiency decreased gradually from 58 to 51% over four cycles. Thus, PGAC and MPPACs showed different removal phenomena as the number of cycles increased. As shown in the kinetic results, the removal of IBP by PPAC was hindered by anionic Cl- while MPPACs could have promoted removal by the Donnan effect as Na+ is accumulated on the surface of magnetite/maghemite. Through fitting data with the pseudo-second-order kinetic model, MPPAC-Fe(3.8%) had the highest K2 (0.76–1.16 g mg-1 min-1) and v0 (4.96–7.5 mg g-1 min-1), respectively. The kinetic comparison showed that K2 of MPPAC-Fe(3.8%) was about 1.9~2.8- and 9.1~15.8-fold higher than MPPAC-Fe(8.6%) and PGAC, respectively. For the case of v0, MPPAC-Fe(3.8%) was 1.8~2.5- and 24.6~35.4-fold higher than MPPAC-Fe(8.6%) and PGAC. g A second phase of repeated adsorption tests using MPPAC-Fe(3.8%) was conducted for 13 cycles with a shortened operational time (15 min). Although, except for the first cycle, IBP was almost depleted at 15 min in each cycle during the second to ninth cycles, a slower adsorption rate was observed compared with the first phase [Fig 7B]. The K2 and v0 obtained for 13 cycles were 0.1~0.18 g mg-1 min-1 and 0.8~1.3 mg g-1 min-1, respectively [Fig 7C and 7D]. Compared with the first phase, the reduction of these values occurred due to a shortened operation time, with which diffused IBP previously adsorbed can block the diffusion of IBP in subsequent cycles. Hypothetically, this happened due to the fact that the rate-determining step of IBP adsorption is pore diffusion, because most sorption sites are at micropores (< 2 nm). To prove this hypothesis, several kinetic models, such as the pseudo-first order, pseudo-second order, simple Elovich, power function, and parabolic diffusion, were applied to find the values of R2 through fitting kinetic data (Table C in S1 File). As a result, the parabolic diffusion had the highest R2 for all cases, suggesting that the adsorption of IBP was diffusion-controlled (Table D in S1 File) [39, 40]. The depletion time was also obtained by fitting data with the pseudo-sec- ond order kinetic model (Fig 7E). As K2 and v0 decreased, the depletion time of IBP increased, to 36-67 min in the 10th-13th cycles. PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water had the highest removal speed for IBP [Fig 7A]. The kinetic results demonstrated that IBP was removed completely by MPPAC-Fe(3.8%) within 6 min, except for the first cycle, which required 10 min for completion. In the case of MPPAC-Fe(8.6%), all IBP was removed completely within 30 min during the second to fourth cycles, but about 0.16 mg L-1 IBP remained at 30 min for the first cycle. This can be explained by the establishment of Na+ layer on the media’s surface during the first cycle. PGAC did not remove IBP completely, and its removal efficiency decreased gradually from 58 to 51% over four cycles. Thus, PGAC and MPPACs showed different removal phenomena as the number of cycles increased. As shown in the kinetic results, the removal of IBP by PPAC was hindered by anionic Cl- while MPPACs could have promoted removal by the Donnan effect as Na+ is accumulated on the surface of magnetite/maghemite. Through fitting data with the pseudo-second-order kinetic model, MPPAC-Fe(3.8%) had the highest K2 (0.76–1.16 g mg-1 min-1) and v0 (4.96–7.5 mg g-1 min-1), respectively. The kinetic comparison showed that K2 of MPPAC-Fe(3.8%) was about 1.9~2.8- and 9.1~15.8-fold higher than MPPAC-Fe(8.6%) and PGAC, respectively. For the case of v0, MPPAC-Fe(3.8%) was 1.8~2.5- and 24.6~35.4-fold higher than MPPAC-Fe(8.6%) and PGAC. A second phase of repeated adsorption tests using MPPAC-Fe(3.8%) was conducted for 13 cycles with a shortened operational time (15 min). Although, except for the first cycle, IBP was almost depleted at 15 min in each cycle during the second to ninth cycles, a slower adsorption rate was observed compared with the first phase [Fig 7B]. The K2 and v0 obtained for 13 cycles were 0.1~0.18 g mg-1 min-1 and 0.8~1.3 mg g-1 min-1, respectively [Fig 7C and 7D]. Compared with the first phase, the reduction of these values occurred due to a shortened operation time, with which diffused IBP previously adsorbed can block the diffusion of IBP in subsequent cycles. Hypothetically, this happened due to the fact that the rate-determining step of IBP adsorption is pore diffusion, because most sorption sites are at micropores (< 2 nm). To prove this hypothesis, several kinetic models, such as the pseudo-first order, pseudo-second order, simple Elovich, power function, and parabolic diffusion, were applied to find the values of R2 through fitting kinetic data (Table C in S1 File). Repeated adsorption The first repeated removals of IBP were conducted to compare the rate of IBP uptake for each medium and cycle. Each cycle was operated for 30 min. Among the media, MPPAC-Fe(3.8%) Table 3. Gibb’s free energy change, enthalpy change and entropy change for IBP adsorption. Media Kelvin (K) Gibbs free energy, ΔG° (kJ mol-1) Isosteric enthalpy, ΔH° (kJ mol-1) Entrophy of adsorption, ΔS° (J mol- 1 K-1) Reference Cork waste AC 298–313 -7.3 8.1 51.6 [32] Clothes AC 298–328 -1.0 60.2 205.4 [37] Artemisia vulgaris AC 298–318 -1.75 to -5.73 57.5 198.2 [38] MPPAC-Fe(3.8%) 298–318 -1.69 to -7.99 6.35 21.61 This study doi:10.1371/journal.pone.0141013.t003 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 12 / 18 alpy change and entropy change for IBP adsorption. Table 3. Gibb’s free energy change, enthalpy change and entropy change for IBP adsorption. Table 3. Gibb’s free energy change, enthalpy change and entropy change f PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 12 / 18 Thermal regeneration and re-adsorption Thermal regeneration and re-adsorption tests were conducted six times (Fig 8). MPPACs showed better re-adsorption capability than PPAC. For each cycle, PPAC and MPPACs showed different adsorption patterns. In the case of PPAC, the sorption capacity was reduced sharply after the second cycle. At the sixth cycle, the sorption capacity was only 13 mg g-1. Both MPPAC-Fe(3.8%) and MPPAC-Fe(8.6%) showed increases in sorption capacities at the second cycle, but the sorption capacities decreased gradually to 70 and 86 mg g-1 at the sixth cycle, respectively, which were 41 and 22% reductions compared with the first sorption values. Based on the results obtained, we suggest that the magnetite/maghemite have thermal cata- lytic effects to reduce the desorption energy [41, 42]. Moreover, the low-temperature desorp- tion of IBP could indicate that the adsorption was linked to physi-sorption rather than chemi- sorption [43]. The reduction of adsorption capacities after several cycles is due to incomplete desorption of IBP adsorbed in micropores or exhaustion of functional groups. However, fur- ther investigations should be performed to determine the optimal regeneration temperature without damaging the carbon pore structures. The magnetic properties of MPPAC were not changed even after six cycles of regeneration. Other regeneration method such as using acid- PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 13 / 18 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water base [44] for desorption would be unfavorable due to dissolution of m condition. Fig 7. Repetitive removal of 5 mg L-1 IBP at pH 7.0 with 20 mg absorbent (a) 4 continuous cycles with MPPAC-Fe(3.8%), M and (b) IBP removal using MPPAC-Fe(3.8%) with 13 successive cycles (Long-dashed lines denote pseudo-second orde adsorption rate (k2), initial adsorption rate (v0) and depleted time (min) was determined through pseudo second order fit doi:10.1371/journal.pone.0141013.g007 Fig 7. Repetitive removal of 5 mg L-1 IBP at pH 7.0 with 20 mg absorbent (a) 4 continuous cycles with MPPAC-Fe(3.8%), MPPAC-Fe(8.6%) and GAC, and (b) IBP removal using MPPAC-Fe(3.8%) with 13 successive cycles (Long-dashed lines denote pseudo-second order kinetic), (c-e) The adsorption rate (k2), initial adsorption rate (v0) and depleted time (min) was determined through pseudo second order fitting. d i 10 1371/j l 0141013 007 doi:10.1371/journal.pone.0141013.g007 base [44] for desorption would be unfavorable due to dissolution of magnetite low pH condition. base [44] for desorption would be unfavorable due to dissolution of magnetite low pH condition. Thermal regeneration and re-adsorption PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 14 / 18 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Fig 8. Adsorption capacities of saturated adsorbents after 6 cycles of thermal regeneration at 623.15 K. doi:10.1371/journal.pone.0141013.g008 Fig 8. Adsorption capacities of saturated adsorbents after 6 cycles of thermal regeneration at 623.15 K. doi:10.1371/journal.pone.0141013.g008 Fig 8. Adsorption capacities of saturated adsorbents after 6 cycles of thermal regeneration at 623.15 K. doi:10.1371/journal.pone.0141013.g008 Fig 8. Adsorption capacities of saturated adsorbents after 6 cycles of thermal regeneration at 623.15 K. doi:10.1371/journal.pone.0141013.g008 doi:10.1371/journal.pone.0141013.g008 PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 Supporting Information S1 File. Media characterizations and adsorption parameters. Physicochemical properties of ibuprofen (Table A). Comparisons of adsorption kinetics of IBP uptake by media at different ionic strength (Table B). Linear plots and equation for 5 different kinetic models (Table C). Determination Coefficient (R2) of GAC, MPPAC-Fe(3.6%) and MPPAC-Fe(8.6%) through data fitting with different kinetic models. Through phase 1, all adsorbent were continuously adsorb for 4 times while in 2nd phase, only MPPAC-Fe(3.8%) were repeated 14 times (Table D). Relationship between Fe contents with pore characteristics results (Fig A). pHZPC of PPAC and MPPAC–Fe(3.8%) with deionized water and 0.1 M NaCl (Fig B). Temperature effect on adsorption isotherm of IBP: MPPAC-Fe(3.8%) at pH 7. Line curve: Langmuir model; Long dashed curve: Freundlich model (Fig C). (DOCX) Acknowledgments The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/qh3zQe. Conclusions Via an economic and scalable preparation method, we successfully synthesized MPPAC where magnetite/maghemite was deposited homogeneously on the outer surface of PPAC. Based on the results of FTIR, it was found that Fe impregnation using ultrasound resulted in oxidation of the surface carbon of PPAC, to form phenolic and C = O groups. Remarkably, isotherm results showed that MPPAC-Fe(3.8%) had about a 2.2-fold higher Qmax (157.3 mg g-1) and a 2.5-fold higher sorption density (0.23 mg m-2) than PPAC. Based on the overall results, the major mechanism of IBP removal was found to be donor-acceptor complexes, with a C = O group and dispersive π-π interaction with a carbon surface. With the repeated adsorption tests, it was found that pore diffusion was the rate-determining step and MPPAC-Fe(3.8%) had a much faster removal capacity than MPPAC-Fe(8.6%) and PGAC. Magnetized activated carbon showed better regeneration than PPAC due to a thermal catalytic effect of magnetite/maghe- mite at a low thermal temperature. PLOS ONE \| DOI:10.1371/journal.pone.0141013 October 23, 2015 15 / 18 Recyclable Magnetized Activated Carbon for Ibuprofen Removal in Water Supporting Information Supporting Information contains the methodology regarding various instrumental analysis, physical and chemical characteristics of IBP, kinetic and isotherm models, and kinetic values for ionic strength effect. Detailed description of data on pore characteristics, adsorption kinet- ics, the relationship between Fe contents with pore characteristics, and temperature effect on adsorption isotherms. Author Contributions Conceived and designed the experiments: KTW YY MJ. Performed the experiments: KTW. Analyzed the data: KTW YY MJ. Contributed reagents/materials/analysis tools: MJ. Wrote the paper: KTW YY MJ. Conceived and designed the experiments: KTW YY MJ. Performed the experiments: KTW. Analyzed the data: KTW YY MJ. Contributed reagents/materials/analysis tools: MJ. Wrote the paper: KTW YY MJ. 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https://openalex.org/W3179036914	https://journal.unhas.ac.id/index.php/fs/article/download/10931/6972	English	null	Analysis and interpretation of forest fire data of Sikkim	Forest and Society	2,021	cc-by	8,862	Kapila Sharma 1,, Gopal Thapa 2 Kapila Sharma 1,, Gopal Thapa 2 1 Assistant Professor, Sikkim Manipal Institute of Technology, India; kapila.s@smit.smu.edu.in 2 Assistant Professor, Sikkim Manipal Institute of Technology, India; gopal.t@smit.smu.edu.in * Correspondence author: kapila.s@smit.smu.edu.in, Tel.: 7908112860 1 Assistant Professor, Sikkim Manipal Institute of Technology, India; kapila.s@smit.smu.edu.in 2 Assistant Professor, Sikkim Manipal Institute of Technology, India; gopal.t@smit.smu.edu.in * Correspondence author: kapila.s@smit.smu.edu.in, Tel.: 7908112860 1 Assistant Professor, Sikkim Manipal Institute of Technology, India; kapila.s@smit.smu.edu.in 2 Assistant Professor, Sikkim Manipal Institute of Technology, India; gopal.t@smit.smu.edu.in * Correspondence author: kapila.s@smit.smu.edu.in, Tel.: 7908112860 Abstract: Forest ecosystems are depleting and heading towards degradation which would adversely affect the world's socio-economic harmony. Various disasters disturb the cordial relationship of the flora and fauna and impose imbalance in the ecology as a whole; forest fire is one of its kind. India has witnessed a 125% rise in forest fire occurrences between the years 2015 and 2017. This paper presents a study of various factors and the analysis of forest fire in Sikkim. The period of 10 years, forest fire incidences, i.e., from the year 2004 to the year 2014 have been considered for the study. The forest fire data was collected from Forest and Environment Department, Government of Sikkim, and preliminary processing was performed to check for anomalies. The study observed that there has been an increased forest fire incidence over the years and highest being in the year 2009. These fire incidences have damaged a total area of 5,047.16 ha of land damaging various flora and fauna. It was observed that the maximum forest fire cases are below an altitude of 1500m, during winter months (December to February extending to March) and in sub-tropical Sal (Shorea robusta) forest. West district of Sikkim recorded the highest number of forest fire incidences and area covered followed by south and east districts; the north district was least affected. As per the visual interpretation of forest fire incidence data and literature review, the main factors responsible for forest fire in Sikkim are low rainfall, dry winter season, and type of vegetation. Also, a linear regression was performed between weather factors like average temperature (°C), relative humidity (%), and wind velocity (Km/h) on incidences of forest fire between the year 2009-2014 (n=389). It was found that the average temperature (r=0.37, Slope=9.59 and SD= ±12.00) and relative humidity (r=-0.6, Slope=-4.52, and SD=±2.68) plays a moderate linear relationship in influencing the incidences of forest fires. Kapila Sharma 1,*, Gopal Thapa 2 However, wind velocity showed almost a flat curve indicating its minimal role in influencing forest fire incidences. Parameter modelling and preparation of forest fire risk zone map would be an effective tool in preventing and managing forest fire in Sikkim. Keywords: Forest fire; Biodiversity; Ecology; Sikkim; Linear regression Keywords: Forest fire; Biodiversity; Ecology; Sikkim; Linear regression Received: 2020-08-01; Accepted: 2021-06-21 Copyright © 2021 by Forest and Society. This work is licensed under a Creative Commons Attribution 4.0 International Licen ; p right © 2021 by Forest and Society. This work is licensed under a Creative Commons Attribution 4.0 International License. Keywords: Forest fire; Biodiversity; Ecology; Sikkim; Linear regression Forest and Society Vol. 5(2): 261-276, November 2021 http://dx.doi.org/10.24259/fs.v5i2.10931 Forest and Society Vol. 5(2): 261-276, November 2021 http://dx.doi.org/10.24259/fs.v5i2.10931 Review Article 1. Introduction This is evident from the fact that there is a significant decreasing trend in the mean maximum temperature during monsoon and a decrease in cold wave conditions, resulting in a rise in average minimum temperature, which is the main driving factor for forest fire (Sharma et al., 2014). However, finding climatic data in Sikkim is a major problem as the data collecting station is absent in most areas of concern. In Sikkim state, the forest fire data and studies have revealed that the fire occurrences are mostly ground fires that destroy the biodiversity of the forests to a great extent. Change in rainfall regime, prolonged dry season, and decrease in rainfall are the major factors responsible for a forest fire in Sikkim (Sharma et al., 2012). From literature, the most forest fire in Sikkim is observed to be due to anthropogenic activities (Sharma et al., 2012). Accidental forest fire is caused mainly when local farmers and cattle herders make fire intending to keep away the wild animals from the agricultural fields and wave out the broom grass to improve soil fertility (Sharma et al., 2014; Stocks et al; 1997). Similar is the case in the Asian context. The 1997 forest fires in South East Asia were completely human-made, which lead to numerous health problems in Singapore, Malaysia, and Brunei (Frankenberg et al., 2005). 95% of such fires occurred in spring and autumn in Mongolia, China (Goldammer, 2001). Land clearing by slash and burn method is attributed as a common cause of forest fire in South East Asia (Varma, 2003; Yong et al., 2016). In Japan, though the climate is humid but yearly, more than 4000 forest fire cases were recorded damaging 4000 ha of forests in the 1980s and 2300 ha in the 1990s (Zorn et. al., 2001), and it was estimated that 99% of these fires are human- made (Nakagoshi et al., 1987). The analysis of forest fire data of south Asia found that moist deciduous forest, followed by dry deciduous forest and semi-evergreen forest, were the most vulnerable forest types in these regions (Reddy et al., 2017). In Indonesia during 1997-98 damaged 5.2 million hectares of forest in East Kalimantan, which was primarily due to the EI-Nino southern oscillation (ENSO) event bringing drought to the area (Hoffmann et al., 1999). 1. Introduction Forests are dynamic complex structures with varied vegetation, trees performing diverse roles like weather adjustments, carbon level maintenance, the dwelling place for various animals and birds, the livelihood of humans, and above all a source of economic growth by providing the resource i.e., wood (Gigović et al., 2018; Saklani, 2008; Sarab et al., 2014). Forest fire is the natural or deliberate burning of the various fuels in the forest bed or trees. Forest fire adversely affects forest diversity. It reduces forest cover, destroys wildlife, depletes environmental conditions, degrades soil quality, and affects forest regeneration (Kalantar et al., 2020). In addition to the loss of forest biodiversity, the emission of pollutant gases like carbon monoxide, nitrogen oxide, and sulfur oxide from the forest fire leads to global warming, climatic changes, and water level imbalance which affects human health (Rather et al., 2018; Joseph et al., 2009). It is estimated that the forest fire emits about 8 billion tons of CO2 per year (Van Der Werf et al., 2017). In India, total CO2 outflow by forest fire was found to be 98.11 Tg in 2014 (Reddy et al., 2017). In Sikkim, as the winters are increasingly becoming warmer and drier, the subtropical Sal forests are becoming vulnerable to ground fires, affecting the regeneration of ground flora. The dry winter season that extends from December to March is the main forest fire season in Sikkim (Sharma et al., Forest and Society. Vol. 5(2): 261-276, November 2021 262 2012). Among all the forest types, East Himalayan Sal (3C/C1a (I)) has the highest forest fire chances. Sub-tropical forests and Sal Forest showed the highest percentage of fire burnt areas as detected using satellite imagery (Sharma et al., 2012). The calorific value is high in oak trees; the fire takes the form of a canopy and burns for several days, affecting the regeneration of these species. Such fire occurred in 1970 in the west district of Sikkim; the coniferous and oak forests have not yet recovered (Sharma et al., 2014). Increasing incidents of forest fires in Sikkim have resulted in forest loss and ecological imbalance. As per the 2009 forest fire data of Sikkim, 905 ha of the area was damaged. The dry season is becoming more dryer and wet wetter. 1. Introduction It was found that ground fires are frequent in Bhawal National Park, Bangladesh, which destroyed substantial vegetation (Alauddin et al., 2020). A study in Madupur Sal forest, Bangladesh, showed that forest fires are the major causes of deforestation, which are mostly due to illegal human activities and sometimes naturally during the summer season (Hossain et al., 2015). Large forest fire occurrences of different intensities are recorded every year in Asian countries. The 2009 fire occurrences in Nepal lead to many deaths and destruction of human settlements (Matin et al., 2017; Berwyn et al., 2018). In 2019, 35,000 fire incidences were reported in Indonesia, which led to various health problems within and in the neighbouring countries due to air pollution. They also cause various socio- economic issues related to land use, property damage, agriculture, and infrastructure loss (Malik et al., 2013). In 2017 wildfire in Canada produced 20 times more aerosols than Pinatubo's volcanic eruption in 1991 (Liu et al., 2017). As per Global Forest Resource Assessment 2010, a forest fire in Australia caused the death of 70 lives in addition to the loss of agriculture and infrastructure (FAO, 2010). The year 2018 and 2019 saw the most devastating forest fire in Australia and California, which covered 6.3 million hectares and 0.72 million hectares of forest area, respectively, and took the lives of 24 humans and millions of animals (Srivastava, 2020). The study reveals that the area burned due to forest fire in the United States increased from 1.62 million ha. In 2002 to 4.05 million ha in 2006, (Satendra & Kaushik, 2014). As per the United Nations Development Program report, the financial losses due to forest fire accounted for Rupees 9000 per hectare per annum (Satendra & Kaushik, 2014). Time of occurrence and area-covered data is crucial for risk assessment and fire zonation (Suryabhagavan et al., 2016; Kappes et al., 2012). Forest and Society. Vol. 5(2): 261-276, November 2021 263 Understanding past forest fire regimes through their locations, burnt area, date, and time plays a crucial role in the prevention and mitigation of major forest fires (Tonini et al., 2020). The present study analyzed and interpreted the historical data of forest fire in the state of Sikkim (2004-2014). 1.1. Factors that contribute to forest fire The vegetation of an area represents the availability of fuel. Fuels are one of the important organic matters that help in fire ignition. The vegetation category, the plant's properties, the species morphology, availability and arrangement of biomass, and cover types are essential for an area's forest fuel characteristics (Keane et al., 2001; Keane et al., 2010). The fuel is available for combustion, the fuel moisture, fuel arrangement, fuel size distribution, and ecosystem influence all aid in fires (Stocks et al., 1997). Dried and decayed vegetation contribute to ground fires and surface fires, respectively. Humus, trees, shrubs, roots, muck, peat, leaves of trees, grasses, weeds, ferns, low bushes, seedlings, saplings of trees, and deadwood are major contributors to ground fires. Standing dried trees, mosses, epiphytic plants, and lichens are aerial fuels (Gupta & Nair, 2012). Different tree types embody various adaptation types to resist forest fire, such as bark thickness, increased moisture level, foliage size, and high oiliness. Wetland vegetation and sparse vegetation are less susceptible to fire (Sharma et al., 2014). Forests mostly consisting of sal, bamboo, pine, chir, deodar, sesame are highly flammable (Rather et al., 2018). Fire generally damages woody plants compared to herbaceous plants because of the growth form and location of the meristematic tissue (Bidwell et al., 2005). Fuels are one of the important organic matters that help fire ignition; hence, the mapping of fuels is essential for forest fire management. Fuel structure in the forest acts as an initiator and facilitator of fire in the forest (Keane et al., 2001). The amount of compressed moisture in the air compared to the total amount that it is capable of holding at that temperature and pressure is called relative humidity, which when reaches 60%, causes chances of forest fire (Malik et al., 2013). Fuels in the forest get dried faster in higher temperatures. As suggested by the intergovernmental panel on climate change 2007, droughts due to drier weather, shortage in rainfall, rise in temperature, and decrease in the level of precipitation are the fundamental reasons for the cause of forest fire in Asia (Berwyn, 2018). There are various changes in the vegetation of the forest, like an increase in dissimilarity index of plant species, increase in supremacy, climatic changes that may increase the vegetation's sensitivity to forest fires (Berwyn, 2018). 1. Introduction The interrelation of the fire incidences in Sikkim with the weather factors like average rainfall (mm), average temperature (°C), relative humidity (%), and wind velocity (Km/h) were also analyzed for the years 2009-2014. This study shall act as a baseline for further studies in these fields with the use of the latest technologies and empirical studies, with the availability of recent year's data. In India, about 6 70,000 km2 of forest land are burnt each year, which is about 2% of the world's forest area, i.e., 35 million hectares of forest area (Rather et al., 2018, Mallik et al., 2013). As per the Forest Survey of India (FSI) Report-1995, about 50% to 54% of Indian forest fires are ground fires (FSI, 1995). In northeastern states, such ground fires occur due to shifting cultivation and human activities (Satendra & Kaushik, 2014). According to FSI report published in 2018, 37,059 fires were detected, of which 64.3% fall under the category of occasional, moderate, and high incidence fire levels, and the rest, 35.71%, have not reported any incidences of fires. India spends nearly 44 million US dollars annually for forest fire patrolling and combating forest fires (Mallik et al., 2013). 1.1. Factors that contribute to forest fire The area burnt is an important parameter for fire trend analysis as it helps estimate carbon emissions by a forest fire (Randerson et al., 2012). Extreme fire can produce a huge amount of CO2 in a very small duration of burning, resulting in global warming, devastating fires, and further warming the land. According to International Energy Agency, the summative emission of CO2 recorded in 2017 was 32.5 billion tons (Berwyn, 2018). Fuel phenological patterns also affect anthropogenic fire seasonal behavior, directly shaping the Spatio- temporal distribution of fire ignitions across the landscape (Bajocco et al., 2017). The slope is another important factor contributing to the forest fire. It is referred to as the gradient of the land expressed in percentage or the land's steepness. With the rise in slope and heating of fuels due to increase heat transmitted by radiation and the transfer of heat from one place to another, the occurrence and spread of fire intensify. The slope acts as a chimney and Forest and Society. Vol. 5(2): 261-276, November 2021 264 accelerates the fire upwards. In the hills where steep slopes are prominent, fire damages vegetation and loosens the organic matter in the soil, leading to erosion and debris flow (Ajin et al., 2016). In steep slopes, where wind speed and closer angle of fire to the ground's surface, convection applies, leading to faster movement of fire. The speed of winds in the downhill is much lesser i.e., 2 to 3mph, than the up slopes i.e., 5 to 10 mph, which intensifies the fire spread (Cannon et al., 2001). The aspect determines the level of sunlight received by the slope. The ignition period is longer in south-facing slopes as they receive more solar radiation; hence, it results in lower relative humidity, less moisture, and high temperature, which tends to dry the fuel. The slope facing north receives lesser sunlight thus is cooler and greener than the southern slope. Therefore, the aspect of an area also plays an essential role in the spread of forest fire. Elevation relatively affects the moisture content of fuel and humidity; the higher the elevation, the land is somewhat cooler hence the lesser chances of fire. Fire usually travels in the direction of the wind, which heats the fuel (Ajin et al., 2016). 1.1. Factors that contribute to forest fire The ill-effects of urbanization and the misuse of forests lead to forest land-use changes, which increase the growth of shrubs and other plants that are highly inflammable. 90% of the fire occurrences in China are due to human activities (Li et al., 2017). The interrelationship between the rise in the ignition and the excessive use of land is a force for the increase in road expansion in North America (Ye et al., 2017). Fire is increasing manifolds with land-use change due to anthropogenic activities, hotter climate, and dry weather. Forest areas nearer to the settlement areas, roads, and cultivated lands are more susceptible to fire, as seen in forested landscapes in tropical, temperate, and subtropical regions of Churia, lowlands in northeast India, and mid-hills (Matin et al., 2017). 2.1 Feature of the Study Area This paper presents a study done on forest fires in the state of Sikkim. The state of Sikkim is located in the northeastern part of the Himalayan Mountain range. It is the second smallest Indian state with an area of 7096 square kilometers. The study site is shown in Figure 1. It lies between the latitude of 27° 5'S to 28° 9' N and longitude of 87° 9'W to 88°56' E that falls in Survey of India topographic sheets of 78A1 to 78A15 series (Sharma et al., 2012). Figure 1. Map showing the location of the state of Sikkim (study area) Figure 1. Map showing the location of the state of Sikkim (study area) 265 Forest and Society. Vol. 5(2): 261-276, November 2021 Figure 2. Map showing Forest types in the state of Sikkim Figure 2. Map showing Forest types in the state of Sikkim The state being small, has been divided into four districts as East, West, North, and South districts. Its altitude ranges from 300 m to over 8586 m above the mean sea level. The annual rainfall ranges between 2700 mm to 3200 mm, and the annual temperature varies from sub-zero during winter to 28 °C during summer (FSI, 2019). Sikkim is bestowed with more than 4000 flowering plants (Sundriyal et al., 2004) and around 30 % of all the birds found in the Indian subcontinent (Ganguli et al., 2011). The Forest of Sikkim is characterized into three major vegetation types viz. Tropical, Temperate, and Alpine (Hooker, 1854). As per the Forest Survey of India’s State of Forest Report 2019, Sikkim has a forest cover of 47.11 % of the state geographic area and has a canopy density class as a very dense forest (15.53 %), moderately dense forest (12.88 %), and open forest (9.70 %). The forests in Sikkim are categorized into six major forest type group viz., Tropical moist deciduous forests, Subtropical broadleaved Hill forests, Montane wet temperate forests, Himalayan moist temperate forests, Subalpine forests and Moist alpine scrub (FSI, 2019). The forest types map is shown in Figure 2. 2.3 Data Preprocessing and interpretation Forest fire data (2004-2014) has been used for the study. After the data was collected, the data was validated and preprocessed to improve the understanding and quality of data. The available forest fire data was segregated concerning; a. Frequency of fire incidences by analyzing the number of fire incidences each year. a. Frequency of fire incidences by analyzing the number of fire incidences each year. b. Year-wise area damaged was analyzed by taking the highest area covered by the forest fire. b. Year-wise area damaged was analyzed by taking the highest area covered by the forest fire. b. Year-wise area damaged was analyzed by taking the highest area covered by the forest fi c. Year-wise area damaged in various forest territorial ranges was segregated, and the range w the highest area damage for each year is analyzed. d. District wise frequency of occurrence of forest fire and area damage by segregating the ye wise data into district wise according to the location of the fire. e. Finally, numerical data tabulated, and a graphical representation of the numerical values performed. f. For map preparation, Arc GIS 10.5.1 was used. Survey of India topographic sheets scale 1:25,000 was geometrically rectified on UTM projection and WGS 84 datum and mosaicking the topographic sheet All ground point locations were collected. Finally, all the ground points were converted into the database and were transferred to the GIS environment (Shapefile format). g. Monthly weather data was collected for all the districts of Sikkim year-wise from (2009-2014). h. Finally, linear regression was performed to determine if the weather factors (2009-2014) like relative humidity (%), average temperature (°C), and wind velocity (km/h) influence fire incidences in Sikkim. h. Finally, linear regression was performed to determine if the weather factors (2009-2014) like relative humidity (%), average temperature (°C), and wind velocity (km/h) influence fire incidences in Sikkim. 2.2 Data collection method The research is based on the secondary data of forest fire (2004-2014). The Forest and Environment Department, Government of Sikkim has collected the GPS location of forest fires immediately after the forest fire incidence. The forest fire data was validated with experts of the Forest and Environment Department, Govt. of Sikkim, and intensive prior study of various related literature and documents of the Forest and Environment Department offices were performed to collect the relevant data for a forest fire. The data's various attributes are date, location, type of fire, time, area damage in a hectare, and species damaged. Weather data was gathered from https://www.worldweatheronline.com. Weather data was available for (2009-2014). The weather Forest and Society. Vol. 5(2): 261-276, November 2021 266 data used for the study were average rainfall (mm), relative humidity (%), average temperature (°C), and wind speed (Km/h). 2.3 Data Preprocessing and interpretation 3. Results Forest fire data for the year 2004-2014 were collected from the Forest and Environment Department, Government of Sikkim. Forest fire incidence places were categorized as four districts of Sikkim depending upon the district the places of fire incidences belonged. 3.1 Visual interpretation of forest fire data (2004-2014) 3.1 Visual interpretation of forest fire data (2004-2014) 3.1 Visual interpretation of forest fire data (2004-2014) Table 1: District-wise total incidences and area damage (2004-20014) for the state of Sikkim. District Incidences (No.) Area damage (ha) East 98 976 West 170 1997 North 16 210.5 South 166 1483 The total fire area was computed district-wise based on the area covered in each place of fire incidence. The segregated data are shown in Table 1. The highest number of fire incidence (170 No.), as well as the area covered (1997 ha), was recorded in the West district of Sikkim followed by the South district with forest fire incidences (166 No.) and area cover (1483 ha). The No. of fire incidence in the East district was comparatively lower than the West and South district of Sikkim i.e., (16 No.) and area damage (976 ha). North district of Sikkim recorded the least number of forest fire incidences (16 No.) and area covered 210.5 (ha). 267 Forest and Society. Vol. 5(2): 261-276, November 2021 Table 2. Major species damage by forest fires (2004-20014) in the state of Sikkim Sl. No. Species damaged No. of fire incidences Common /Local name Scientific name 1 Ground bushes - 261 2 Sal Shorea robusta 112 3 Chilauna Schima wallichii 27 4 Bamboo - 21 5 Teak Tectona grandis 14 6 Dhalne Katus Castanpsis indica 6 7 Patle Katus Castanpsis hystrix 9 8 Salimbo Cynodon dactylon 14 9 Siris Albizia Spp. 8 10 Lampatey Duabangaa sonneratioides 8 11 Thakal Cycas pectinate 8 12 Dhuppi Cryptomeriajaponica 7 13 Champ Mangolia lanuginosa 6 14 Pareng Cephalostachyum hookeriana 5 15 Uttis Alnus nepalensis 4 16 Simul Bombax ceiba 4 17 Gurash Rhodendron arboretum 14 18 Juniper Juniperus recurva 3 19 Kaijal Bischofia javanica 3 20 Panisaj Terminalia myriocarpa 2 21 Chiuri Bassia butyracea 5 22 Odal Sterculia villosa 1 23 Mauwa Engelhardtia spicata 4 24 Chir pine Pinus roxburghii 3 Table 2. Major species damage by forest fires (2004-20014) in the state of Sikkim Species damaged Table 2 depicts major species damaged in the forest fire incidences over the period from the year 2004 to 2014 in the state of Sikkim. In most incidences (58%) ground bushes have been damaged. 24.88% of incidences involved damage of Sal (Shorea robusta) species. Damages of Schima wallichii in 4.88% of incidences, Rhododendron arboretum in 3.11% of incidences, Bamboo and Cynodon dactylon in 3.11% of incidences were observed which are considerable. 3.1 Visual interpretation of forest fire data (2004-2014) 268 Forest and Society. Vol. 5(2): 261-276, November 2021 Figure 3. Map showing the forest fire incidences recorded between 2004-2014 in the state of Sikkim. Figure 3. Map showing the forest fire incidences recorded between 2004-2014 in the state o Sikkim. Figure 3 depicts forest fire area wise and district wise respectively the forest fire incidences in the state of Sikkim. Figure 3 indicates that the maximum forest fire incidences have occurred in the low altitude areas of the South, East, and West district which is also corresponding to the larger area damaged by the forest fire. Also, the incidences are more in the fragmented forest areas in comparison to the continuous unfragmented forests. The forest fire incidences are comparatively low in the Wildlife Protected Areas. Figure 4. Year-wise Forest fire incidences in Sikkim. Figure 4. Year-wise Forest fire incidences in Sikkim. Forest and Society. Vol. 5(2): 261-276, November 2021 269 Figure 4 depicts the yearly number of incidences of forest fires in Sikkim. From Figure 4, it is evident that the forest fire frequency has increased over the years. However, it cannot be said that the frequency of the forest fires in Sikkim is continuously increasing as some years like the year 2007, 2010, and 2013 has shown a decrease in forest fire incidences than the preceding year. The year 2009 shows an abrupt increase of forest fire incidences (31.4%) in the total number of incidences that occurred between 2004-2014, while the year 2004 showed the lowest incidences (1.8%) on the total number of incidences between the year 2004-2014. Figure 5. Year-wise fire area coverage due to the forest fire in Sikkim. Figure 5. Year-wise fire area coverage due to the forest fire in Sikkim. Figure 5 shows the total area damaged due to a forest fire during 2004-2014. The maximum area cover was recorded in 2009 i.e. (36.6%) of the total area covered by the forest fire. The least area covered was recorded in 2004 i.e. (4.24 %) of the total area covered by the forest fire. This directly corresponds to the number of forest fire incidences as shown in Figure 4. The year 2004, 2007, 2010 and 2012 has shown a decrease in forest fire area damaged while the year 2006, 2008, 2009, 2011, 2013 and 2014 has shown increase in forest fire damage area than the preceding year. Figure 6. 3.1 Visual interpretation of forest fire data (2004-2014) District wise frequency of forest fire in Sikkim. Figure 6. District wise frequency of forest fire in Sikkim. Figure 6 represents the number of forest fire incidences in each district of Sikkim. The highest incidences were recorded for the East district (33 numbers) in 2009, West district (32 numbers) in 2014, South district (33 numbers) in 2009. The highest incidences (2004-2014) were recorded in the West district (37.7 %) of the total fire incidences, followed by the South district (36.8%) of the total fire incidences. East district of Sikkim showed 21.7% and the North district of Sikkim showed the Figure 6. District wise frequency of forest fire in Sikkim. Figure 6 represents the number of forest fire incidences in each district of Sikkim. The highest incidences were recorded for the East district (33 numbers) in 2009, West district (32 numbers) in 2014, South district (33 numbers) in 2009. The highest incidences (2004-2014) were recorded in the West district (37.7 %) of the total fire incidences, followed by the South district (36.8%) of the total fire incidences. East district of Sikkim showed 21.7% and the North district of Sikkim showed the Forest and Society. Vol. 5(2): 261-276, November 2021 270 least forest fire incidences of 3.55% of the total forest fire incidences between the year 2004 and 2014 least forest fire incidences of 3.55% of the total forest fire incidences between the year 2004 and 2014. 2014. Figure 7. District-wise area coverage by the forest fire in Sikkim. Figure 7. District-wise area coverage by the forest fire in Sikkim. Figure 7 represents the area covered by the forest fire in each district of Sikkim. West district of Sikkim was found to have the maximum area covered by forest fire i.e., 42.7% of total forest fire covered area between the year 2004-2014. South district of Sikkim recorded 31.7% of the total forest fire area covered and the East district of Sikkim recorded 20.9% of the total forest fire covered area. North district of Sikkim showed the least forest fire covered area of 4.5% of the total forest fire covered area between the years 2004 and 2014.7 Figure 8. Forest area damaged in the various forest ranges of the state of Sikkim. Figure 8. Forest area damaged in the various forest ranges of the state of Sikkim. Figure 8 shows the forest area damaged in the various forest ranges of the state. 3.1 Visual interpretation of forest fire data (2004-2014) The figure shows that most forest fire occurrences are in the Soreng forest territorial range, followed by the Gyalshing forest territorial range and the Tashiding forest territorial range in the west district. The South district of Sikkim encountered frequent forest fires in Melli and Namthang forest territorial Range. Figure 8 shows that the most vulnerable territorial forest range to forest fires in the East district is Singtam and Pakyong territorial range followed by Gangtok and Ranipool range. The Forest territorial ranges in the North district are the least forest fire-affected ranges. Singtam forest territorial range recorded the highest forest fire-covered area with 245 Ha in the year 2009. Forest and Society. Vol. 5(2): 261-276, November 2021 271 3.2 Linear regression plots 10 12 14 16 18 20 0 50 100 150 200 250 Fire incidences (counts) Average temperature (o C) 4 6 8 10 12 14 16 18 0 50 100 150 200 250 Fire incidences (counts) Wind velocity (Km/h) 3.2 Linear regression plots Figure 9. Linear regression plot concerning No. of forest fire incidences for average temperature (on the left), wind velocity (on the middle), and relative humidity (on the right). 10 12 14 16 18 20 0 50 100 150 200 250 Fire incidences (counts) Average temperature (o C) 4 6 8 10 12 14 16 18 0 50 100 150 200 250 Fire incidences (counts) Wind velocity (Km/h) 35 40 45 50 55 60 65 70 0 50 100 150 200 250 Fire incidences (counts) Relative humidity (%) 3.2 Linear regression plots 35 40 45 50 55 60 65 70 0 50 100 150 200 250 Fire incidences (counts) Relative humidity (%) Figure 9. Linear regression plot concerning No. of forest fire incidences for average temperature (on the left), wind velocity (on the middle), and relative humidity (on the right). From Figure 9 (on the left), it is evident that there is an increasing trend of forest fire incidences concerning an increase in temperature (r=0.37, Slope=9.59, SD= ±12.00). Figure 9 (on the middle) shows the linear plot for wind velocity and No. of forest fire incidences which shows almost a flat curve (r=0.11, Slope=2.32, SD=±9.05). Figure 9 (on the right) shows a linear regression plot of relative humidity and No. of fire incidences wherein the best fit was found for relative humidity (r=-0.6, Slope=-4.52, and SD=±2.68). 4. Discussion The forest fire data of the year 2004-2014 upon interpretation shows that the forest fire incidences and area damage have increased over years. Lower hill forests like Zoom Reserve Forest, Sipsoo Reserve Forest, Sirithang Reserve Forest, and Sudhir Khasmal Forest recorded frequent forest fires in the West district. Simultaneously, middle hill forests like Sakyong Reserve Forest also encountered repeated forest fire cases in Gyalshing Forest Territorial Range. The forest fire was mostly seen to be occurring in Sal (Shorea robusta), Teak (Tectona grandis) and Chir (Pinus sp.) forest in lower hills whereas miscellaneous forest with Oak, Alder (Alnus nepaulensis), Mauwa (Engelhardtia spicata), Odal (Sterculia villosa), and Chewri (Bassia butyracea) also recorded frequent forest fires in Gyalshing and Tashiding Forest Territorial Range. In the South district of Sikkim, Salghari Reserve Forest, Majhitar Reserve Forest, Narak Reserve Forest, and Mamring Reserve Forest recorded a maximum number of forest fire incidences and area coverage. The species involved are Sal (Shorea robusta), Thakal (Cycas pectinate), Teak (Tectona grandis), and associate species in lower hill forest and species like Chilauney (Schima wallichi), Panisaj (Terminalia myriocarpa), Salimbo (grass), Katus (Castanopsis sp.), bamboo, etc. in the middle hill forest. In the Singtam Forest Territorial Range in the East district of Sikkim, Reserve Forests like Tumlabong, Salingay, Sittey, and Khanikhola encountered major forest fires whereas, in Pakyong Forest Territorial Range, forest like Bhasmey, Damlakha, Linkey, Pachey, Bagpani encounters most fire cases. In Gangtok and Ranipool Forest Territorial Range, there are fewer forest fire cases at forests of Syari, Namnang, Kambal, Ranka, etc. Kyongnosla Forest Territorial Range encounters rare forest fire cases, but the area coverage is large once there is a fire incidence. This must account for dense grooves of small bamboo of Ningal (Arundinaria sp.) and Malingo (Arundinaria maling) in those areas (Tamang et al., 2013). Table 2 depicts that there is an increased fire incidence damaging ground bushes but it cannot be made out from the data whether these damages of ground bushes account for forest fire incidences in any specific type of forest. Table 2 also depicts that the Sal (Shorea robusta) species was damaged in the greatest number of incidences followed by Schima wallichii, Bamboo, Tectona grandis, and Rhododendron arboretum. Cynodon dactylon which is again a grass species has been damaged with a considerable number of incidences. 4. Discussion From Figure 8 it is obvious that the Soreng Forest Territorial Range in the West district of Sikkim encounters repeated forest fires and it exhibits the highest forest fire coverage area in most of the Forest and Society. Vol. 5(2): 261-276, November 2021 272 years. In 2009, Singtam Forest Territorial Range in the East District of Sikkim encountered the largest forest fire area coverage within the studied years. In the South district of Sikkim, the Melli Forest Territorial Range encounters repeated forest fires and larger area coverage followed by Namthang Forest Territorial Range. Though the North District of Sikkim encounters the least forest fire cases, Phodong Forest Territorial Range exhibited the largest forest fire coverage in the year 2008. Forest fire is rare in the upper hills because of low temperature and altitude (Sharma et al., 2014). However, forest fire incidences have been recorded in 2009, 2010, 2013, and 2014 in Mangan, Phodong, and Dzongu Forest Territorial Range in the miscellaneous forests of lower elevations. The highest area covered by forest fire in the North district was in 2013 i.e., 188 ha. This indicates how global warming and climate change have made the high altitude of the North district also vulnerable to forest fire (Sharma et al; 2014). Table 1 depicts that the forest fire cases are comparatively higher in the West, South, and East districts than that of the North district in Sikkim. This accounts for the higher temperature of >20°C and lowers the humidity of <50% during fire season i.e., January to March (https://www.worldweatheronline.com) in these districts. On the contrary North district of Sikkim exhibit comparatively lower temperature < 10°C and higher humidity > 50% (https://www.worldweatheronline.com). Moreover, North Sikkim represents less low altitude areas and Sal (Shorea robusta) forests. From the data, it was also revealed that the highest number of forest fire incidences from 2009-2014 was recorded in March (240 No.), followed by February (57 No.), January (48 No.), and April (41 No.) and an average rainfall <300 mm was recorded during these months (Das et al., 2017). So, it can be concluded that the rate of rainfall and forest fire incidences are inversely related. Forest fires in India are human-made in around 95% of cases (Srivastava et al., 2020). Most cases of forest fires in Sikkim are either accidental or intentional. 4. Discussion The outbreak generally starts from the roadside due to live bidi/cigarette butt thrown by the passer-by (Sharma et al., 2014). Other reasons are intentional fire left out by the villagers to clear agricultural debris and better regeneration of grasses. Sometimes fire gets broken accidentally because of the torch (pultho) villagers use while passing through the forest areas. Winter is also a season for picnickers where they use fire for cooking and leave it live. Also, accidental forest fire happens due to faulty electricity lines and transformers (Sharma et al., 2014). While as per the villagers, the natural forest fire breaks due to falling boulders and rattling of bamboo in the groove. Figure 4 shows that the forest fire incidences were abruptly higher in the year 2009. This corresponds with the weather condition in that particular year where there was an extended drought season and the rainfall was scanty (Sharma et al., 2012). The year 2009 was the warmest year on record since 1901 (until 2014). The year 2009 experienced low precipitation <2751.62 mm and lowest rainfall 2548.86 mm (Rahman et al., 2012) whereas, higher annual rainfall during 2010 coupled with higher winter rain also resulted in lesser forest fire incidences in 2010 (Sharma et al., 2014). In 2009 Barsey Rhododendron Sanctuary, West Sikkim alone, encountered the damage of 200 ha though the forest fires are not very frequent in this area. Similar was the case in Fambonglho Wildlife Sanctuary in East Sikkim, where a single forest fire incident damaged 240 ha of miscellaneous forest. From figure 5 it can be interpreted that every succeeding year of low area coverage has shown an increase in forest fire area coverage. In the year 2009, forest fire recorded the highest area coverage of over 1625 ha with around 157 Nos. of incidences recorded. Though 2006, 2011, and 2014 recorded similar forest fire incidences, the year 2014 showed comparatively less area coverage. Figures 6 & 7 indicate that the number of fire incidences and corresponding area covered is highest in West and South districts covered by low-lying Sal Forest. This low-lying area extends from Mamring, Jorethang to Salghari in the South district of Sikkim and Nayabazar, Sipsoo to Tashiding in the West district of Sikkim. This must account for the scanty rainfall in the said area because these areas fall in the rain-shadow of Darjeeling hills. 4. Discussion Amongst all the forest types, subtropical forest and Sal (Shorea robusta) found mostly in these two districts, have the highest percentage of burnt areas detected using satellite imagery (Sharma et al., 2012). Forest and Society. Vol. 5(2): 261-276, November 2021 273 The analysis and interpretation of forest data for 2004-2014 reveal that the maximum forest fire cases in Sikkim are below 1500 m during the winter months. Sub-tropical forest and Sal (Shorea robusta) forest in Sikkim have the highest frequency of forest fires and the highest percentage of forest fire burnt area. Very negligible forest fire incidences were observed in the alpine and temperate forests. Also, middle hill to high hill miscellaneous forests showed a smaller number of forest fires in comparison. The number of forest fires is greater in southern and western aspects than in northern aspects, which could be due to a longer period of exposure to sunlight. Figure 9 (i.e., on the left and the right) shows that relative humidity (%) and average temperature (°C) have a moderate effect on the no. of fire incidences than the wind velocity (on the middle) for the state of Sikkim. Figure 9 (on the right) shows the best fit plot for relative humidity (%) on a number of forest fire incidences (r=-0.6, slope=-4.52, SD= ±2.68) having a moderate negative linear relationship which can be interpreted as more the relative humidity the lesser will be the chances of forest fire incidences. Figure 9 (on the left) shows the moderate positive linear relationship for average temperature (°C) on a number of forest fire incidences (r=0.37, slope=9.59 and SD= ±12.00) which can be interpreted as more the average temperature the more will be the chances of forest fire incidences. Figure 9 (on the middle) shows almost a flat curve indicating that the wind velocity (km/h) has a minimal influence over the occurrence of forest fire incidences. Conflicts of Interest: The authors declare no conflict of interest. Acknowledgement: The authors acknowledge the Forest and Environment Department, Government of Sikkim for all the necessary help and resources made available to the authors, and thank Ms. Anjali Sharma and Mrs. Hemlata Chamling (technical officials of GIS cell) for technical support in the preparation of maps. 5. Conclusions In this study, apart from visual interpretation of forest fire data, a linear regression analysis was performed between the average temperature, relative humidity, and wind velocity with the number of fire incidences. This study shall act as a baseline for various such parametric studies with the use of the latest technology and empirical studies, with the availability of data in the future. This study will also help the researchers and forest managers analyze the trend of forest fire in Sikkim. From this study, it is evident that the main reason for the forest fire incidences in Sikkim is anthropogenic, and natural forest fire cases are very rare. In Sikkim, factors like humidity, temperature, vegetation type, and rainfall play a crucial role in influencing forest fire incidences. AA The Disaster Management Act (2005) recognizes forest fire as one of the major natural disasters in the country. In comparison to other natural disasters like landslides or earthquakes, a forest fire is far more predictable and thus can be prevented. This study would also allow the authority to prepare an administrative and management plan like road approach, water facilities, deployment of resources, equipment, etc., well in time, thus helping prevent and mitigate forest fires in the state. AA However, parameter modeling and preparation of forest fire risk zone map with the application of Geo-spatial and other latest technologies is the need of the hour to enable the concerned authority to deploy appropriate resources at places prone to forest fires. Conflicts of Interest: The authors declare no conflict of interest. Ajin, R. S., Loghin, A. M., Vinod, P. G., & Jacob, M. K. (2016). Forest fire risk zone mapping using RS and GIS techniques: a study in Achankovil Forest Division, Kerala, India. Journal of Earth, Environment and Health Sciences, 2(3), 109. https://doi.org/10.4103/2423-7752.199288 References Ajin, R. S., Loghin, A. M., Vinod, P. G., & Jacob, M. K. (2016). Forest fire risk zone mapping using RS and GIS techniques: a study in Achankovil Forest Division, Kerala, India. Journal of Earth, Environment and Health Sciences, 2(3), 109. https://doi.org/10.4103/2423-7752.199288 Alauddin, M., Hossain, M.N., Islam, M.B., Islam, S., and Islam, M.K. (2020). Management Strategies for Sustainable Forest Biodiversity Conservation in Protected Areas of Bangladesh: A Study of Bhawal National Park, Gazipur. Grassroots Journal of Natural Resources, 3(3): 56-72. https://doi.org/10.33002/nr2581.6853.03035 Alauddin, M., Hossain, M.N., Islam, M.B., Islam, S., and Islam, M.K. (2020). Management Strategies for Sustainable Forest Biodiversity Conservation in Protected Areas of Bangladesh: A Study of Bhawal National Park, Gazipur. Grassroots Journal of Natural Resources, 3(3): 56-72. https://doi.org/10.33002/nr2581.6853.03035 Assessment, G. F. R. (2010). Main report. Food and Agriculture Organization of the United Nations, Rome. Assessment, G. F. R. (2010). Main report. Food and Agriculture Organization of the United Nations, Rome. Forest and Society. Vol. 5(2): 261-276, November 2021 274 Bajocco, S., Koutsias, N., & Ricotta, C. (2017). Linking fire ignitions hotspots and fuel phenology: The importance of being seasonal. Ecological Indicators, 82, 433-440. https://doi.org/10.1016/j.ecolind.2017.07.027 Bajocco, S., Koutsias, N., & Ricotta, C. (2017). Linking fire ignitions hotspots and fuel phenology: The importance of being seasonal. Ecological Indicators, 82, 433-440. https://doi.org/10.1016/j.ecolind.2017.07.027 Berwyn B. (2018). 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https://openalex.org/W2532992378	http://mural.maynoothuniversity.ie/13651/1/MD-Perceived-2017.pdf	English	null	Perceived weight discrimination mediates the prospective relation between obesity and depressive symptoms in U.S. and U.K. adults.	Health psychology	2,017	cc-by	10,415	Perceived Weight Discrimination Mediates the Prospective Relation Between Obesity and Depressive Symptoms in U.S. and U.K. Adults Angelina Sutin Florida State University College of Medicine Eric Robinson University of Liverpool Michael Daly University of Stirling and University College Dublin Objective: Obesity has been shown to increase risk of depression. Persons with obesity experience discrimination because of their body weight. Across 3 studies, we tested for the first time whether experiencing (perceived) weight-based discrimination explains why obesity is prospectively associated with increases in depressive symptoms. Method: Data from 3 studies, including the English Longitudinal Study of Ageing (2008/2009–2012/2013), the Health and Retirement Study (2006/2008–2010/2012), and Midlife in the United States (1995/1996–2004/2005), were used to examine associations between obesity, perceived weight discrimination, and depressive symptoms among 20,286 U.S. and U.K. adults. Results: Across all 3 studies, Class II and III obesity were reliably associated with increases in depressive symptoms from baseline to follow-up. Perceived weight-based discrimination predicted increases in depressive symptoms over time and mediated the prospective association between obesity and depressive symptoms in all 3 studies. Persons with Class II and III obesity were more likely to report experiencing weight-based discrimination, and this explained approximately 31% of the obesity-related increase in depressive symptoms on average across the 3 studies. Conclusion: In U.S. and U.K. samples, the prospective association between obesity (defined using body mass index) and increases in depressive symptoms in adulthood may in part be explained by perceived weight discrimination. Keywords: obesity, depression, obesity stigma, discrimination, weight stigma Supplemental materials: http://dx.doi.org/10.1037/hea0000426.supp Supplemental materials: http://dx.doi.org/10.1037/hea0000426.supp strength of the association between obesity and depression, whereby persons with Class II obesity and above are most likely to suffer from depressive symptoms (Onyike, Crum, Lee, Lyketsos, & Eaton, 2003; Preiss, Brennan, & Clarke, 2013; Vogelzangs et al., 2010). Although the prospective relation between obesity and depression has now been confirmed, the mechanisms explaining why persons with obesity are at an increased risk of developing depressive symptoms remain unclear (Luppino et al., 2010; Preiss et al., 2013). Moreover, the majority of studies that have examined potential mechanisms linking obesity to depression have relied on cross-sectional designs and/or nonrepresentative samples (Preiss et al., 2013). Health Psychology 2017, Vol. 36, No. 2, 112–121 Health Psychology 2017, Vol. 36, No. 2, 112–121 © 2016 The Author(s) 0278-6133/17/$12.00 http://dx.doi.org/10.1037/hea0000426 © 2016 The Author(s) 0278-6133/17/$12.00 http://dx.doi.org/10.1037/hea0000426 Sample Participants were drawn from ELSA, an ongoing prospective cohort study established in 2002 to study the health and ageing of community dwelling older adults (50 years). The initial ELSA sample was recruited from three waves of the Health Survey for England (1998, 1999, 2001), an annual cross-sectional survey based on a stratified random sample of English households. Inter- view data are collected every 2 years, and a clinical assessment is conducted every 4 years. In the current analyses, we calculate body mass index (BMI) from height and weight measurements collected as part of the Wave 4 (2008–2009) health assessment and examine longitudinal change in depressive symptoms over the 4-year period from Wave 4 to Wave 6 (2012–2013). Participants completed a measure of discrimination as part of the Wave 5 (2010–2011) interview. To be included in the current analyses, participants needed to have provided complete demographic, body mass index (BMI), and depressive symptom data as well as the perceived weight discrimination measure (N 6,000). Sample characteris- tics are detailed in Table 1. Participants in all three studies pro- vided informed consent and ethical approval was obtained for each study. A recent cross-sectional study of English older adults showed that perceived weight discrimination is associated with lower quality of life and more depressive symptoms (Jackson, Beeken, & Wardle, 2015a). Although cross-sectional studies that link weight- based discrimination to adverse psychological outcomes are infor- mative, they are also limited as it is plausible that reverse causality may explain these associations; those suffering from depression may be particularly likely to perceive weight-based discrimination (Jackson et al., 2015a), which has been shown to further propagate weight gain (Sutin & Terracciano, 2013). To date, there have been no examinations of the prospective associations among obesity, perceived weight discrimination, and depression. The aim of the current research was to examine whether experiencing (perceived) weight-based discrimination mediates the prospective association between obesity and subsequent changes in depressive symptoms in three large cohort studies of U.S. and U.K. adults. We predicted that experiencing weight discrimination would in part explain why persons with obesity show increases in depressive symptoms over time. A further aim of the current research was to examine whether gender moderated this effect. Sample We reasoned that women may be more likely to experience increases in depressive symptoms as a result of experiencing weight-based discrimination because of the importance attached to female thinness in our current social cli- mate (Thompson & Stice, 2001). Study 1: English Longitudinal Study of Ageing (ELSA) Our first aim was to make use of data from the ELSA to examine whether there is evidence that perceived weight discrim- ination mediates the prospective association between obesity and depressive symptoms among older U.K. adults. OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION 113 is highest (Dutton et al., 2014; Jackson et al., 2015b; Spahlholz, Baer, Konig, Riedel-Heller, & Luck-Sikorski, 2016). For example, recent data from a representative survey of German participants indicate that 19% and 38% of participants with Class II and Class III obesity report experiencing weight-based discrimination (Sikorski, Spahlholz, Hartlev, & Riedel-Heller, 2016). In addition, a number of theoretical models suggest that experiencing weight discrimination is likely to act as a form of psychological stressor (Major, Eliezer, & Rieck, 2012; Tomiyama, 2014), which could reduce self-worth and increase negative affect among persons with obesity (Crocker, Cornwell, & Major, 1993; Sikorski, Luppa, Luck, & Riedel-Heller, 2015). Thus, the experience of weight- based stigma may be an important factor explaining why obesity is associated with increased depressive symptoms. is highest (Dutton et al., 2014; Jackson et al., 2015b; Spahlholz, Baer, Konig, Riedel-Heller, & Luck-Sikorski, 2016). For example, recent data from a representative survey of German participants indicate that 19% and 38% of participants with Class II and Class III obesity report experiencing weight-based discrimination (Sikorski, Spahlholz, Hartlev, & Riedel-Heller, 2016). In addition, a number of theoretical models suggest that experiencing weight discrimination is likely to act as a form of psychological stressor (Major, Eliezer, & Rieck, 2012; Tomiyama, 2014), which could reduce self-worth and increase negative affect among persons with obesity (Crocker, Cornwell, & Major, 1993; Sikorski, Luppa, Luck, & Riedel-Heller, 2015). Thus, the experience of weight- based stigma may be an important factor explaining why obesity is associated with increased depressive symptoms. Perceived Weight Discrimination Mediates the Prospective Relation Between Obesity and Depressive Symptoms in U.S. and U.K. Adults There is convincing evidence for a bidirectional link between obesity and depression (de Wit et al., 2010; Luppino et al., 2010): Depression is associated with future weight gain (Grundy, Cotter- chio, Kirsh, & Kreiger, 2014; Luppino et al., 2010), and persons with obesity are at greater risk of developing depressive symptoms than are their “normal” weight counterparts (Faith et al., 2011; Herva et al., 2006; Roberts, Deleger, Strawbridge, & Kaplan, 2003). There is evidence that the severity of obesity predicts the This article was published Online First October 17, 2016. Eric Robinson, Department of Psychological Sciences, Institute of Psy- chology, Health & Society, University of Liverpool; Angelina Sutin, Flor- ida State University College of Medicine; Michael Daly, Behavioural Science Centre, Stirling Management School, University of Stirling and UCD Geary Institute, University College Dublin. A number of studies have shown that obesity is stigmatized, and a substantial portion of persons with obesity report being treated unfairly because of their weight, otherwise known as perceived weight discrimination (Jackson, Steptoe, Beeken, Croker, & Wardle, 2015b; Puhl & Heuer, 2009; Sutin & Terracciano, 2013). Recent findings have linked experiencing weight-based discrimi- nation with a variety of adverse health outcomes. For example, individuals who report experiencing discrimination because of their weight are more likely to suffer ill health as indexed by both self-report and physiological measures (Chen et al., 2007; Fettich & Chen, 2012; Sutin, Stephan, Carretta, & Terracciano, 2015; Sutin, Stephan, Luchetti, & Terracciano, 2014). Moreover, per- ceived weight discrimination is most common among persons with Class II obesity and above, among whom risk of future depression This article has been published under the terms of the Creative Com- mons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any me- dium, provided the original author and source are credited. Copyright for this article is retained by the author(s). Author(s) grant(s) the American Psychological Association the exclusive right to publish the article and identify itself as the original publisher. Correspondence concerning this article should be addressed to Eric Robinson, Department of Psychological Sciences, Institute of Psychology, Health & Society, University of Liverpool, Bedford Street, South Liver- pool, L69 7ZA UK. E-mail: eric.robinson@liv.ac.uk 112 g g y g g y a Score ranging from 0 to 8, with higher scores indicating greater depressive symptoms. b Score ranging from 0 to 9, with higher scores indicating greater depressive symptoms. c Score ranging from 0 to 7, with higher scores indicating greater depressive symptoms. Note. ELSA English Longitudinal Study of Ageing; HRS Health and Retirement Study; MIDUS Midlife in the United States. b ng from 0 to 8, with higher scores indicating greater depressive symptoms. b Score ranging from 0 to 9, with higher scores mptoms. c Score ranging from 0 to 7, with higher scores indicating greater depressive symptoms. Measures We used this method because our perceived weight dis- crimination mediator variable was dichotomous, and path a coef- ficients (independent variable to dichotomous mediator) derived Mediation analyses. Across all three studies, mediation anal- ysis was used to identify whether weight status at baseline (i.e., overweight, Class I, II, and III obesity relative to normal weight) had an indirect effect on depressive symptoms (standardized to have a mean of 0 and a standard deviation of 1) at follow-up through perceived weight discrimination. All mediation analyses were adjusted for initial depressive symptoms and covariates that may confound the relationship between obesity and depression: age, age2 (to account for a potential nonlinear relationship), gen- der, education, marital status, and employment status. We first established the preconditions necessary for successful mediation (Baron & Kenny, 1986). This involved establishing an association between (a) weight status categories and depressive symptoms (total effect, path c), (b) weight status categories and perceived weight discrimination (path a), and (c) perceived weight discrim- ination and depressive symptoms (path b) in a model that included baseline weight status. When the conditions for mediation were met, we conducted further analyses of the potential indirect effects (path a b) identified using the khb command in STATA (Ver- sion 13; Karlson, Holm, & Breen, 2012; Kohler, Karlson, & Holm, 2011). We used this method because our perceived weight dis- crimination mediator variable was dichotomous, and path a coef- ficients (independent variable to dichotomous mediator) derived Depressive symptoms. A validated eight-item version of the Center for Epidemiology Depression Scale (CES-D; Radloff, 1977; Turvey, Wallace, & Herzog, 1999) was administered to assess depressive symptoms at baseline and at follow-up. The short form CES-D uses a yes/no response format to assess feelings over the last week, including sadness, lethargy, loneliness, happiness, and enjoyment of life. Positively worded items were reverse scored, and a total sum score was generated ranging from 0 to 8, with higher scores indicating greater depressive symptoms. The CES-D demonstrated sufficiently high levels of reliability (Cron- bach’s .79 in both waves) and a moderate degree of stability across study waves (r .50, p .001). Measures During Wave 5 of ELSA, the frequency of five forms of unfair treatment was assessed (“you are treated with less respect or courtesy,” “you are threatened or harassed,” “you re- ceive poorer service than other people in restaurants and stores,” “people act as if they think you are not clever,” “you receive poorer service or treatment than other people from doctors or hospitals”) on a 6-point scale ranging from never to almost every day. Next, participants who reported having experienced discrim- ination in daily life were asked to select the reason(s) they believed they were discriminated against from a list that included weight. Participants could choose as many or as few attributions for the unfair treatment as necessary. In fitting with other studies that have examined the association between perceived weight discrimination and health outcomes (Jackson et al., 2015a; Sutin et al., 2015), perceived weight discrimination (dichotomous variable) was de- fined as those who reported experiencing discrimination and indi- cated they believed that weight was a reason for this discrimina- tion. Rates of perceived weight discrimination across body weight categories are detailed in Table 2. Mediation analyses. Across all three studies, mediation anal- ysis was used to identify whether weight status at baseline (i.e., overweight, Class I, II, and III obesity relative to normal weight) had an indirect effect on depressive symptoms (standardized to have a mean of 0 and a standard deviation of 1) at follow-up through perceived weight discrimination. All mediation analyses were adjusted for initial depressive symptoms and covariates that may confound the relationship between obesity and depression: age, age2 (to account for a potential nonlinear relationship), gen- der, education, marital status, and employment status. We first established the preconditions necessary for successful mediation (Baron & Kenny, 1986). This involved establishing an association between (a) weight status categories and depressive symptoms (total effect, path c), (b) weight status categories and perceived weight discrimination (path a), and (c) perceived weight discrim- ination and depressive symptoms (path b) in a model that included baseline weight status. When the conditions for mediation were met, we conducted further analyses of the potential indirect effects (path a b) identified using the khb command in STATA (Ver- sion 13; Karlson, Holm, & Breen, 2012; Kohler, Karlson, & Holm, 2011). Measures BMI. As part of the Wave 4 health assessment, trained nurses weighed participants to the nearest 0.1 kg using Tanita THD-305 portable electronic scales. Standing height was measured to the nearest millimeter using a portable stadiometer. Participants stood on the center of a baseplate looking straight ahead in order to gauge height accurately and consistently. BMI was derived as Table 1 Basic Demographic Characteristics and Descriptive Statistics for Participants in Studies 1, 2, and 3 Table 1 Basic Demographic Characteristics and Descriptive Statistics for Participants in Studies 1, 2, and 3 Study 1: ELSA (N 6,000) Study 2: HRS (N 9,908) Study 3: MIDUS (N 4,378) Variable M % SD M % SD M % SD Age (years) 64.75 8.60 66.97 9.72 46.68 12.45 Female (%) 55.4 60.1 53.2 White (%) 97.8 85.2 93.8 BMI baseline (kg/m2) 28.29 5.17 29.39 5.83 26.62 5.16 Weight status (%) BMI 25 kg/m2 26.60 22.88 41.69 Overweight 42.13 36.97 37.62 Class I obese 21.30 24.60 13.98 Class II obese 7.00 10.40 4.66 Class III obese 2.97 5.15 2.06 Depressive symptoms (baseline) 1.21a 1.78 1.69b 2.09 .70c 1.83 Depressive symptoms (follow-up) 1.21a 1.78 1.78b 2.13 .61c 1.72 Note. ELSA English Longitudinal Study of Ageing; HRS Health and Retirement Study; MIDUS Midlife in the United States. a Score ranging from 0 to 8, with higher scores indicating greater depressive symptoms. b Score ranging from 0 to 9, with higher scores indicating greater depressive symptoms. c Score ranging from 0 to 7, with higher scores indicating greater depressive symptoms. Table 1 Basic Demographic Characteristics and Descriptive Statistics for Participants in Studies 1, 2, and 3 emographic Characteristics and Descriptive Statistics for Participants in Studies 1, 2, and 3 ROBINSON, SUTIN, AND DALY 114 kg/m2 and defined as normal weight (BMI 25), overweight (BMI 25–29.9), Class I (BMI 30–34.9), Class II (BMI 35–39.9), and Class III obesity (BMI 40 and above). kg/m2 and defined as normal weight (BMI 25), overweight (BMI 25–29.9), Class I (BMI 30–34.9), Class II (BMI 35–39.9), and Class III obesity (BMI 40 and above). Covariates. We based our choice of covariates on recorded variables likely to be associated with depression and/or obesity (Preiss et al., 2013; Luppino et al., 2010). Participants reported demographic information at baseline (Wave 4, 2008–2009) in- cluding their age, gender, ethnicity (White vs. Measures other), education level (1 no qualifications, 7 degree level qualification or above), marital status (married, cohabiting, other), and employ- ment status (employed/self-employed, unemployed, homemaker, retired, permanently sick or disabled). Participants also reported details relating to their health and health behavior. Specifically, participants indicated whether they had a longstanding illness, whether they were a current smoker, the frequency of their alcohol consumption in the last week (0 drank on none of the last 7 days, 7 drank on all days in the last week), and the frequency they engage in moderate and vigorous physical activity (1 more than once a week, 4 hardly ever, or never). Class III obesity (BMI 40 and above). Perceived weight discrimination. In all three studies, partic- ipants completed an adapted version of the Perceived Everyday Experiences With Discrimination Scale (Williams, Yan, Jackson, & Anderson, 1997). Participants first reported how frequently they perceived a set of discriminatory experiences to occur in their day-to-day lives. During Wave 5 of ELSA, the frequency of five forms of unfair treatment was assessed (“you are treated with less respect or courtesy,” “you are threatened or harassed,” “you re- ceive poorer service than other people in restaurants and stores,” “people act as if they think you are not clever,” “you receive poorer service or treatment than other people from doctors or hospitals”) on a 6-point scale ranging from never to almost every day. Next, participants who reported having experienced discrim- ination in daily life were asked to select the reason(s) they believed they were discriminated against from a list that included weight. Participants could choose as many or as few attributions for the unfair treatment as necessary. In fitting with other studies that have examined the association between perceived weight discrimination and health outcomes (Jackson et al., 2015a; Sutin et al., 2015), perceived weight discrimination (dichotomous variable) was de- fined as those who reported experiencing discrimination and indi- cated they believed that weight was a reason for this discrimina- tion. Rates of perceived weight discrimination across body weight categories are detailed in Table 2. Perceived weight discrimination. In all three studies, partic- ipants completed an adapted version of the Perceived Everyday Experiences With Discrimination Scale (Williams, Yan, Jackson, & Anderson, 1997). Participants first reported how frequently they perceived a set of discriminatory experiences to occur in their day-to-day lives. Results and Conclusion Participants in the Class II and III obesity categories were at an increased risk of developing more depressive symptoms from baseline to follow-up (p .01), as shown in Table 3. As expected, the proportion of participants experiencing weight discrimination increased markedly across weight categories (i.e., overweight, obesity Classes I, II, III; see Table 2). For example, among normal weight and overweight participants, fewer than 1% reported weight discrimination, whereas 20% of Class II and III obese participants reported experiencing weight discrimination. Per- ceived weight discrimination was found to be a significant predic- tor of increased depressive symptoms from baseline to follow-up ( .188, p .001) in models adjusting for weight status at baseline, as outlined in Table 3. Robustness tests. We conducted supplementary mediation analyses in which each model was adjusted for health behavior and health status. We considered this an additional stringent test of the study hypotheses, given that health-related variables may act as either confounding factors and/or additional pathways from per- ceived discrimination to depressive symptoms. If including these variables in our regressions did not notably change the indirect association between obesity and depressive symptoms through perceived discrimination, we considered the relationship to be unlikely to be affected by health-related variables. We also tested whether the mediation results were notably different if a continu- ous measure of body weight (i.e., BMI) was used as the predictor variable or if a dichotomous indicator of clinically significant depression was used as the outcome measure. Specifically, we tested whether weight discrimination mediated the longitudinal association between BMI (treated continuously) and changes in depressive symptoms and whether weight discrimination ex- plained the link between weight categories and changes in the presence of clinically significant depression levels over time. For the latter analyses, we used scale specific cut-off scores for clin- ically significant depression scores to identify those meeting the criteria for depression (see Table S1 in the online supplemental We found a significant indirect effect between Class II ( .036, SE .012, p .01, 95% CI .013–.059) and Class III obesity ( .057, SE .019, p .01, 95% CI .020–.095) and longitudinal change in depressive symptoms through perceived weight discrimination, as shown in Table 3. OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION 115 materials for scale cut off scores in each study and depression rates). from logistic regression cannot be multiplied directly with the ordinary least squares path b coefficients (dichotomous mediator to continuous dependent variable, path b) using the standard prod- uct of coefficients approach (Preacher & Hayes, 2008). The khb method decomposes the total effect of obesity on depression into a direct effect and an indirect effect through perceived weight discrimination. It also provides estimates of the magnitude and statistical significance level of the indirect effect and proportion of the total association accounted for by this pathway. from logistic regression cannot be multiplied directly with the ordinary least squares path b coefficients (dichotomous mediator to continuous dependent variable, path b) using the standard prod- uct of coefficients approach (Preacher & Hayes, 2008). The khb method decomposes the total effect of obesity on depression into a direct effect and an indirect effect through perceived weight discrimination. It also provides estimates of the magnitude and statistical significance level of the indirect effect and proportion of the total association accounted for by this pathway. Note. Models use z scores for depressive symptoms as the outcome variable. Models are adjusted for baseline depressive symptoms, age, age2, gender, ethnicity (White vs. other), educational attainment, marital status (married, cohabiting, other) and employment categories (employed/self-employed, unemployed, homemaker, retired, permanently sick or disabled). ELSA English Longitudinal Study of Ageing; IV independent variable; DV dependent variable. p .01. Measures Table 2 Percentage of Participants Reporting Experiencing Weight-Based Discrimination by Weight Status in Studies 1, 2, and 3 Table 2 Percentage of Participants Reporting Experiencing Weight-Based Discrimination by Weight Status in Studies 1, 2, and 3 Study 1: ELSA (N 6,000)a Study 2: HRS (N 9,908)b Study 3: MIDUS (N 4,378)c Weight status % (n/total) % (n/total) % (n/total) Normal weight (BMI 25 kg/m2) .9 (14/1,596) 1.9 (42/2,268) 4.9 (89/1,825) Overweight .9 (22/2,528) 2.5 (91/3,663) 8.4 (138/1,647) Class I obese 5.9 (75/1,278) 9.1 (221/2,437) 21.2 (130/612) Class II obese 20.5 (86/420) 20.8 (214/1,030) 38.7 (79/204) Class III obese 32.6 (58/178) 36.5 (186/510) 58.9 (53/90) Note. ELSA English Longitudinal Study of Ageing; HRS Health and Retirement Study; MIDUS Midlife in the United States. a Perceived weight discrimination among those reporting experiences of discrimination attributable to weight in the 2008/2009 wave of ELSA. b Perceived weight discrimination among those reporting experiences of discrimination attributable to weight in the 2006/2008 wave of HRS. c Perceived weight discrimination among those reporting experiences of discrimination attributable to weight/height in 1995/1996 or 2004/2005 waves of MIDUS. a Perceived weight discrimination among those reporting experiences of discrimination attributable to weight in the 2008/2009 wave of ELSA. b Perceived weight discrimination among those reporting experiences of discrimination attributable to weight in the 2006/2008 wave of HRS. c Perceived weight discrimination among those reporting experiences of discrimination attributable to weight/height in 1995/1996 or 2004/2005 waves of MIDUS. Table 3 Mediation Models of the Indirect Effect of Obesity on Changes in Depressive Symptoms Through Perceived Weight Discrimination in Study 1 (ELSA; N 6,000) Results and Conclusion In total, 18.1% of the total effect of Class II obesity and 20.6% of the effect of Class III obesity on depressive symptoms was mediated through perceived weight discrimination. Our robustness tests indicated that per- ceived weight discrimination explained approximately 28% of the association between Class II and III obesity and depressive symp- toms in models adjusting for the presence of a longstanding limiting illness, whether the participant smoked, and the frequency with which the participant drank and exercised (see Table S2 in the Mediation Models of the Indirect Effect of Obesity on Changes in Depressive Symptoms Through Perceived Weight Discrimination in Study 1 (ELSA; N 6,000) Analyses Point estimate SE 95% CI Effect ratio Class III obesity Weight Status ¡ Discrimination (IV to mediator, path a) 3.892 .320 Discrimination ¡ Depression (mediator to DV, path b) .188 .059 Weight Status ¡ Depression (total effect, path c) .278 .068 Weight Status ¡ Depression (direct effect, path c=) .220 .070 Weight Status ¡ Depression (indirect effect, path a b) .057 .019 [.020.095] .206 Class II obesity Weight Status ¡ Discrimination (IV to mediator, path a) 3.321 .298 Discrimination ¡ Depression (mediator to DV, path b) .188 .059 Weight Status ¡ Depression (total effect, path c) .197 .047 Weight Status ¡ Depression (direct effect, path c=) .161 .048 Weight Status ¡ Depression (indirect effect, path a b) .036 .012 [.013.059] .181 Class I obesity Weight Status ¡ Discrimination (IV to mediator, path a) 2.021 .297 Discrimination ¡ Depression (mediator to DV, path b) .188 .059 Weight Status ¡ Depression (total effect, path c) .031 .032 Weight Status ¡ Depression (direct effect, path c=) .021 .032 Weight Status ¡ depression (indirect effect, path a b) Note. Models use z scores for depressive symptoms as the outcome variable. Models are adjusted for baseline depressive symptoms, age, age2, gender, ethnicity (White vs. other), educational attainment, marital status (married, cohabiting, other) and employment categories (employed/self-employed, unemployed, homemaker, retired, permanently sick or disabled). ELSA English Longitudinal Study of Ageing; IV independent ROBINSON, SUTIN, AND DALY 116 1999). Participants rated nine items (yes/no) that measured depres- sive symptoms during the last week (e.g., “I felt depressed”), which were summed for a total depressive symptoms score. online supplemental materials). Results and Conclusion We used the same analysis strategy as in Study 1. In an initial model unadjusted for perceived weight discrimination, individuals of Class I, II and III obesity were at an elevated risk of increased depressive symptoms from baseline to follow-up, as detailed in Table 4. The numbers of participants experiencing weight discrim- ination increased as BMI increased. For example, among normal weight and overweight participants around 2% reported experienc- ing weight discrimination, whereas 20% of Class II and III obese participants reported weight discrimination (see Table 2). Those who reported perceived weight discrimination showed a signifi- cant increase in depressive symptoms over the 4-year period from baseline to follow-up ( .141, p .001), as shown in Table 4. We observed significant indirect effects of obesity Classes I ( .011, SE .003, 95% CI .005–.016, p .01), II ( .026, SE .006, 95% CI .013–.038, p .01) and III ( .046, SE .011, 95% CI .024–.069, p .01) on depressive symptoms through perceived weight discrimination. Effect ratios showed that perceived weight discrimination explained approximately 34% of the effect of Classes I, II, and III obesity on longitudinal changes in depressive symptoms, as shown in Table 4. Results and Conclusion We interpret this as evidence that the contribution of perceived weight discrimination to explaining the obesity–depression link is unlikely to be due to confounding by health or health behavior in this study. Covariates. Demographic information was provided at base- line (2006–2008) and included age, age2, gender, ethnicity (White vs. other), years of education, marital status (married, separated/ divorced, widowed, never married), and employment categories (employed, unemployed, homemaker, retired, temporary leave, disabled). Health and health behavior were assessed using a mea- sure of disease burden at baseline (a sum of eight diagnosed chronic conditions), history of ever smoking, frequency of vigor- ous physical activity, and average alcohol consumption in a week over the last 3 months. In addition, we found that 22.9% of the total effect of BMI (continuous variable) on increases in depressive symptoms (B .011, SE .002, p .01) was mediated by weight discrimination (B .002, SE .0001, p .01), as shown in Table S3 of the online supplemental materials. Weight discrimination predicted increases in clinically significant depression levels over time (OR 1.51, p .05, 95% CI 1.04–2.19) and mediated 22.3% of the link between Class II and Class III obesity and clinically significant depression on average, as shown in Tables S4 and S5 of the online supplementary materials. These supplementary analyses show that the role of perceived weight discrimination in mediating the link between body weight and depression is not markedly different from our main analyses when either a continuous BMI measure or a dichotomous measure of clinically significant depres- sion was used. Sample A total of 9,908 participants were drawn from the HRS, a longitudinal study of Americans over the age of 50 and their spouses. In 2006, HRS implemented an enhanced face-to-face interview that included a standardized measurement of weight and height and a psychosocial questionnaire that participants com- pleted at home and mailed back to the University of Michigan. Half of the HRS sample participated in the enhanced interview in 2006; the other half participated in 2008. These two samples were combined as baseline. Participants completed the same assessment again 4 years later, in 2010 and 2012, respectively. These assess- ments were combined as the follow-up to give each participant a 4-year follow-up interval. See Table 1 for sample demographic information. Robustness tests. As in Study 1, we also tested the effect of perceived weight discrimination on the relation between obesity and change in depressive symptoms while controlling for other health and health behavior variables (i.e., disease burden, physical activity, smoking and alcohol consumption). This analysis con- firmed that perceived weight discrimination significantly mediated the relation between obesity (Classes I, II, and III) and change in depressive symptoms while controlling for a range of potential confounding variables, explaining approximately 35% of this as- sociation (see Table S2 of the online supplemental materials). As in Study 1, we found that weight discrimination explained a substantial portion (38.6%) of the longitudinal link between BMI (continuous variable) and increases in depressive symptoms (total effect: B .005, SE .001, p .01; indirect effect: B .002, SE .0004, p .01), as shown in Table S6 of the online supplemental materials. Once again, weight discrimination pre- dicted increases in the presence of clinically significant depression from baseline to follow-up (OR 1.50, p .01, 95% CI 1.22–1.84) and partially mediated of the link between Class I, II, and III obesity and clinically significant depression (26.4% ex- plained on average), as shown in Tables S4 and S7 of the online supplemental materials. Study 2: Health and Retirement Study (HRS) In Study 1, we found evidence that the relation between obesity and depressive symptoms is mediated by perceived weight dis- crimination among older English adults. A potential limitation of Study 1 was that the mediator variable (perceived weight discrim- ination) was measured after the baseline measures of BMI and depression. We were able to address this in Study 2. Moreover, given that the relation between obesity and depression has been suggested to be particularly strong among Americans (Luppino et al., 2010), in Study 2 we aimed to replicate the findings of Study 1 in a large sample of older U.S. adults. Sample Data were drawn from the MIDUS study, a national longitudinal study of the psychosocial factors that influence the health and well-being of Americans from midlife to old age (for comprehen- sive sample information see Brim, Ryff, & Kessler, 2004). The main sample was recruited via random digit dialing and the total sample includes siblings within recruited households and a sample of twin pairs. In total 7,108 noninstitutionalized adults aged 25 to 74 were first interviewed in 1995 and 1996. Those included in the current analyses needed to have provided complete demographic information and to have completed both the baseline discrimina- tion measure and a measure of depression at baseline (1995) and follow-up 10 years later (2004 through 2005). Demographic data for those individuals (N 4,283) who met these criteria and were included in the sample are outlined in Table 1. Depressive symptoms. The World Health Organization Com- posite International Diagnostic Interview-Short Form (Kessler, Andrews, Mroczek, Ustun, & Wittchen, 1998) was used to gauge the presence of depressive symptoms at baseline and follow-up. Participants first indicated if they “felt sad, blue, or depressed” or “lost interest in most things” for 2 weeks in the last 12 months. Those who endorsed either of these items then responded to seven (yes/no) follow-up questions assessing depressive symptoms relat- ing to how they felt during this period (e.g., “feel down in yourself, no good, or worthless”). A rating was derived from the two measures, ranging from 0 to 7 (0 no 2-week period of depressed affect or anhedonia in the last year, 7 highest depressive symptom score). Depressive symptoms. The World Health Organization Com- posite International Diagnostic Interview-Short Form (Kessler, Andrews, Mroczek, Ustun, & Wittchen, 1998) was used to gauge the presence of depressive symptoms at baseline and follow-up. Participants first indicated if they “felt sad, blue, or depressed” or “lost interest in most things” for 2 weeks in the last 12 months. Those who endorsed either of these items then responded to seven (yes/no) follow-up questions assessing depressive symptoms relat- ing to how they felt during this period (e.g., “feel down in yourself, no good, or worthless”). A rating was derived from the two measures, ranging from 0 to 7 (0 no 2-week period of depressed affect or anhedonia in the last year, 7 highest depressive symptom score). Measures BMI. As part of the enhanced face-to-face interview, trained staff measured and weighed participants. BMI was derived as kg/m2 and categorized into categories as in Study 1. Perceived weight discrimination. Participants completed the Perceived Everyday Experiences With Discrimination Scale as described in Study 1 (Williams et al., 1997) at baseline. Depressive symptoms. At baseline and follow-up, partici- pants completed a short version of the CES-D scale (Turvey et al., OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION 117 Table 4 Mediation Models of the Indirect Effect of Obesity on Changes in Depressive Symptoms Through Perceived Weight Discrimination in Study 2 (HRS; N 9,908) Analyses Point estimate SE 95% CI Effect ratio Class III obesity Weight Status ¡ Discrimination (IV to mediator, path a) 3.289 .186 Discrimination ¡ Depression (mediator to DV, path b) .141 .033 Weight Status ¡ Depression (total effect, path c) .107 .040 Weight Status ¡ Depression (direct effect, path c=) .061 .042 Weight Status ¡ Depression (indirect effect, path a b) .046 .011 [.024.069] .433 Class II obesity Weight Status ¡ Discrimination (IV to mediator, path a) 2.612 .177 Discrimination ¡ Depression (mediator to DV, path b) .141 .033 Weight Status ¡ Depression (total effect, path c) .067 .031 Weight Status ¡ Depression (direct effect, path c=) .041 .032 Weight Status ¡ Depression (indirect effect, path a b) .026 .006 [.013.038] .389 Class I obesity Weight Status ¡ Discrimination (IV to mediator, path a) 1.732 .173 Discrimination ¡ Depression (mediator to DV, path b) .141 .033 Weight Status ¡ Depression (total effect, path c) .053 .024 Weight Status ¡ Depression (direct effect, path c=) .043 .024 Weight Status ¡ Depression (indirect effect, path a b) .011 .003 [.005.016] .197 Note. Models use z scores for depressive symptoms outcome variable. Models are adjusted for baseline depressive symptoms, age, age2, gender, ethnicity (White vs. other), educational attainment, marital status (married, separated/divorced, widowed, never married) and employment categories (employed, unemployed, homemaker, retired, temporary leave, disabled). HRS Health and Retirement Study; IV independent variable; DV dependent variable. p .05. p .01. Table 4 Mediation Models of the Indirect Effect of Obesity on Changes in Depressive Symptoms Through Perceived Weight Discrimination in Study 2 (HRS; N 9,908) p .05. p .01. Study 3: Midlife in the United States (MIDUS) Perceived weight discrimination. Weight discrimination was derived from the measure of everyday discrimination as in Studies 1 and 2 (Williams et al., 1997). At baseline and follow-up, partic- ipants were asked to indicate how frequently they experienced nine forms of discriminatory treatment, which included similar items to those used in Study 1 and Study 2 (“you are treated with . . . less courtesy than other people,” “. . . less respect than other people,” “you receive poorer service than other people,” “people act as if they . . . think you are not smart,” “. . . are afraid of you,” “. . . think you are dishonest,” “. . . think you are not as good as they are,” “you are . . . called names or insulted,” “. . . threatened or harassed”). After making these ratings, participants were asked to select the reason(s) for this discrimination from a list, including “weight or height.” Perceived weight discrimination (dichotomous variable) was defined as those who identified weight or height as a reason for having experienced discrimination. In the third study we sought to replicate the findings of Study 1 and Study 2 in a sample with a more diverse age range. Note. Models use z scores for depressive symptoms outcome variable. Models are adjusted for age, age2, gender, ethnicity (White vs. other), educational attainment, marital status (married, separated, divorced, wid- owed, never married), and employment categories (employed, self-employed, unemployed, laid off, homemaker, student, retired, on leave, permanently disabled, other). MIDUS Midlife in the United States; IV independent variable; DV dependent variable. p 05 p 01 Measures BMI. Participants reported their height and weight as part of the MIDUS baseline survey. As in Studies 1 and 2, BMI was derived as kg/m2 and divided into overweight, obesity Classes I, II, and III categories. Self-reported BMI and objectively verified BMI recorded during a physical exam were available for a subset of 900 MIDUS participants and found to be highly correlated in this sample (r .92, p .001; Robinson, Hunger, & Daly, 2015). 118 ROBINSON ROBINSON, SUTIN, AND DALY 118 Covariates. Additional covariates included age, age2, gender, ethnicity (White vs. other), educational level (1 no school/some grade school, 12 PhD/MD level), marital status and (married, separated, divorced, widowed, never married), and employment status (employed, self-employed, unemployed, laid off, home- maker, student, retired, on leave, permanently disabled, other). Health and health behavior were gauged by the presence of a chronic health condition at baseline, current regular smoking, the frequency of moderate and vigorous physical activity in the last month, and alcohol consumption in the last month. Robustness tests. As in Studies 1 and 2, we tested the indirect effect of perceived weight discrimination on the relation between obesity and change in depressive symptoms while controlling for health and health behavior variables. Once again, these analyses confirmed that perceived weight discrimination significantly me- diated the relation between obesity and change in depressive symptoms, explaining approximately 30% of this association (see Table S2 in the online supplemental materials). Similarly, our supplementary analyses confirmed that weight discrimination me- diated the association between continuous BMI and depressive symptoms (explaining 54.2% of this link) and mediated the link between Class II and Class III obesity and clinically significant depression (explaining 38.3% of the association), as shown in Tables S8 and S9 of the online supplemental materials. Results and Conclusion We used the same analysis strategy as in Studies 1 and 2. In the first model unadjusted for perceived weight discrimination, de- pressive symptoms among individuals of Class II and III obesity increased from baseline to follow-up 10 years later (see Table 5). Once again, perceived weight discrimination increased markedly in line with weight status, as shown in Tables 2 and 5. Perceived weight discrimination was a significant predictor of increased depressive symptoms from baseline to follow-up ( .152, p .001), and the inclusion of perceived weight discrimination re- duced the strength of the associations between Classes II and III obesity and depressive symptoms at follow-up (see Table 5). Mediation analyses confirmed significant indirect effects of Class II ( .052, SE .017, 95% CI .018–.086, p .01) and Class III ( .081, SE .026, 95% CI .028–.132, p .01) obesity on depressive symptoms through perceived weight discrimination. An examination of the effect ratios indicated that perceived weight discrimination explained over 31% of the total effect of obesity (Classes II and III) on depressive symptoms. Additional mediation analysis. In our main analyses for Study 3, we combined perceived weight discrimination scores measured at baseline and follow-up. However, further analyses also showed that obesity at baseline predicted increases in weight discrimination from baseline to follow-up, and this increase ex- plained changes in depressive symptoms over time. More specif- ically, in unadjusted analyses obesity Classes I, II, and III showed a strong graded associated with increases in weight discrimination from baseline to follow-up (Class I: OR 5.39, 95% CI 3.60–8.07; Class II: OR 8.07, 95% CI 4.92–13.23; Class III: OR 24.47. 95% CI 13.06–45.84). In analyses adjusting for baseline weight discrimination and covariates, we found that only obesity Class III predicted longitudinal increases in depressive symptoms (total effect: .220, p .05). Results and Conclusion Including changes in weight discrimination between baseline and follow-up in this model explained 25.5% of the longitudinal association between Table 5 Mediation Models of the Indirect Effect of Obesity on Changes in Depressive Symptoms Through Perceived Weight Discrimination in Study 3 (MIDUS; N 4,378) Table 5 Mediation Models of the Indirect Effect of Obesity on Changes in Depressive Symptoms Through Perceived Weight Discrimination in Study 3 (MIDUS; N 4,378) Analyses Point estimate SE 95% CI Effect ratio Class III obesity Weight Status ¡ Discrimination (IV to mediator, path a) 3.455 .259 Discrimination ¡ Depression (mediator to DV, path b) .152 .048 Weight Status ¡ Depression (total effect, path c) .293 .101 Weight status ¡ Depression (direct effect, path c=) .212 .104 Weight Status ¡ Depression (indirect effect, path a b) .081 .026 [.028.132] .273 Class II obesity Weight Status ¡ Discrimination (IV to mediator, path a) 2.751 .193 Discrimination ¡ Depression (mediator to DV, path b) .152 .048 Weight Status ¡ Depression (total effect, path c) .147 .069 Weight Status ¡ Depression (direct effect, path c=) .094 .071 Weight Status ¡ Depression (indirect effect, path a b) .052 .017 [.018.086] .356 Class I obesity Weight Status ¡ Discrimination (IV to mediator, path a) 2.040 .157 Discrimination ¡ Depression (mediator to DV, path b) .152 .048 Weight Status ¡ Depression (total effect, path c) .001 .044 Weight Status ¡ Depression (direct effect, path c=) .027 .045 Weight Status ¡ Depression (indirect effect, path a b) Note. Models use z scores for depressive symptoms outcome variable. Models are adjusted for age, age2, gender, ethnicity (White vs. other), educational attainment, marital status (married, separated, divorced, wid- owed, never married), and employment categories (employed, self-employed, unemployed, laid off, homemaker, student, retired, on leave, permanently disabled, other). MIDUS Midlife in the United States; IV independent variable; DV dependent variable. p .05. p .01. Additional Analyses Gender. Because women may be judged more critically than men because of their weight, we examined gender differences in each of the key study variables (i.e., obesity, weight discrimina- tion, depressive symptoms) and tested whether gender moderated the relation between perceived weight discrimination and depres- sive symptoms. We did this by including Gender Perceived Weight Discrimination interactions in the earlier reported regres- sion models for Studies 1 through 3 and examined whether this explained further variance in depressive symptoms. Because of the observational nature of the present work, we cannot make strong claims about the causal influence that per- ceived weight discrimination has on the development of depressive symptoms. However, experimental work suggests that experienc- ing weight-based stigma increases negative affect (Himmelstein, Incollingo Belsky, & Tomiyama, 2015; Schvey, Puhl, & Brownell, 2011), and the present work adds to this emerging literature. Moreover, a number of theoretical models suggest that experienc- ing weight discrimination is likely to be stressful and may reduce self-worth (Crocker et al., 1993; Sikorski et al., 2015; Tomiyama, 2014), both of which are likely to increase depressive symptoms. Obesity is viewed negatively by large proportions of society and realizing that one is part of a stigmatized social group is likely to be psychologically distressing (Hunger & Major, 2015; Hunger, Major, Blodorn, & Miller, 2015). Experiencing weight discrimi- nation may therefore reinforce negative beliefs about how a person with obesity believes they are viewed by others. Understanding the pathways by which experiencing weight-based discrimination is associated with increased depressive symptoms will now be im- portant. Experiencing weight-based discrimination could also con- tribute to depressive symptoms by limiting employment opportu- nities, increasing body dissatisfaction (Wardle, Waller, & Rapoport, 2001), internalization of weight stigma (Durso & Lat- ner, 2008), damaging self-esteem (Myers & Rosen, 1999) and/or by increasing feelings of loneliness (Lewis et al., 2011). Regard- less of the pathways by which experiencing weight-based discrim- ination is associated with depressive symptoms, challenging dis- crimination based on weight will now be important and policies which challenge the derogation of persons with obesity or outline the damaging effects of weight stigma may be ways of achieving this. Across the three studies, we found little evidence that rates of obesity differed between men and women. However, women showed larger increases in depressive symptoms than men in all studies, as shown in Table S10 of the online supplemental mate- rials. OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION OBESITY, PERCEIVED WEIGHT DISCRIMINATION AND DEPRESSION 119 discrimination with impaired well-being and depressive symptoms (Chen et al., 2007; Jackson et al., 2015a). However, the present work is the first to show that there is a prospective association between perceived weight-based discrimination and increased de- pressive symptoms. To date, there has also been little research explaining potential mechanisms linking heavier body weight to longitudinal increases in depressive symptoms (Preiss et al., 2013; Remigio-Baker et al., 2014); our findings suggest that among U.S. and U.K. adults, perceived weight-based discrimination may be an important factor explaining this link. In Study 3, we observed that the effects on depressive symptoms of experiencing weight-based discrimination were more detrimental to women than to men, but this finding was not observed in either Study 1 or Study 2, so the replicability of this gender effect is unclear and warrants further attention. obesity Class III and subsequent changes in depressive symptoms (indirect effect: .056, p .05). Thus, the association between obesity and longitudinal change in depressive symptoms is in part explained by experiencing weight discrimination when changes in perceived weight discrimination over time are examined as a mediator. General Discussion We used three large samples of predominantly White U.S. and U.K. adults to test the hypothesis that experiencing weight-based discrimination mediates the prospective effect of obesity on de- pressive symptoms. In line with previous research (Preiss et al., 2013; Vogelzangs et al., 2010), we found consistent evidence that obesity (Classes II and III) was associated with increases in de- pressive symptoms over several years. Moreover, across all three samples the prospective association between obesity and depres- sive symptoms was in part explained by perceived weight discrim- ination; adults with obesity were more likely to report experienc- ing weight-based discrimination, which in turn predicted increases in depressive symptoms over time. On average, perceived weight discrimination was linked to an increase in depressive symptoms (0.16SD change), and on average explained 31% of the total effect of obesity Classes II and III on depressive symptoms. Results and Conclusion Analyses Point estimate SE 95% CI Effect ratio Class III obesity Weight Status ¡ Discrimination (IV to mediator, path a) 3.455 .259 Discrimination ¡ Depression (mediator to DV, path b) .152 .048 Weight Status ¡ Depression (total effect, path c) .293 .101 Weight status ¡ Depression (direct effect, path c=) .212 .104 Weight Status ¡ Depression (indirect effect, path a b) .081 .026 [.028.132] .273 Class II obesity Weight Status ¡ Discrimination (IV to mediator, path a) 2.751 .193 Discrimination ¡ Depression (mediator to DV, path b) .152 .048 Weight Status ¡ Depression (total effect, path c) .147 .069 Weight Status ¡ Depression (direct effect, path c=) .094 .071 Weight Status ¡ Depression (indirect effect, path a b) .052 .017 [.018.086] .356 Class I obesity Weight Status ¡ Discrimination (IV to mediator, path a) 2.040 .157 Discrimination ¡ Depression (mediator to DV, path b) .152 .048 Weight Status ¡ Depression (total effect, path c) .001 .044 Weight Status ¡ Depression (direct effect, path c=) .027 .045 Weight Status ¡ Depression (indirect effect, path a b) Additional Analyses Women were also more likely than were men to experience weight-based discrimination in Studies 2 and 3. In Study 3 (MIDUS), women experienced a particularly increased risk of weight discrimination (OR 2.207, 95% CI 1.750–2.784, p .01) and depressive symptoms ( .167, SE .031, p .01), potentially pointing to a gender difference in the mediating role of weight discrimination in that study. There was no evidence that gender moderated the prospective association between perceived weight discrimination and depres- sive symptoms in Studies 1 and 2 (ps .05). In Study 3, we identified a significant interaction that indicated perceived weight discrimination was more closely linked to change in depression among women. Supplementary mediation analyses showed that while obesity was linked to higher rates of perceived weight discrimination in both men and women, discrimination only acted as a pathway from obesity (Classes II and II) to depressive symp- toms for women in Study 3 (explaining 43% of this association, see Table S11 of the online supplemental materials). Limitations and Future Directions Coping with obesity stigma affects depressed mood in African-American and white candidates for bariatric ( ) p g y g depressed mood in African-American and white candidates for bariatric surgery. Obesity, 20, 1118–1121. http://dx.doi.org/10.1038/oby.2012.12 depressed mood in African-American and white candidates for bariatric surgery. Obesity, 20, 1118–1121. http://dx.doi.org/10.1038/oby.2012.12 surgery. Obesity, 20, 1118–1121. http://dx.doi.org/10.1038/oby.2012.12 Grundy, A., Cotterchio, M., Kirsh, V. A., & Kreiger, N. (2014). Associa- tions between anxiety, depression, antidepressant medication, obesity and weight gain among Canadian women. PLoS ONE, 9(6), e99780. http://dx.doi.org/10.1371/journal.pone.0099780 Hebl, M. R., Ruggs, E. N., Singletary, S. L., & Beal, D. J. (2008). Perceptions of obesity across the lifespan. Obesity, 16(Suppl. 2), S46– S52. http://dx.doi.org/10.1038/oby.2008.458 S52. http://dx.doi.org/10.1038/oby.2008.458 Herva, A., Laitinen, J., Miettunen, J., Veijola, J., Karvonen, J. T., Läksy, K., & Joukamaa, M. (2006). Obesity and depression: Results from the longitudinal Northern Finland 1966 Birth Cohort Study. International Journal of Obesity, 30, 520–527. http://dx.doi.org/10.1038/sj.ijo .0803174 Himmelstein, M. S., Incollingo Belsky, A. C., & Tomiyama, A. J. (2015). The weight of stigma: Cortisol reactivity to manipulated weight stigma. Obesity, 23, 368–374. http://dx.doi.org/10.1002/oby.20959 Hunger, J. M., & Major, B. (2015). Weight stigma mediates the association between BMI and self-reported health. Health Psychology, 34, 172–175. http://dx.doi.org/10.1037/hea0000106 Hunger, J. M., Major, B., Blodorn, A., & Miller, C. T. (2015). Weighed down by stigma: How weight-based social identity threat contributes to weight gain and poor health. Social and Personality Psychology Com- pass, 9, 255–268. http://dx.doi.org/10.1111/spc3.12172 Conclusions In U.S. and U.K. samples, the prospective association between obesity and increases in depressive symptoms in adulthood may in part be explained by perceived weight discrimination. Jackson, S. E., Beeken, R. J., & Wardle, J. (2015a). Obesity, perceived weight discrimination, and psychological well-being in older adults in England. Obesity, 23, 1105–1111. http://dx.doi.org/10.1002/oby.21052 Jackson, S. E., Steptoe, A., Beeken, R. J., Croker, H., & Wardle, J. (2015b). Perceived weight discrimination in England: A population-based study of adults aged 50 years. International Journal of Obesity, 39, 858–864. http://dx.doi.org/10.1038/ijo.2014.186 References Baron, R. M., & Kenny, D. A. (1986). The moderatormediator variable distinction in social psychological research: Conceptual, strategic, and statistical considerations. Journal of Personality and Social Psychology, 51, 1173–1182. http://dx.doi.org/10.1037/0022-3514.51.6.1173 Karlson, K. B., Holm, A., & Breen, R. (2012). Comparing regression coefficients between same-sample nested models using logit and probit a new method. Sociological Methodology, 42, 286–313. http://dx.doi .org/10.1177/0081175012444861 Brim, O. G., Ryff, C. D., & Kessler, R. C. (2004). The MIDUS national survey: An overview. In O. G. Brim, C. D. Ryff, & R. C. Kessler (Eds.), How healthy are we? A national study of well-being at midlife (pp. 1–36). Chicago, IL: University of Chicago Press. Kessler, R. C., Andrews, G., Mroczek, D., Ustun, B., & Wittchen, H.-U. (1998). The World Health Organization Composite International Diag- nostic Interview short-form (CIDI-SF). International Journal of Meth- ods in Psychiatric Research, 7, 171–185. http://dx.doi.org/10.1002/ mpr.47 Chen, E. Y., Bocchieri-Ricciardi, L. E., Munoz, D., Fischer, S., Katterman, S., Roehrig, M., . . . Le Grange, D. (2007). Depressed mood in class III obesity predicted by weight-related stigma. Obesity Surgery, 17, 669– 671. http://dx.doi.org/10.1007/s11695-007-9112-4 Kohler, U., Karlson, K. B., & Holm, A. (2011). Comparing coefficients of nested nonlinear probability models. The Stata Journal, 11, 420–438. Lewis, S., Thomas, S. L., Blood, R. W., Castle, D. J., Hyde, J., & Komesaroff, P. A. (2011). How do obese individuals perceive and respond to the different types of obesity stigma that they encounter in their daily lives? A qualitative study. Social Science & Medicine, 73, 1349–1356. http://dx.doi.org/10.1016/j.socscimed.2011.08.021 Crocker, J., Cornwell, B., & Major, B. (1993). The stigma of overweight: Affective consequences of attributional ambiguity. Journal of Person- ality and Social Psychology, 64, 60–70. http://dx.doi.org/10.1037/0022- 3514.64.1.60 de Wit, L., Luppino, F., van Straten, A., Penninx, B., Zitman, F., & Cuijpers, P. (2010). Depression and obesity: A meta-analysis of community-based studies. Psychiatry Research, 178, 230–235. http://dx .doi.org/10.1016/j.psychres.2009.04.015 Lewis, T. T., Cogburn, C. D., & Williams, D. R. (2015). Self-reported experiences of discrimination and health: Scientific advances, ongoing controversies, and emerging issues. Annual Review of Clinical Psychol- ogy, 11, 407–440. http://dx.doi.org/10.1146/annurev-clinpsy-032814- 112728 Durso, L. E., & Latner, J. D. (2008). Understanding self-directed stigma: Development of the Weight Bias Internalization Scale. Obesity, 16 (Suppl. 2), S80–S86. http://dx.doi.org/10.1038/oby.2008.448 Luppino, F. S., de Wit, L. M., Bouvy, P. F., Stijnen, T., Cuijpers, P., Penninx, B. W., & Zitman, F. G. (2010). Limitations and Future Directions Our focus in the present work was on middle age and older adulthood, so we do not know whether the same pattern of results would be observed among younger adults. Given that experiencing weight-based and other forms of discrimination have been associ- ated with adverse health outcomes among younger age groups (Puhl & Heuer, 2009; Schmitt, Branscombe, Postmes, & Garcia, 2014; Wott & Carels, 2010) and obesity may be stigmatised most among younger age groups (Hebl et al., 2008), weight based discrimination may also play a role in explaining the link between obesity and depression in younger age groups. However, further The results of the present research are consistent with previous cross-sectional findings linking the experience of weight-based ROBINSON, SUTIN, AND DALY 120 work is now needed to test whether this process holds among younger adults. Further work would also benefit from considering the importance of personality variables when considering per- ceived weight discrimination and depressive symptoms, as it is plausible that factors such as neuroticism may increase the likeli- hood that a person perceives an experience as discriminatory and/or exacerbate the damaging psychological effects of discrim- ination. It should be noted that associations between experiencing discrimination and mental health in other studies tend to be robust, irrespective of adjusting for personality characteristics (Lewis, Cogburn, & Williams, 2015). A limitation of the present work was that we did not have very large numbers of participants with Class II and III obesity in each study, although we still observed con- sistent findings across studies and when BMI was used as a continuous predictor rather than weight status categories. Our samples also predominantly consisted of White participants and the lack of racial diversity could have influenced our results. It is therefore not clear whether experiencing weight discrimination is prospectively linked to increased depressive symptoms among other ethnic groups. Some final limitations concern Study 3: Be- cause of practical constraints, only self-reported BMI data were available, and the measure of perceived weight discrimination was derived from participants’ reports of being discriminated against because of their size more generally (e.g., weight or height), as opposed to only their weight. Faith, M. S., Butryn, M., Wadden, T. A., Fabricatore, A., Nguyen, A. M., & Heymsfield, S. B. (2011). Evidence for prospective associations among depression and obesity in population-based studies. Obesity Reviews, 12, e438–e453. http://dx.doi.org/10.1111/j.1467-789X.2010 .00843.x Fettich, K. C., & Chen, E. Y. (2012). ies. 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https://openalex.org/W2321792313	https://zenodo.org/records/1625325/files/article.pdf	English	null	THE SOLUBILITY OF ACETANILIDE, PHENACETINE, CAFFEINE AND SALOL IN SEVERAL SOLVENTS.	Journal of the American Chemical Society	1,907	public-domain	2,152	THE SOLUBILITY OF ACETANILIDE, PHENACETINE, CAFFEINE AND SALOL I N SEVERAL SOLVENTS. Kecci\.cd >lay, 2 , I,'I:. 1;s . ~ ~ l l ~ . l ~ . r ~ ~ s G I 1;s . ~ ~ l l ~ . l ~ . r ~ ~ s G I Kecci\.cd >lay, 2 , The experiinents described iii this paper ]\'ere uiiclertaken for thc purpose of obtaining the necessary data iipoii \vliicli to liase a satisfac- tor), method for the separatioii of home of the ct!iisiittieiits of lieadache poivders. The object vas to fiiitl solvciits i!i whicli the solubilit!- of oiic or more of the above iiained substaiiccs differed iiiost from that of the others uiider the saiiie coiiditioiis. The tleterniiiiatioiis \Yere tliereforc not made with tlie care \vhicii s1io:ild be devoted to result:; siibiiiitted priniarilj- a s solubilit!, constaiits, siiice it \vas recognized that onl!- ivitlc differences iti the actual :iiiioLiiit,s of :lie se:-era1 suhitaiices clissolvetl b>- 2 single solvent, could be utilized for the cluaiititati\~e qiaratioii of mix- tures of these conipouiicls. A search of the literature indicated that comparative!y few solubility results ivliich coulcl be used for the purpose. were available. T h e usual reference books upon piiai-maceutical and organic clieiiiistrJ- give approsi- mate deteriiiiiiatioiis for each of these substances in the more coii~iiioii solvents such as water, alcohol, ether. cliloroforiii. ctc. 111 adclitioii to data of this character there were found results upoii the solubility of acetanilide in methyl alcohol, ethyl alcoliol, aiicl chloroform ~ > y Spe>-ers' and in iiiistures of etlij-1 alcohol and water 11~. Holletiiaii and Aliituscli.' The material used for the deteriiiiiiatioiis here recorded was in all cases that sold uiider tlie designation c'. 1'. or 7:. S. P. The esperiiiieiits ivci-e made at room temperature arid therefore the temperature..; re- corded are probably accurate in iiiost cases to within a degree or tlvo. .\bout 30 cc. of tlie 501- vent and an excess of the solid iii each case irere placed iii glass stoppcreil cylinders ivliich were then attached to ail axle ivhicli \vas rotated b y iiieaiis of a water motor. A11 deteriiiiiiatioiis iii any oiic soli-eiit Ivere made simultaneously and hence are comparable in so far as temperature, time of shaking etc. are concerned. The time of rotation was at least six hours in all cases, usuallJ. iiiuch longer. IOSS IOSS ATHERTON SEIDELI, [COSTRIBUTIOS FROM THP: 1)RI:G ~,hHORATORY. III-RE.\U OF CI3I:lf I S T R Y . ]'VI;- 1,:SHELI BY PI~R3IISSIOS O F THE: SECi<F:TAX\- O F ;\C:RICT~I.TI~II~.]. Ho:le:iin:i and Antuicli, Rec. trav. chin:,, 13, 2 9 3 , I iS94). Spcyers, . h i . J. Sci. ( 4 1 . 14, 294, ( 1932). H l ii i d A t i li R hi 13 I iS94) Spcyers, . h i . J. Sci. ( 4 1 . 14, 294, ( 1932). Ho:le:iin:i and Antuicli, Rec. trav. chin:,, 13, 2 9 3 , I iS94 THE SOLUBILITY OF ACETANILIDE, PHENACETINE, CAFFEINE AND SALOL I N SEVERAL SOLVENTS. The removal of a clear portion of the solution was effected by drawing it up with the aid of suction iiito a pipette provided with a filter of ahsorbciit cotton at the opeiiiiig through which tlie liquid eiitered. The solution was then transferred to a pycnometer and after being weighed was washed (usually with alcohol) into a tared platinum dish. The solveiit was then re- moved by evaporatioii at rooiii temperature with tlie aid of ail electric The method of deteriiiiiiatioii was as follows : S i SOLCBILITY OF ACETAKILIDE, ETC. 1089 fan. The residue remaining in the dish was alternately weighed and dried at 60' until no further loss occurred. fan. The residue remaining in the dish was alternately weighed and dried at 60' until no further loss occurred. In the case of benzaldehyde, the benzoic acid formed during the evap- oration was determined by titration with standard alkali and its amount deducted from the total weight of residue found. With salol in benzal- dehyde, however, a determination could not be made since no solid separated even on continued evaporation of the solution by means of the current of air. The first series of determinations were made in aqueous ethyl alcohol solutions and the results are presented in Table I together with those of Holleman and Antusch upon similar aqueous alcoholic solutions, deter- mined at 25'. Eight saturated solutions were prepared and rotated at the temperature of the room which held closely at 30' for several hours previous to the withdrawal of portions for the analysis. T.4BLE I. SOLUBILITT O F ACETANILIDE I N 3IIXTURES O F ETHYL ALCOHOL AND WATER. Results at zjOl Results at 30° Weight _ h -___- _---A__- 7 Alcohol per roo gins of per IM) gms. of in Mixtures Sat. Solution Solution Sat. Solution Solution Percent Gms. C,;HaXHOCsH, Sp gr. Gins. CBH5NHOC2H3 Sp. pr. 0 0.54 0.997 IO 0.93 0.995 2 0 1 . 2 s 0.973 30 2.30 0.962 40 4.Sj 0.950 50 S.S7 0.939 60 14.17 0.92s io 19.84 0.918 so 25.17 0.90j 85 26.93 0.899 90 27.6j 0.890 95 2 6 . S ~ 0 Si4 I00 24.77 0.SjI 0.69 1.000 1.00 0.984 2.20 0.970 4.80 0.9j6 9.40 0.945 15.40 0.934 2 2 . 0 0 0.926 2 7.60 0.917 31.20 0 . W 31.70 0.900 31.60 0.893 29.00 0.876 33.s3 0.SSj T.4BLE I. THE SOLUBILITY OF ACETANILIDE, PHENACETINE, CAFFEINE AND SALOL I N SEVERAL SOLVENTS. SOLUBILITT O F ACETANILIDE I N 3IIXTURES O F ETHYL ALCOHOL AND WATE The weight percents of alcohol in the eight water alcohol mixtures used were calculated from their specific gravities determined by the pycnometer method. The dissolved acetanilide was determined by weigh- ing the residue left after the evaporation of the solvent from weighed portions of the saturated solutions. The interpolated values shown in the table were read from the curve drawn through the eight points plotted on cross section paper. For this curve, the weight percents of alcohol in the alcohol-water mixtures were taken as abscissas and the grams of acetanilide per IOO grams of solution as ordinates. The curve plotted in the same manner from the 25' results of Holleman and Antusch lies some- what below the 30' curve but in all other respects the two are entirely similar. An examination of these curves shows that the amount of dis- 'Holleman and Antusch. X TH E R T 0 S S E IDEL I. 1090 solvecl acetanilide increases at first slo\vl?- nith iiicrease iii coiiceiitratioii of alcohol, and then quite rapitll!. 111) to the inasiina. lvliicli iii the caic of tlie 2 jo curve is at 90 \\-eight percent alcohol aiid iii the case of tile 30' curl-e is at 8 j percent alcohol. *iboi.e these coiiceiitratioiis of alco- hol the solubility diminishes in each case. A satisfactory esplaiiatioii for this increased solubilit!, of acctaiiilide iii alcohol coiitniniiig I O ~ w r cent water (and eve11 inore at a liiglier temperature) is :it present iiot appare11t. Tlic solubility of acetanilide in methyl alcohol, etlij.1 alcohol aiid ill iii chloroform as determined by Speyers' is reported iii terms of gram iiiolecales of acetanilide per IOO #rani molecules of the sol\.eiits at ir- regular temperatures, and in addition the weights of tlie saturated solii- tioiis are given for other temperatures than those at which the solubility determinations were made. Xltliough 110 new results for these solvents li31.e been determiiied by the present writer it was tlionght of interest to recalculate those of Spej-ers to tlie basis selected for presenting all the other solubility determiiiatioiis included herewith. This has been done aiicl the results given in Table 11. g T.IBLE 11. CHLOROIXIK~I ( SPEYERS). SUI.LlBIl,ITY 01' ACET.INII,IDE IS 3IETHY1, AI.COHOI., ETHYI, ALCOHOL ,491) IX Tem- per. a- titre. THE SOLUBILITY OF ACETANILIDE, PHENACETINE, CAFFEINE AND SALOL I N SEVERAL SOLVENTS. 0 I O 20 3" 40 50 60 111 lfetlivl Alcohol Gnis. C,,H;.UHOC,H3 U't of per 100 pms. I cc. Soliiticn Solution IS. j 0. b6o 2 3 . 1 0.864 29. I 0. 87 j .:j.I 0.S92 42.9 u.911 51.7 0.912 59.2 0.957 Iii Ethyl Alcohol Gms.C,;H;SHOC?H.I Wt. of _ _ - _ _ A _____ per IOO gms. I cc. Soiiltion Solution 12,s 0.842 16. j 0.844 21.>? O . S j O 26.5 0.S60 32.9 o.s;4 39.4 0. 895 46.4 0.920 In Chlorcform Gins C,.H;SHOC?H3 \Yt of __-_ per loo gms. I cc. Sclutiotl Sollition 3.53 I . j03 j.24 1.475 10.7 1.440 14.5 I. 39s 1S.7 1.254 23.7 1.314 29. I 1 . 2 7 2 I T bl I11 i h h i l bili i f ilid e I11 is shown the comparative solubilities of acetanilide, In Table I11 is shown the comparative solubilities of acetanilide, phenacetine, caffeine and salol iii twelve different solvents. Although this number is not as large as would be desirable in order to fully accoin- plish the purpose mentioned in the first part of this paper, it is believed that a fair degree of success can be attained in the separation of mixtures of at least three of the above named substances on the basis of the results shown in the table. Thus it may be expected that with proper nianipulation either toluene, benzene or xylene will remove salol when mixed with the other three compounds, and amyl alcohol or acetate will effect a separation of caffeine from a mixture of this substance with acetanilide and phenacetine. It is seen that with none of the solvents is there a wide difference between the solubility of acetanilide and phen- acetine. ' ' !oc. cit. ' !oc. cit. ACETANILIDE I N HEADACHE POWDERS ACETANILIDE I N HEADACHE POWDERS 1091 1091 TABLE 111. SOLCBILITY OF ACETAXILIDE, PHENACETINE, CAFFEINE AND SALOL I N ORGANIC SOLVESTS. Grains per IOO grams saturated solution : sp. gl.. ----_A Solvent. of solvent. t') Acelanilide. Phenacetine. Caffeine. Salol. 'Water 0.997 25 0.56 0.11 2.14 insoluble 'Ether 0.716 '' 7.7 1.56 0 . 2 7 - 'Chloroform Acetone Benzene Benzaldehyde Amylacetate Aniline Aniylalcohol Acetic acidY Xylene Toluene 1.476 0.527 0.S 7 2 I.0jj 0.S60 0 SI4 0.547 0.S62 1.02 1.055 30-3 I C ' 25 21. THE SOLUBILITY OF ACETANILIDE, PHENACETINE, CAFFEINE AND SALOL I N SEVERAL SOLVENTS. j 32.5 25.0 16.6 4.76 31.75 (0.902) 10.68 2.46 (0.S75) 0.65 (0.573) 18.83 (1.06s) 5.44 (1.063) 10.46 (o.SS2) 2.42 (0.865) 19.38 (1.034) 9.46 (r.025) 14.00 3.5r (0.519) 33.21 13.65 (1.064) 1.6j (0.847) 1.25 (0.847) 0.jo ( 0 . S 6 2 ) 0.30 (0.863) 11.0 2.18 (0.E32) 1.22 (0.S7j) 11.62 (1.087) 0.72 (0.862) 22.89 (1.080) 0.49 (0.810) 2.44 1.11 (0.S47) o 57 (0.861) - 90.99 8S.jj (1.145) ? S5.29 (1.136) very soluble 20.44 (0.569) 63.24 (1.143) s7.14- 83.62 (1.12s) Figures in parentheses are the specific gravities of the saturated solutions. TABLE 111. SOLCBILITY OF ACETAXILIDE, PHENACETINE, CAFFEINE AND SALOL I N SOLVESTS. Grains per IOO grams saturated solution : \\.'bile continuing the investigations described in the previous paper a suggestion was obtained which has led to the following method for deter- Results from U. s. P., 8th Revision. TABLE 111. gures in parentheses are the specific gravities of the saturated solutions. Fairly good results have been obtained by the author in separating mixtures of different amounts of salol, caffeine, and acetanilide by digesting first in toluene for the removal of salol and then in amyl alcohol for the removal of the acetanilide. As is shown by the table, none of the four compounds are completely insoluble in any of the solvents, and consequently it cannot be expected that a very sharp separation can be made. This is especially true for mixtures containing relatively small amounts of either of the substances. I t is also to be mentioned that the influence of the presence of one sub- stance upcn the solubility of the others in the mixture has not been investigated and therefore conclusions based upon the solubility of single substances in a given solvent may be considerably in error when applied to mixtures of two or more substances acted upon by the same solvent. The author regrets that circumstances have prevented the extension of these determinations to a larger number of solvents and the applica- tion of the results to a more accurate method of separating and deter- mining the constituents of headache powders. [CONTRIBUTION FROM THE DRUG LABORATORY, BVREAU OF CHEMISTRY, PUB- LISHED BY PERMISSION O F THE SECRETARY O F AGRICULTURE]. [CONTRIBUTION FROM THE DRUG LABORATORY, BVREAU OF CHEMISTRY, PUB- A RAPID METHOD FOR THE QUANTITATIVE DETERMINATION OF ACETANILIDE IN HEADACHE POWDERS. LISHED BY PERMISSION O F THE SECRETARY O F AGRICULTURE]. BY ATHERTON SEIDELL. Received May 2. 1902. BY ATHERTON SEIDELL. Received May 2. 1902. BY ATHERTON SEIDELL. Received May 2. 1902. \\.'bile continuing the investigations described in the previous paper a suggestion was obtained which has led to the following method for deter- Results from U. s. P., 8th Revision.
https://openalex.org/W4309287838	https://www.researchsquare.com/article/rs-2011977/latest.pdf	English	null	Selection of red fluorescent protein for genetic labeling of mitochondria and intercellular transfer of viable mitochondria	Scientific reports	2,022	cc-by	5,845	Selection of red fluorescent protein for genetic labeling of mitochondria and intercellular transfer of viable mitochondria Isamu Taiko Nihon University Chika Takano Nihon University Masayuki Nomoto Nihon University Shingo Hayashida Nihon University Kazunori Kanemaru Nihon University Toshio Miki (  miki.toshio@nihon-u.ac.jp ) Nihon University Article Keywords: Posted Date: September 8th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2011977/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Selection of red fluorescent protein for genetic labeling of mitochondria and intercellular transfer of viable mitochondria Isamu Taiko Nihon University Chika Takano Nihon University Masayuki Nomoto Nihon University Shingo Hayashida Nihon University Kazunori Kanemaru Nihon University Toshio Miki (  miki.toshio@nihon-u.ac.jp ) Nihon University Article Keywords: Posted Date: September 8th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2011977/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License nsed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Abstract The phenomenon of intercellular mitochondrial transfer has attracted great attention in various fields of research, including stem cell biology. Elucidating the mechanism of mitochondrial transfer from healthy stem cells to cells with mitochondrial dysfunction may lead to the development of novel stem cell therapies to treat mitochondrial disorders, among other advances. To visually evaluate and analyze the mitochondrial transfer process, dual fluorescent labeling systems are often used to distinguish the mitochondria of donor and recipient cells. Although enhanced green fluorescent protein (EGFP) has been well-characterized for labeling mitochondria, other colors of fluorescent protein have been less extensively evaluated in the context of mitochondrial transfer. Here, we generated different lentiviral vectors with mitochondria-targeted red fluorescent proteins, including DsRed, mCherry mKOκ, and TurboRFP. Among these proteins, mitochondria-targeted DsRed and its variant mCherry often generated bright aggregates in the lysosome while other proteins did not. We further validated that TurboRFP- labeled mitochondria were successfully transferred from amniotic epithelial cells, one of the candidates for donor stem cells, to mitochondria-damaged recipient cells without losing the membrane potential. Our study provides new insight into the genetic labeling of mitochondria with red fluorescent proteins, which may be utilized to analyze the mechanism of intercellular mitochondrial transfer. Results Selection of mitochondria-targeted fluorescent proteins and design of lentiviral vector constructs Selection of mitochondria-targeted fluorescent proteins and design of lentiviral vector constructs To evaluate different red fluorescent proteins, the following four commonly used red fluorescent proteins derived from different organisms were selected; DsRed derived from coral Discosoma sp. and its monomeric variant mCherry14,15, TurboRFP derived from the sea anemone Entacmaea quadricolor, and the monomeric orange-shifted fluorescent protein mKOκ derived from the stony coral Verrillofungia concinna16,17 (Table 1). DNA sequences encoding these red fluorescent proteins were inserted into a common lentiviral vector (pLKO) backbone (Fig. 1). We also generated the same backbone lentiviral vector with the EGFP gene for comparison. To prevent extrinsic protein overloading in mitochondria, we employed a human phosphoglycerate kinase (hPGK) promoter for a moderate level of gene expression18,19. Two human COX8A gene sequences were used as a tandem MTS to prevent the leakage of fluorescent protein into the cytosol20. Differential intracellular impact of the mitochondria-targeted fluorescent proteins Lentivirally transduced fluorescent proteins had no notable cytosolic leakage and localized to mitochondria in immortalized hAECs (iAECs) (Fig. 2). We examined the precise subcellular localization of the mitochondria-targeted proteins by costaining with MitoTracker dye. Mitochondria-targeted EGFP, TurboRFP, and mKOκ were costained with MitoTracker dye and did not form visible aggregates or cause abnormal mitochondrial structure in most cells (Fig. 2). In contrast, mitochondria-targeted mCherry and DsRed formed MitoTracker-unstained granular aggregates around the nucleus in 37 ± 4.5 % and 54 ± 5.5% of cells, respectively (Fig. 2B). These aggregates had higher fluorescence intensity than mitochondria-localized fluorescent proteins. These cells also had normal mitochondrial morphology. In some of the cells, both aggregated and accurately mitochondria-targeted mCherry and DsRed coexisted. In other cells, only aggregates were present. These aggregates emerged even at the low viral dose and were not significantly changed in a viral dose-dependent manner (Fig. 3). To investigate whether the mCherry aggregates were present in the lysosome, lysosomes were visualized with LysoTracker. As expected, most of the aggregated mCherry was costained with LysoTracker, while mitochondria-localized mCherry was completely distinct (Fig. 4). Introduction The mislocalization of fluorescent proteins may be responsible for failure to detect mitochondrial transfer, especially by flow cytometric analysis or fluorescence-activated cell sorting. Thus, appropriate red fluorescent proteins are essential for the precise detection of mitochondrial transfer. causing mislocalization of the proteins13. The mislocalization of fluorescent proteins may be responsible for failure to detect mitochondrial transfer, especially by flow cytometric analysis or fluorescence-activated cell sorting. Thus, appropriate red fluorescent proteins are essential for the precise detection of mitochondrial transfer. In this study, we generated four lentiviral vectors with different red fluorescent proteins targeted to mitochondria by MTS and assessed the intracellular fluorescein localizations in hAECs. DsRed and its derivative mCherry formed prominent perinuclear localized punctate aggregates, whereas TurboRFP and mKOκ rarely formed aggregates. Furthermore, we successfully detected mitochondrial transfer by using TurboRFP. Introduction Mitochondria are ATP-producing organelles responsible for cellular energetics and metabolism. Mitochondrial dysfunction causes critical cellular defects that are closely related to various mitochondrial disorders. Currently, there is no highly effective treatment or cure for mitochondrial disorders. Recent studies revealed that mitochondria are actively transported from healthy donor cells to cells with damaged mitochondria 1,2. This phenomenon is called intercellular mitochondrial transfer and may be one of the innate survival systems of eukaryotic cells. It is hypothesized that this phenomenon can be applied to treat diseases associated with mitochondrial damage. Intercellular mitochondrial transfer from and between tissue stem cells has been reported to have therapeutic potential3. Thus, efforts to elucidate the mechanism of mitochondrial transfer have attracted great attention in the field of stem cell biology. Human amniotic epithelial cells (hAECs) are a type of placental stem cell that possess pluripotent stem cell-like differentiation potential, immunomodulatory, and anti-inflammatory properties and are therefore considered to have prospects for cell-based therapy4. Since the placenta is the youngest donor tissue, placental mitochondria are considered to be less damaged by aging and environmental factors. Thus, hAECs could be ideal donor stem cells for therapeutic mitochondrial transfer. To conduct further investigations of mitochondrial transfer from hAECs, reliable tools for mitochondrial visualization are desired. Imaging fluorescently labeled mitochondria with small molecules such as MitoTracker is a promising technique that can visualize mitochondrial transfer, thereby contributing to an understanding of the mechanisms underlying this process. However, small molecules often leak from labeled organelles, which can lead to false-positive results5. Moreover, the covalent labeling of mitochondrial matrix proteins with fluorescent molecules is sometimes harmful to mitochondrial respiration 6,7. These problems can be addressed by genetically expressing fluorescent proteins with a mitochondrial targeting signal (MTS)8. Genetically encoded fluorescent tags enable the long-term observation of mitochondria even in vivo9. Enhanced green fluorescent protein (EGFP) is the most widely used fluorescent protein tag for studying various cellular functions, including labeling mitochondria. In addition to EGFP, distinct color variations of fluorescent proteins are required to study mitochondrial transfer between two different cells. Red fluorescent protein is the second most widely used fluorescent protein tag for labeling organelles in living cells. DsRed and its variants are often used for labeling mitochondria in studying mitochondrial transfer10–12. However, DsRed and some other red fluorescent proteins can oligomerize, which can affect protein folding and translocation to the mitochondria, Page 2/16 Page 2/16 causing mislocalization of the proteins13. Intracellular transfer of viable TurboRFP-labeled mitochondria We tested whether mitochondrial transfer can be detected by labeling mitochondria with TurboRFP. As described above, hAECs should have the potential to transfer intact mitochondria into cells with damaged mitochondria. We utilized mitochondria-targeted TurboRFP in iAECs to label donor mitochondria. HEK293T cells were employed as recipient cells. We transduced HEK293T cells with cytosolic EGFP to distinguish donor iAECs. We adopted a hydrogen peroxide (H2O2)-induced damage model for mitochondrial dysfunction. Because mitochondrial DNA is more susceptible than nuclear DNA to H2O2, the short-term treatment of cells with an appropriate concentration of H2O2 can selectively damage mitochondrial DNA and thus induce mitochondrial dysfunction. Donor HEK393T cells were exposed to hydrogen peroxide for 60 min prior to the experiments. Damaged HEK293T cells were cocultured with iAECs expressing mitochondria-targeted TurboRFP for 20 hours. Then, these cells were analyzed by confocal microscopy to properly distinguish the transferred intracellular red mitochondria from those overlaid on EGFP-positive cells. We found that TurboRFP-labeled mitochondria were transferred into EGFP-labeled HEK293T cells (Fig. 7). To evaluate the membrane potential of the transferred TurboRFP-positive mitochondria, the samples were further stained with MitoTracker Deep Red. 3D-reconstructed confocal microscopy imaging revealed that the TurboRFP- positive mitochondria in the EGFP-positive cells were costained with MitoTracker Deep Red, which indicated that the transferred mitochondria maintained the active membrane potential (Fig. 7C). Toxicity and photostability of red fluorescent proteins We next examined the toxicity of these red fluorescent proteins when expressed in mitochondria. We assessed the viability of cells stably transduced with mitochondria-targeted red fluorescent protein. Unexpectedly, the cell growth rates were almost comparable to each other and even to the control cells (Fig. 5A). All fluorescent proteins were maintained for one month. The fluorescence intensity and retained cell rate gradually decreased, except for that of EGFP, which exhibited no noticeable decrease even after one month (Fig. 5B). Since studies of mitochondrial transfer may require long-term observation of the transferred mitochondrion, the labeling fluorescent protein should possess sufficient brightness and photostability. We compared the fluorescence intensity and photobleaching profiles of these red fluorescent proteins in mitochondria. Among the four tested red fluorescent proteins, TurboRFP showed the highest fluorescence intensity (12831± 917 A.U.) under the optical setups widely used to observe RFP (Fig. 6A). The photostability was calculated from the change in fluorescent intensity after exposure. Although the Page 3/16 Page 3/16 fluorescence intensity of TurboRFP decreased quickly, it maintained approximately 50% intensity for up to 900 seconds of exposure (Fig. 6B). summary, we concluded that TurboRFP was the most suitable red fluorescent protein for In summary, we concluded that TurboRFP was the most suitable red fluorescent protein for exploring mitochondrial transfer. In summary, we concluded that TurboRFP was the most suitable red fluorescent protein for exploring mitochondrial transfer. Intracellular transfer of viable TurboRFP labeled mitochondria Discussion The results of this study showed that lentivirally transduced mitochondria-targeted DsRed and mCherry proteins formed a large number of bright punctate aggregates that were not localized in mitochondria. Aggregate formation did not affect the cell viability in this study, and thus the experiment could be carried out without noticing aggregation, which could lead to false conclusions. We further showed that these aggregates were localized mainly in lysosomes. Lysosomes are known to be able to release their contents by exocytosis machinery. The extracellularly released DsRed and mCherry aggregates could be internalized by other cells, including donor cells, through endocytosis or phagocytosis. These internalized aggregates may be mistaken for transferred mitochondria. This is an especially critical issue in the quantitative evaluation of intercellular mitochondrial transfer by fluorescence-activated flow cytometric analysis, which cannot easily distinguish internalized aggregates from transferred mitochondria based on their shape. Thus, although DsRed and mCherry are currently used in most mitochondrial transfer experiments, interpretation of the data requires care. This finding provides a first warning of the need to select appropriate red fluorescent protein for each experimental design. We do not have compelling evidence to explain the mechanisms underlying the aggregate formation of MTS-fused DsRed and mCherry proteins in lysosomes. One possible reason is that the MTS or the linker peptide used in our experiments might affect protein folding, maturation, or localization. However, other proteins used here and in other experiments with this MTS and linker were precisely localized in the mitochondria23. Alternatively, excessive expression of the proteins might induce aggregate formation in lysosomes. However, we showed that aggregates appeared even with a low MOI and did not increase with the viral dose. Furthermore, since the hPGK promoter is a weak promoter in most mammalian cell types, the proteins were unlikely to be expressed in excessive amounts. Katayama et al. reported that cytoplasmic DsRed proteins can accumulate in lysosomes, possibly by autophagocytosis21. Costantini et al. also reported that Golgi complex targeting sequence fused mCherry proteins accumulated in lysosomes22. Taken together, lysosomal aggregation seems like an inherent feature of DsRed and mCherry proteins because of their origin. Our comparison study indicated that TurboRFP is suitable as an alternative to DsRed and mCherry for mitochondrial transfer experiments in AECs. TurboRFP is a bright and rapidly maturing red fluorescent protein and is also widely used. Cell culture HEK293T cells were purchased from GeneHunter and cultured on collagen-coated dishes (Nunc) in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and streptomycin, and 2.5 ng/ml amphotericin B (Gibco). Immortalized hAEC line cells were previously established in our laboratory and used as donor cells in this study. Briefly, primary human amniotic epithelial cells (hAEC) were isolated from donated healthy human placentae. These hAECs were immortalized by using an SV40 Lentiviral vector (pLenti-SV40-T+t, Applied Biological Materials Inc., Richmond, BC Canada)28. Immortalized hAECs (iAECs) were cultured in DMEM supplemented with 10% fetal bovine serum (Nichirei), 200 μM L- glutamine (Gibco), 1 × nonessential amino acids (Gibco), 5.5 µM 2-mercaptoethanol (Gibco), 5 ng/ml EGF (PeproTech), 100 U/ml penicillin and streptomycin, and 2.5 ng/ml amphotericin B. All cells were cultured in 5% CO2 at 37 °C. Discussion TurboRFP Page 4/16 Page 4/16 has appropriate excitation and emission wavelengths that overlap minimally with green fluorescent molecules such as GFP or FITC and deep red fluorescent molecules such as Cy5 dye and is thus ideal for multicolor labeling strategies16. We showed that TurboRFP possesses brightness and photostability comparable to those of mCherry and exhibits no notable toxicity. Most importantly, we could detect mitochondrial transfer by labeling mitochondria with TurboRFP. The investigation of dynamics and morphology is essential for mitochondrial research. Our comparison study of mitochondrial labeling is also informative for other mitochondrial experiments. In this study, we showed for the first time that hAECs can donate their mitochondria to hydrogen peroxide-damaged cells. The ability of mitochondria to transfer into damaged cells has been reported in various cell types, including MSCs3. However, until now, there has been no report that hAECs can transfer mitochondria. As a donor of mitochondria transfer, hAECs have certain advantages associated with their unique characteristics4. First, hAECs contain healthy, virtually undamaged mitochondria. Since hAECs are placenta-derived neonatal cells, they have minimal exposure to aging and environmental damage. Second, large quantities of hAECs can be easily and inexpensively isolated from a placenta with minimum ethical concerns24. The placenta is usually discarded as clinical waste after delivery. Thus, no additional invasive procedures are required to obtain hAECs. Third, hAECs can be safely transplanted into other individuals. Due to their genomic stability, hAECs lack tumorigenicity and do not form any type of tumor upon transplantation25,26. Moreover, hAECs have immunomodulatory properties, which can benefit the control of immune-mediated inflammation and rejection after cell transplantation27. Taken together hAECs can be considered one of the best donor cells for mitochondrial transfer Our study paves the way for the cell properties, which can benefit the control of immune-mediated inflammation and rejection after cell transplantation27. Taken together, hAECs can be considered one of the best donor cells for mitochondrial transfer. Our study paves the way for the cell- based therapeutic application of hAECs to treat damaged mitochondria. In summary, we characterized various red fluorescent proteins for mitochondrial labeling. Among them, mCherry and DsRed proteins tended to accumulate in the lysosome. Moreover, we showed that hAECs whose mitochondria were labeled with TurboRFP possessed the ability to transfer functional mitochondria. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Imaging To image mitochondria-targeted proteins, iAECs were seeded on a 24-well plate (Nunc) at a density of 50,000 cells/well and infected with lentivirus after 12 hours. Cells were seeded on glass bottom dishes (Matsunami) 2 days before imaging. Cells were loaded with 100 nM MitoTracker green or Orange CMTMRos (Life Technologies) and 500 ng/ml Hoechst 33342 for 30 min in culture medium at 37 °C. For lysosome staining, cells were loaded with 100 nM LysoTracker Deep Red (Life Technologies) in addition to the above. Then, the cells were washed twice with medium and imaged in 5% CO2 at 37 °C in the culture medium. For the mitochondrial transfer experiment, HEK293T cells transduced with cytosolic GFP were seeded on collagen- and poly-L- lysine-coated glass-bottom dishes for two days. The cells were treated with 300 μM hydrogen peroxide for 60 minutes at 37 °C and then cocultured with iAECs transduced with mitochondria-targeted TurboRFP in fresh iAE medium. After 20 hours of culture, the cells were loaded with MitoTracker Deep Red (Life Technologies) and imaged at room temperature. Fluorescence images were captured using a confocal microscope (TCS SP8, Leica) equipped with a 63× objective (NA 1.40, HC PL APO, Leica) with excitation/emission wavelengths (nm) of 405/415–455 for Hoechst 33342, 488/496–543 for MitoTracker Green and EGFP, 555/565–620 for MitoTracker Red and the red fluorescent proteins, and 638/570–700 for LysoTracker Deep Red and MitoTracker Deep Red. Cell growth and flow cytometry experiments iAECs were seeded on a 24-well plate at a density of 5x104 cells/well and infected with lentivirus at an MOI of 5. After 4 days of infection, the cells were passaged and cultured for 6 days to exclude virus toxicity. Then, the cells were reseeded on 96-well plates (Nunc) at a density of 5,000 cells/well. Cell proliferation was measured 12, 24, 48, 72, and 120 hours after seeding by using a Cell Counting Kit-8 (Dojindo) according to the manufacturer's protocol. For flow cytometry analysis, cells were analyzed by a FACS Aria (BD) equipped with 488 (for EGFP) or 561 (for DsRed, mCherry, TurboRFP and mKOκ) nm laser and 530/30 (for EGFP) or 582/15 (for DsRed, mCherry, TurboRFP and mKOκ) nm emission filter. Cells were analyzed 6, 14, 21, 28 and 42 days after infection. Cells were passaged at intervals of 1 week. Acknowledgments This study was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers JP 20K17522 (T.M), JP 22K19574 (T.M), JP 22K06868 (I.T), a Nihon University School of Medicine 50th Anniversary Fund Research Grant (2021) (C.T), a Nihon University Research Grant-in-aid for Early-Career Scientists (I.T) and a research grant from the Chairperson and the President of Nihon University (2021–2023) (C.T, T.M). Lentiviral vector production Lentiviral vectors (transfer plasmids) were constructed by cloning each fluorescent gene into a backbone of a common second generation lentiviral vector plasmid with hPGK promoter (pLKO). Two human COXA8 genes were inserted under the promoter as a tandem MTS (Fig. 1). For lentiviral particle production, HEK293T cells were seeded on collagen-coated 10 cm dishes at 70–80% confluency. Cells were cotransfected with 4 μg of lentiviral vector plasmid along with 4 μg psPAX2 and 2 μg of pMD2. G (packaging plasmids, Addgene plasmids 12260 and 12259). Transfection mixtures were prepared in 500 μL of Opti-MEM I (Life Technologies) Page 5/16 containing the DNA and 27 μL of Lipofectamine 3000 (Life Technologies) according to the manufacturer's protocol. The medium was changed 12 h after transfection, and lentiviral supernatants were collected after 24 and 48 hr. Collected lentiviral supernatants were concentrated via ultracentrifugation, and the medium was exchanged with phosphate buffered saline. I i containing the DNA and 27 μL of Lipofectamine 3000 (Life Technologies) according to the manufacturer's protocol. The medium was changed 12 h after transfection, and lentiviral supernatants were collected after 24 and 48 hr. Collected lentiviral supernatants were concentrated via ultracentrifugation, and the medium was exchanged with phosphate buffered saline. Imaging Corresponding author Correspondence to Toshio Miki Correspondence to Toshio Miki References 1. Spees, J. 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Author information Contributions Page 6/16 Page 6/16 I.T., C.T., S.H., and K.K. carried out the cell biology experiments. I.T. and K.K. carried out the imaging experiments and analysis, and constructed figures. I.T., C.T., K.K. and T.M. designed the study and wrote the manuscript. All authors discussed the results and approved the submission of the manuscript. References Isolation of Amniotic Epithelial Stem Cells. Curr. Protoc. Stem Cell Biol.3, 1E.3.1-1E.3.9 (2007). 24. Miki, T., Marongiu, F., Ellis, E. & C. Strom, S. Isolation of Amniotic Epithelial Stem Cells. Curr. Protoc. Stem Cell Biol.3, 1E.3.1-1E.3.9 (2007). 25. Miki, T., Lehmann, T., Cai, H., Stolz, D. B. & Strom, S. C. Stem Cell Characteristics of Amniotic Epithelial Cells. Stem Cells23, 1549–1559 (2005). 26. Ilancheran, S. et al. Stem Cells Derived from Human Fetal Membranes Display Multilineage Differentiation Potential. Biol. Reprod.77, 577–588 (2007). 27. Miki, T. Stem cell characteristics and the therapeutic potential of amniotic epithelial cells. Am. J. Reprod. Immunol.80, e13003 (2018). 28. Miki, T., Takano, C., Garcia, I. M. & Grubbs, B. H. Construction and Evaluation of a Subcutaneous Splenic Injection Port for Serial Intraportal Vein Cell Delivery in Murine Disease Models. Stem Cells Int.2019, e5419501 (2019). 28. Miki, T., Takano, C., Garcia, I. M. & Grubbs, B. H. Construction and Evaluation of a Subcutaneous Splenic Injection Port for Serial Intraportal Vein Cell Delivery in Murine Disease Models. Stem Cells Int.2019, e5419501 (2019). 29. Cormack, B. P., Valdivia, R. H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33– 38 (1996). 30. Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7, 1006–1012 (2006). 30. Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7, 1006–1012 (2006). References M., Burgess, R. W. & Lichtman, J. W. Imaging axonal transport of mitochondria in vivo. Nat. Methods4, 559–561 (2007). 10. Gao, L., Zhang, Z., Lu, J. & Pei, G. Mitochondria Are Dynamically Transferring Between Human Neural Cells and Alexander Disease-Associated GFAP Mutations Impair the Astrocytic Transfer. Front. Cell. Neurosci.13, (2019). 11. Dong, L.-F. et al. Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells. eLife6, e22187. 12. Moschoi, R. et al. Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy. Blood128, 253–264 (2016). 13. Campbell, R. E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci.99, 7877–7882 (2002). 14. Bevis, B. J. & Glick, B. S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol.20, 83–87 (2002). 15. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol.22, 1567–1572 (2004). 15. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol.22, 1567–1572 (2004). 16. Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods4, 555–557 (2007). 16. Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods4, 555–557 (2007). Page 7/16 Page 7/16 Page 7/16 17. Tsutsui, H., Karasawa, S., Okamura, Y. & Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat. Methods5, 683–685 (2008). 18. Wrobel, L. et al. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature524, 485–488 (2015). 19. Boos, F. et al. Mitochondrial protein-induced stress triggers a global adaptive transcriptional programme. Nat. Cell Biol.21, 442–451 (2019). 20. Filippin, L. et al. Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix. Cell Calcium37, 129–136 (2005). 21. Katayama, H., Yamamoto, A., Mizushima, N., Yoshimori, T. & Miyawaki, A. GFP-like Proteins Stably Accumulate in Lysosomes. Cell Struct. Funct.33, 1–12 (2008). 22. Costantini, L. M. et al. A palette of fluorescent proteins optimized for diverse cellular environments. Nat. Commun.6, 7670 (2015). 23. Suzuki, J. et al. Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat 23. Suzuki, J. et al. Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat. Commun.5, 4153 (2014). 24. Miki, T., Marongiu, F., Ellis, E. & C. Strom, S. Tables Table 1 Reported properties of fluorescent proteins. Table 1 Reported properties of fluorescent proteins. Protein Oligomerization Origin Excitation maximum (nm) Emission maximum (nm) Extinction coefficient (M-1 cm-1) Quantum yield pKa reference EGFP Weak dimer Aequorea victoria 488 507 55,900 0.6 6.0 29, 30 DsRed Tetramer Discosoma sp 558 583 57,000 0.79 4.7 13, 14 mCherry Monomer Discosoma sp 587 610 72,000 0.22 4.5 15 TurboRFP Dimer Entacmaea quadricolor 553 574 92,000 0.67 4.4 16, 31 mKOκ Monomer Verrillofungia concinna 551 563 105,000 0.61 4.2 17 Page 8/16 Figure 1 Figure 1 Scheme of a mitochondria-targeted fluorescent protein expression vector. The selected fluorescent protein genes were inserted between BamHI and KpnI restriction enzyme sites of pLKO-based backbone, each preceded by a human phosphoglycerate kinase (hPGK) promoter and a tandem mitochondrial targeting signal (MTS) of the human COX8A gene. Page 9/16 Figure 2 Aggregate formation of mitochondria targeted mCherry and DsRed proteins. (A) Representative images of iAECs transduced with each mitochondria-targeted protein MitoTracker and Hoechst. Merged images of each color are shown. Blue: Hoechst 3334 mKOκ and uninfected control, green: MitoTracker Green, red: each fluorescent protein. F Figure 2 Figure 2 Figure 2 Aggregate formation of mitochondria targeted mCherry and DsRed proteins. Aggregate formation of mitochondria targeted mCherry and DsRed proteins. Aggregate formation of mitochondria targeted mCherry and DsRed proteins. (A) Representative images of iAECs transduced with each mitochondria-targeted protein or uninfected control stained with MitoTracker and Hoechst. Merged images of each color are shown. Blue: Hoechst 33342. For DsRed, mCherry, TurboRFP mKOκ and uninfected control, green: MitoTracker Green, red: each fluorescent protein. For EGFP, green: EGFP, red: MitoTracker Orange. Scale bar = 10 µm. (A) Representative images of iAECs transduced with each mitochondria-targeted protein or uninfected control stained with MitoTracker and Hoechst. Merged images of each color are shown. Blue: Hoechst 33342. For DsRed, mCherry, TurboRFP mKOκ and uninfected control, green: MitoTracker Green, red: each fluorescent protein. For EGFP, green: EGFP, red: MitoTracker Orange. Scale bar = 10 µm. (B) Proportion of cells with aggregates in the mitochondria-targeted fluorescent protein-transduced cell population. Data are shown as the mean ± SD, n = 3 biological replicates. Each replicate included at least 80 cells from 7 optical fields. *: p < 0.01, : p < 0.05 n.s.: not significant (p > 0.05); Tukey's HSD test. Page 10/16 Figure 3 Aggregate formation is not dependent on the virus dose. (A) Representative images of iAECs transduced with serially diluted lentivirus containing mitochondria-targeted mCherry (red) stained with MitoTracker Green (green) and Hoechst 33342 (blue). The top-left numbers in the images indicate the virus MOI. Scale bar = 10 µm. (B) The proportion of cells with aggregates is plotted against the virus titer. Data are shown as the mean ± SD, n = 3 biological replicates. Each replicate included at least 80 cells from 7 visual fields. n.s.: not significant (p > 0.05); Tukey's HSD test. Figure 3 Aggregate formation is not dependent on the virus dose. Figure 3 Aggregate formation is not dependent on the virus dose. (A) Representative images of iAECs transduced with serially diluted lentivirus containing mitochondria-targeted mCherry (red) stained with MitoTracker Green (green) and Hoechst 33342 (blue). The top-left numbers in the images indicate the virus MOI. Scale bar = 10 µm. (A) Representative images of iAECs transduced with serially diluted lentivirus containing mitochondria-targeted mCherry (red) stained with MitoTracker Green (green) and Hoechst 33342 (blue). The top-left numbers in the images indicate the virus MOI. Scale bar = 10 µm. (B) The proportion of cells with aggregates is plotted against the virus titer. Data are shown as the mean ± SD, n = 3 biological replicates. Each replicate included at least 80 cells from 7 visual fields. n.s.: not significant (p > 0.05); Tukey's HSD test. (B) The proportion of cells with aggregates is plotted against the virus titer. Data are shown as the mean ± SD, n = 3 biological replicates. Each replicate included at least 80 cells from 7 visual fields. n.s.: not significant (p > 0.05); Tukey's HSD test. Page 11/16 Figure 4 Colocalization of mCherry aggregates with lysosomes. Representative images of iAECs transduced with mitochondria targeting mCherry or GFP or uninfected controls stained with MitoTracker Green (green, for mCherry and control) or Orange (red, for GFP) and LysoTracker Deep Red (blue). Scale bar = 10 Figure 4 Figure 4 Colocalization of mCherry aggregates with lysosomes. Representative images of iAECs transduced with mitochondria targeting mCherry or GFP or uninfected controls stained with MitoTracker Green (green, for mCherry and control) or Orange (red, for GFP) and LysoTracker Deep Red (blue). Scale bar = 10 µm. Representative images of iAECs transduced with mitochondria targeting mCherry or GFP or uninfected controls stained with MitoTracker Green (green, for mCherry and control) or Orange (red, for GFP) and LysoTracker Deep Red (blue). Scale bar = 10 µm. Representative images of iAECs transduced with mitochondria targeting mCherry or GFP or uninfected controls stained with MitoTracker Green (green, for mCherry and control) or Orange (red, for GFP) and LysoTracker Deep Red (blue). Scale bar = 10 µm. Page 12/16 Page 12/16 Page 12/16 Page 12/16 Figure 5 Cytotoxicity of mitochondria-targeted proteins. (A) Growth curve of iAECs transduced with the mitochondria-targeted protein or uninfected control. Data are shown as the mean ± SD, n = 3, n.s.: not significant (p > 0.05); Tukey's HSD test. Figure 3 (B) Time course of depletion of fluorescent positive cells analyzed by flow cytometry Figure 5 Cytotoxicity of mitochondria-targeted proteins. (A) Growth curve of iAECs transduced with the mitochondria-targeted protein or uninfected control. Data are shown as the mean ± SD, n = 3, n.s.: not significant (p > 0.05); Tukey's HSD test. (A) Growth curve of iAECs transduced with the mitochondria-targeted protein or uninfected control. Data are shown as the mean ± SD, n = 3, n.s.: not significant (p > 0.05); Tukey's HSD test. (B) Time course of depletion of fluorescent positive cells analyzed by flow cytometry. Page 13/16 Page 13/16 Figure 6 Brightness and photostability of the mitochondria-targeted proteins. (A) Average fluorescence intensity of mitochondria-targeted fluorescent proteins. n = 3 biological replicates. Each replicate Figure 6 Brightness and photostability of the mitochondria-targeted proteins. Brightness and photostability of the mitochondria-targeted proteins. (A) Average fluorescence intensity of mitochondria-targeted fluorescent proteins. n = 3 biological replicates. Each replicate included at least 80 cells from 7 visual fields. n.s.: not significant (p > 0.05); Tukey's HSD test. (A) Average fluorescence intensity of mitochondria-targeted fluorescent proteins. n = 3 biological replicates. Each replicate included at least 80 cells from 7 visual fields. n.s.: not significant (p > 0.05); Tukey's HSD test. (B) Photobleaching of fluorescent proteins in iAECs during confocal imaging with a 488 nm (for EGFP) or a 555 nm (for other fluorescent proteins) excitation laser. The data are the average of at least three cells. (B) Photobleaching of fluorescent proteins in iAECs during confocal imaging with a 488 nm (for EGFP) or a 555 nm (for other fluorescent proteins) excitation laser. The data are the average of at least three cells. Page 14/16 Page 14/16 Figure 7 Mitochondria transfer from AE cells to damaged cells. Mitochondria transfer from AE cells to damaged cells. (A) Representative images of transferred mitochondria. Stacked confocal microscopy images are shown. White arrows indicate transferred mitochondria. The white dotted line indicates a boundary of cocultured donor cell. (B) 3D reconstruction of z-slices of the corresponding regions shown in (A). (B) 3D reconstruction of z-slices of the corresponding regions shown in (A). Middle: One of the z-slice images shown in (A). Upper: An optical section of the corresponding region of the vertical bar shown in the middle panel. Right: An optical section of the corresponding region of the horizontal bar shown in the middle panel. Both horizontal and vertical bars shown in the upper or right panel indicate sectioned positions of the middle. (C) The 3D reconstructed image shown in (B). Page 15/16 Page 15/16 Page 15/16 Top: Full view, bottom: an enlarged image of the squared area at the top. Black arrows indicate transferred mitochondria, as shown in (A). Note that TurboRFP-labeled mitochondria were inside the recipient cell and were stained by MitoTracker Deep Red, whose accumulation is dependent on mitochondrial membrane potential. Scale bar = 10 µm. Top: Full view, bottom: an enlarged image of the squared area at the top. Black arrows indicate transferred mitochondria, as shown in (A). Note that TurboRFP-labeled mitochondria were inside the recipient cell and were stained by MitoTracker Deep Red, whose accumulation is dependent on mitochondrial membrane potential. Scale bar = 10 µm. Note that TurboRFP-labeled mitochondria were inside the recipient cell and were stained by MitoTracker Deep Red, whose accumulation is dependent on mitochondrial membrane potential. Scale bar = 10 µm. Page 16/16
https://openalex.org/W2884768727	https://europepmc.org/articles/pmc6070941?pdf=render	English	null	Effects of Cold Rolling and Annealing Prior to Dealloying on the Microstructure of Nanoporous Gold	Nanomaterials	2,018	cc-by	7,141	Received: 8 June 2018; Accepted: 10 July 2018; Published: 18 July 2018 Abstract: The properties of nanoporous gold (NPG) were known to be dependent on the microstructure of NPG. In this study, the effects of cold rolling and annealing of the original Ag0.7Au0.3 alloy on the microstructure of NPG produced by dealloying under free corrosion condition were investigated. Ag0.7Au0.3 alloy samples were cold-rolled to different strain levels/thickness reductions up to 98% and annealed at 900 ◦C for 3 h before dealloying. It was found that cold rolling and annealing of the original alloy can lead to reduced ligament and pore sizes of NPG. Moreover, post-deformation annealing of the original alloy was found to facilitate the formation of a homogeneous and continuous NPG structure. The minima of pore and ligament sizes (both being ~8 nm) with uniform distribution were obtained in the annealed sample with a thickness reduction of 60% for a dealloying time of 7 h. The present study indicated the signiﬁcant effect of a pre-dealloying treatment of the original alloy (by plastic deformation and annealing) on the formation and optimization of the NPG microstructure produced by dealloying. Keywords: nanoporous gold; microstructure; dealloying; cold rolling; annealing nanomaterials nanomaterials nanomaterials Effects of Cold Rolling and Annealing Prior to Dealloying on the Microstructure of Hanyu Hui 1, Re Xia 1,2 ID , Juying Li 3, Qingsong Mei 1,, Ye Ma 1, Feng Chen 1 and Yan Le Hanyu Hui 1, Re Xia 1,2 ID , Juying Li 3, Qingsong Mei 1,, Ye Ma 1, Feng Chen 1 and Yan Lei 1 1 School of Power and Mechanical Engineering, Wuhan University, Wuhan 430072, China; huihanyu0927@163.com (H.H.); xiare@whu.edu.cn (R.X.); mayer@whu.edu.cn (Y.M.); fengc1004@163.com (F.C.); yappee@126.com (Y.L.) Hanyu Hui 1, Re Xia 1,2 ID , Juying Li 3, Qingsong Mei 1,, Ye Ma 1, Feng Chen 1 and Y 1 School of Power and Mechanical Engineering, Wuhan University, Wuhan 430072, China; huihanyu0927@163.com (H.H.); xiare@whu.edu.cn (R.X.); mayer@whu.edu.cn (Y.M.); fengc1004@163.com (F.C.); yappee@126.com (Y.L.) Hanyu Hui 1, Re Xia 1,2 ID , Juying Li 3, Qingsong Mei 1,, Ye Ma 1, Feng Chen 1 and Yan Lei 1 1 School of Power and Mechanical Engineering, Wuhan University, Wuhan 430072, China; huihanyu0927@163.com (H.H.); xiare@whu.edu.cn (R.X.); mayer@whu.edu.cn (Y.M.); fengc1004@163.com (F.C.); yappee@126.com (Y.L.) g y pp 2 Key Laboratory of Hydraulic Machinery Transients, Ministry of Education, Wuhan University, Wuhan 430072, China 3 School of Mechanical Engineering, Wuhan Polytechnic University, Wuhan 430023, China; jylimei@163. * Correspondence: qsmei@whu.edu.cn; Tel.: +86-27-68772252 Nanomaterials 2018, 8, 540; doi:10.3390/nano8070540 1. Introduction Due to their chemical stability, strength, and high speciﬁc surface area, nanoporous metals have attracted great interests in many applications, including catalysis, fuel cells, sensors, and more [1,2]. Current popular methods for the preparation of nanoporous metals include template methods [3] and dealloying [4]. Nanoporous metals, including Au, Cu, Ni, Ag, and Pt, have been successfully fabricated by chemical or electrochemical dealloying [4–8], among which much attention has been paid to the prototypical Ag–Au system. The dealloying of an Ag–Au alloy can produce nanoporous gold (NPG) with an open, three-dimensional bicontinuous interpenetrating ligament-channel structure with nanometer length scales [4,5,7]. g The properties of NPG were found to depend on the microstructure of NPG, such as length scales of ligaments and pores [9–11]. Studies have revealed that the sizes of pores and ligaments of NPG produced by a dealloying of Ag–Au alloy were inﬂuenced by corrosion time, corrosion temperature, and an annealing of samples after corrosion [12–14]. The original Ag–Au alloy was usually prepared in various states before dealloying [15–21], for which the different fabrication and processing methods and states of the original alloy were found to have signiﬁcant effects on the morphology of the NPG microstructure [16,17]. www.mdpi.com/journal/nanomaterials Nanomaterials 2018, 8, 540; doi:10.3390/nano8070540 www.mdpi.com/journal/nanomaterials 2 of 7 Nanomaterials 2018, 8, 540 Plastic deformation and annealing are common methods used for the processing and microstructure modiﬁcation of metals. It is well known that plastic deformation can result in an increase of lattice defects and even grain reﬁnement in metals, and a subsequent annealing can lead to the recovery and recrystallization of the deformed structure by formation of more homogenous and reﬁned grains with reduced lattice defects [22–24]. Thus, it is expected that plastic deformation and annealing prior to dealloying can have signiﬁcant effects on the dealloying process and hence the NPG microstructure, for which experimental evidences are still lacking. In this work, the original Ag–Au alloy was subjected to cold rolling and annealing to investigate their effects on the microstructure of NPG produced by subsequent dealloying. 2. Experimental The Ag0.7Au0.3 (wt. %, purity, 99.99%) alloy ingot used in this study was purchased from China New Metal (Beijing, China). The ingot was cut into 7 × 7 × 1 mm3 pieces as original samples. The cold rolling of the Ag0.7Au0.3 alloy was performed with thickness reductions up to 98% at the speed of 1 rpm (referred to as CR samples). The annealing of cold-rolled samples was carried out at 900 ◦C for 3 h under argon atmosphere followed by furnace cooling (referred to as AN samples). Samples were polished with abrasive paper and then cleaned with dehydrated alcohol in an ultrasonic cleaning bath before dealloying. The dealloying was performed by immersing samples in a 68% nitric acid solution at room temperature under free corrosion conditions for 3 h or 7 h. The as-dealloyed samples were rinsed repeatedly using dehydrated alcohol before further investigation. The microstructure was observed using a ZEISS SIGMA ﬁeld emission scanning electron microscope (FE-SEM) (Carl Zeiss Microscopy Ltd, Cambridge, United Kingdom), operated at an accelerating voltage of 5 kV. The pore and ligament sizes, deﬁned as the width and diameter of the pore, as well as the wall thickness of the band, respectively, were averaged by manually measuring about 100 data points of SEM images. 3. Results and Discussion SEM images of NPG produced by dealloying of CR samples with different thickness reduc (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples and without post-deformation annealing with the same dealloying time of 3 h. Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h 100nm 100nm 100nm 100nm (a) (b) (c) (d) Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h. 100nm 100nm 100nm 100nm (a) (b) (c) (d) Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h. Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h. 100nm 100nm 100nm 100nm (a) (b) (c) (d) 100nm 100nm (b) (d) 100nm 100nm (b) (d) 100nm 100nm (a) (c) 100nm 100nm (a) (c) (a) (a) (b) (b) (c) (c) Figure 1. 3. Results and Discussion SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h. Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h. Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h Figure 2. SEM images of NPG produced by dealloying of AN samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% cold rolled and annealed for a dealloying time of 7 h and (d) 98% cold rolled and annealed for a dealloying time of 3 h. 100 nm 100 nm 100 nm 100 nm (a) (b) (c) (d) 20 nm Figure 2. SEM images of NPG produced by dealloying of AN samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% cold rolled and annealed for a dealloying time of 7 h and (d) 98% cold rolled and annealed for a dealloying time of 3 h. 100 nm 100 nm 100 nm 100 nm (a) (b) (c) (d) 20 nm Figure 2. SEM images of NPG produced by dealloying of AN samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% cold rolled and annealed for a dealloying time of 7 h and (d) 98% cold rolled and annealed for a dealloying time of 3 h. 3. Results and Discussion As shown in Figure 1, after dealloying the CR samples, the NPG structure was produced with average ligament sizes of 8~20 nm, and average pore sizes of 8~16 nm, respectively, depending on the degree of plastic deformation. It was found that both the ligament and pore sizes of NPG produced by the dealloying of the CR samples decrease with increasing thickness reduction, which can be attributed to the increased lattice defects, such as point defects and dislocations in the CR samples induced by plastic deformation. It is known that formation of NPG by dealloying of Ag–Au alloy is due to the selective corrosion of Ag [20,25]. During the dealloying, lattice defects (point defects and dislocations) can act as preferred sites for corrosion. Therefore, an increase of the lattice defects can lead to increased corrosion sites, and hence a reduction in NPG pore size. It is noted that some areas of the NPG samples (Figure 1) are still discontinuous, which is mainly due to the large number of defects caused by cold rolling and the microscopical heterogeneity of plastic strain in the sample. The CR samples were further annealed at 900 ◦C for 3 h and subjected to dealloying (AN samples). Figure 2 shows the corresponding NPG microstructure for the AN samples. As shown in Figure 2, an open, bicontinuous interpenetrating ligament-channel structure of NPG with an average ligament sizes of 8~20 nm and an average pore sizes of 8~14 nm, respectively, was produced in the AN samples, depending on the thickness reduction of the cold rolling. It is clear that compared with the CR samples (Figure 1), the AN samples have a more uniform and better connected nanoporous structure (Figure 2). 3 of 7 Nanomaterials 2018, 8, 540 Figure 1. SEM images of NPG produced by dealloying of CR samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% for a dealloying time of 7 h and (d) 98%, for which a shorter dealloying time of 3 h was used to avoid over-corrosion of the sample and to compare the NPG structure of samples with and without post-deformation annealing with the same dealloying time of 3 h. 100nm 100nm 100nm 100nm (a) (b) (c) (d) Figure 1. 3. Results and Discussion 100 nm 100 nm (b) (d) 20 nm 100 nm 100 nm (b) (d) 20 nm (b) (b) 100 nm (c) 100 nm (c) g g p y y g p reductions: (a) 0%; (b) 60% and (c) 90% cold rolled and annealed for a dealloying time of 7 h and (d) 98% cold rolled and annealed for a dealloying time of 3 h. Figure 2. SEM images of NPG produced by dealloying of AN samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% cold rolled and annealed for a dealloying time of 7 h and (d) 98% cold rolled and annealed for a dealloying time of 3 h. Figure 2. SEM images of NPG produced by dealloying of AN samples with different thickness reductions: (a) 0%; (b) 60% and (c) 90% cold rolled and annealed for a dealloying time of 7 h and (d) 98% cold rolled and annealed for a dealloying time of 3 h. Figure 3 depicts the average pore and ligament sizes as functions of thickness reduction for CR and AN samples as indicated. The detailed numerical data are shown in Table 1. As shown in Figure 3, 4 of 7 or CR Nanomaterials 2018, 8, 540 Figure 3 depicts the AN samples have smaller pore and ligament sizes than those of the CR samples with a thickness reduction of 60%, while larger ones with a thickness reduction of 90%, i.e., the minima of pore and ligament sizes (both are ~8 nm) were obtained in AN sample with a thickness reduction of 60% for a dealloying time of 7 h. Similarly, a more uniform and continuous NPG structure is observed in the AN sample than those in the CR sample, with a thickness reduction of 98% and a dealloying time of 3 h under the same free corrosion condition, despite a slight increase of the pore size (~12 nm) in the AN sample compared to that of the CR sample (~8 nm). This can be attributed to the faster corrosion rate in the AN sample with a larger thickness reduction: in the same corrosion time, the structure of NPG is more quickly stabilized, and a single nanopore coarsening stage occurs in the AN sample with a larger thickness reduction. 3. Results and Discussion Compared with previous studies of NPG produced by means of a free corrosion method with pore and ligament sizes mostly in the range of ~6–60 nm [4,12,15,20,21,26], the ligaments and pores of NPG produced in this study have almost reached the minima. It should be noted that simply by pre-dealloying deformation and annealing, one may not get the best NPG microstructure over all other methods, since the ﬁnal formation of NPG can be inﬂuenced by many other factors, such as original alloy composition and microstructure, dealloying condition, heat treatment after dealloying, and so on. p g 3, the AN samples have smaller pore and ligament sizes than those of the CR samples with a thickness reduction of 60%, while larger ones with a thickness reduction of 90%, i.e., the minima of pore and ligament sizes (both are ~8 nm) were obtained in AN sample with a thickness reduction of 60% for a dealloying time of 7 h. Similarly, a more uniform and continuous NPG structure is observed in the AN sample than those in the CR sample, with a thickness reduction of 98% and a dealloying time of 3 h under the same free corrosion condition, despite a slight increase of the pore size (~12 nm) in the AN sample compared to that of the CR sample (~8 nm). This can be attributed to the faster corrosion rate in the AN sample with a larger thickness reduction: in the same corrosion time, the structure of NPG is more quickly stabilized, and a single nanopore coarsening stage occurs in the AN sample with a larger thickness reduction. Compared with previous studies of NPG produced by means of a free corrosion method with pore and ligament sizes mostly in the range of ~6–60 nm [4,12,15,20,21,26], the ligaments and pores of NPG produced in this study have almost reached the minima. It should be noted that simply by pre-dealloying deformation and annealing, one may not get the best NPG microstructure over all other methods, since the final formation of NPG can be influenced by many other factors, such as original alloy composition and microstructure, dealloying condition, heat treatment after dealloying, and so on. Figure 3. 3. Results and Discussion Average ligament and pore sizes of NPG of CR and AN samples as functions of thickness edu tio 0 20 40 60 80 100 5 10 15 20 25 30 35 40 Size (nm) Thickness reduction (%) Ligament (CR Sample) Pore (CR Sample) Ligament (AN Sample) Pore (AN Sample) Figure 3. Average ligament and pore sizes of NPG of CR and AN samples as functions of thickness reduction. Thickness reduction (%) Figure 3. Average ligament and pore sizes of NPG of CR and AN samples as functions of thickne d ti Figure 3. Average ligament and pore sizes of NPG of CR and AN samples as functions of thickness reductio Figure 3. Average ligament and pore sizes of NPG of CR and AN samples as functions of thickness reduction Figure 3. Average ligament and pore sizes of NPG of CR and AN samples as functions of thickness reduction. reduction. Table 1. Average ligament and pore sizes of NPG of CR and AN samples for different thickness reduction. Parameters CR Samples AN Samples Thickness reduction 0% 60% 90% 98% 0% 60% 90% 98% Pore size (nm) 16.1 ± 3.4 13.7 ± 3.8 9.4 ± 2.5 8.0 ± 2.0 10.5 ± 2.0 7.5 ± 1.7 14.1 ± 3.3 12.0 ± 2.4 Ligament size (nm) 20.2 ± 4.7 16.0 ± 2.6 11.5 ± 2.4 8.1 ± 1.5 20.3 ± 4.3 7.9 ± 1.2 17.7 ± 3.0 13.0 ± 2.2 To further characterize the uniformity of the pores, the full width at half maxima (FWHM) of the Gauss function fitting curves of the pore size distribution were measured, as shown in Figure 4. Naturally, the smaller the value of FWHM, the more uniform the pores of the NPG sample. Figure 5 plots FWHM as a function of thickness reduction for CR and AN samples, as indicated. As shown in Figure 5, the FWHM shows a clear decrease in the CR samples as compared to that of the undeformed sample, indicating a more uniform microstructure of NPG induced by the cold rolling of the original sample. Moreover, one can see from Figure 5 that AN samples show evidently smaller FWHM than those of CR samples i e the distribution of pores is more uniform in AN samples From Figures 3 Table 1. Average ligament and pore sizes of NPG of CR and AN samples for different thickness reduction. 3. Results and Discussion Parameters CR Samples AN Samples Thickness reduction 0% 60% 90% 98% 0% 60% 90% 98% Pore size (nm) 16.1 ± 3.4 13.7 ± 3.8 9.4 ± 2.5 8.0 ± 2.0 10.5 ± 2.0 7.5 ± 1.7 14.1 ± 3.3 12.0 ± 2.4 Ligament size (nm) 20.2 ± 4.7 16.0 ± 2.6 11.5 ± 2.4 8.1 ± 1.5 20.3 ± 4.3 7.9 ± 1.2 17.7 ± 3.0 13.0 ± 2.2 ( ) Ligament size (nm) 20.2 ± 4.7 16.0 ± 2.6 11.5 ± 2.4 8.1 ± 1.5 20.3 ± 4.3 7.9 ± 1.2 17.7 ± 3.0 13.0 ± 2.2 To further characterize the uniformity of the pores, the full width at half maxima (FWHM) of the Gauss function fitting curves of the pore size distribution were measured, as shown in Figure 4. Naturally, the smaller the value of FWHM, the more uniform the pores of the NPG sample. Figure 5 plots FWHM as a function of thickness reduction for CR and AN samples, as indicated. As shown in Figure 5, the FWHM shows a clear decrease in the CR samples as compared to that of the undeformed sample, indicating a more uniform microstructure of NPG induced by the cold rolling of the original sample. Moreover, one can see from Figure 5 that AN samples show evidently smaller FWHM than h f CR l i h di ib i f i if i AN l F Fi 3 To further characterize the uniformity of the pores, the full width at half maxima (FWHM) of the Gauss function ﬁtting curves of the pore size distribution were measured, as shown in Figure 4. Naturally, the smaller the value of FWHM, the more uniform the pores of the NPG sample. Figure 5 plots FWHM as a function of thickness reduction for CR and AN samples, as indicated. As shown in Figure 5, the FWHM shows a clear decrease in the CR samples as compared to that of the undeformed sample, indicating a more uniform microstructure of NPG induced by the cold rolling of the original sample. Moreover, one can see from Figure 5 that AN samples show evidently smaller FWHM than those of CR samples, i.e., the distribution of pores is more uniform in AN samples. 3. Results and Discussion Parameters CR Samples AN Samples Thickness reduction 0% 60% 90% 98% 0% 60% 90% 98% Pore size (nm) 16.1 ± 3.4 13.7 ± 3.8 9.4 ± 2.5 8.0 ± 2.0 10.5 ± 2.0 7.5 ± 1.7 14.1 ± 3.3 12.0 ± 2.4 Ligament size (nm) 20.2 ± 4.7 16.0 ± 2.6 11.5 ± 2.4 8.1 ± 1.5 20.3 ± 4.3 7.9 ± 1.2 17.7 ± 3.0 13.0 ± 2.2 To further characterize the uniformity of the pores, the full width at half maxima (FWHM) of the Gauss function ﬁtting curves of the pore size distribution were measured, as shown in Figure 4. Naturally, the smaller the value of FWHM, the more uniform the pores of the NPG sample. Figure 5 plots FWHM as a function of thickness reduction for CR and AN samples, as indicated. As shown in Figure 5, the FWHM shows a clear decrease in the CR samples as compared to that of the undeformed sample, indicating a more uniform microstructure of NPG induced by the cold rolling of the original sample. Moreover, one can see from Figure 5 that AN samples show evidently smaller FWHM than those of CR samples, i.e., the distribution of pores is more uniform in AN samples. From Figures 3 and 5, one can see that the AN sample with a thickness reduction of 60% and Table 1 Average ligament and pore sizes of NPG of CR and AN samples for different thickness Table 1. Average ligament and pore sizes of NPG of CR and AN samples for different thickness reduction. Table 1. Average ligament and pore sizes of NPG of CR and AN samples for different thickness reduction. Parameters CR Samples AN Samples Thickness reduction 0% 60% 90% 98% 0% 60% 90% 98% P i ( ) 16 1 3 4 13 7 3 8 9 4 2 5 8 0 2 0 10 5 2 0 7 5 1 7 14 1 3 3 12 0 2 4 Table 1. Average ligament and pore sizes of NPG of CR and AN samples for different thickness reduction. 3. Results and Discussion From Figures 3 and 5, one can see that the AN sample with a thickness reduction of 60% and ( ) Ligament size (nm) 20.2 ± 4.7 16.0 ± 2.6 11.5 ± 2.4 8.1 ± 1.5 20.3 ± 4.3 7.9 ± 1.2 17.7 ± 3.0 13.0 ± 2.2 To further characterize the uniformity of the pores, the full width at half maxima (FWHM) of the Gauss function fitting curves of the pore size distribution were measured, as shown in Figure 4. Naturally, the smaller the value of FWHM, the more uniform the pores of the NPG sample. Figure 5 plots FWHM as a function of thickness reduction for CR and AN samples, as indicated. As shown in Figure 5, the FWHM shows a clear decrease in the CR samples as compared to that of the undeformed sample, indicating a more uniform microstructure of NPG induced by the cold rolling of the original sample. Moreover, one can see from Figure 5 that AN samples show evidently smaller FWHM than h f CR l i h di ib i f i if i AN l F Fi 3 To further characterize the uniformity of the pores, the full width at half maxima (FWHM) of the Gauss function ﬁtting curves of the pore size distribution were measured, as shown in Figure 4. Naturally, the smaller the value of FWHM, the more uniform the pores of the NPG sample. Figure 5 plots FWHM as a function of thickness reduction for CR and AN samples, as indicated. As shown in Figure 5, the FWHM shows a clear decrease in the CR samples as compared to that of the undeformed sample, indicating a more uniform microstructure of NPG induced by the cold rolling of the original sample. Moreover, one can see from Figure 5 that AN samples show evidently smaller FWHM than those of CR samples, i.e., the distribution of pores is more uniform in AN samples. From Figures 3 and 5, one can see that the AN sample with a thickness reduction of 60% and 5 of 7 5 of 7 Nanomaterials 2018, 8, 540 Nanomaterials 2018, 8, x FO a dealloying time of 7 h has both the smallest pore and ligament sizes and FWHM, indicating an optimal condition for the production of NPG with reﬁned pore size and uniform distribution by dealloying. 3. Results and Discussion and 5, one can see that the AN sample with a thickness reduction of 60% and a dealloying time of 7 h has both the smallest pore and ligament sizes and FWHM, indicating an optimal condition for the production of NPG with refined pore size and uniform distribution by dealloying. p y y g Figure 4. Pore size distribution with corresponding Gauss fit for different samples as indicated. (a–d) CR samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively; (e–h) AN samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively. 3 6 9 12 15 18 21 24 0.0 0.1 0.2 0.3 0.4 0.5 Relative Frequency Pore Size (nm) 0%-AN Sample Dealloyed 7h 3 6 9 12 15 0.0 0.2 0.4 0.6 0.8 Relative Frequency Pore Size (nm) 60%-AN Sample Dealloyed 7h 6 9 12 15 18 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 90%-AN Sample Dealloyed 7h 6 9 12 15 18 21 24 0.0 0.2 0.4 0.6 Relative Frequency Pore Size (nm) 98%-AN Sample Dealloyed 3h 6 9 12 15 18 21 24 27 0.0 0.1 0.2 0.3 0.4 Relative Frequency Pore Size (nm) 0%-CR Sample Dealloyed 7h 6 9 12 15 18 21 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 60%-CR Sample Dealloyed 7h 3 6 9 12 15 18 21 0.0 0.2 0.4 0.6 Relative Frequency Pore Size/nm 90%-CR Sample Dealloyed 7h 3 6 9 12 15 18 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 98%-CR Sample Dealloyed 3h Figure 4. Pore size distribution with corresponding Gauss fit for different samples as indicated. (a–d) CR samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively; (e–h) AN samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively. 3. Results and Discussion 6 9 12 15 18 21 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 60%-CR Sample Dealloyed 7h ( ) 3 6 9 12 15 18 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 98%-CR Sample Dealloyed 3h 3 6 9 12 15 18 21 24 0.0 0.1 0.2 0.3 0.4 0.5 Relative Frequency Pore Size (nm) 0%-AN Sample Dealloyed 7h 3 6 9 12 15 0.0 0.2 0.4 0.6 0.8 Relative Frequency Pore Size (nm) 60%-AN Sample Dealloyed 7h 6 9 12 15 18 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 90%-AN Sample Dealloyed 7h 6 9 12 15 18 21 24 0.0 0.2 0.4 0.6 Relative Frequency Pore Size (nm) 98%-AN Sample Dealloyed 3h Pore Size/nm Pore Size (nm) 3 6 9 12 15 0.0 0.2 0.4 0.6 0.8 Relative Frequency Pore Size (nm) 60%-AN Sample Dealloyed 7h ( ) 6 9 12 15 18 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative Frequency Pore Size (nm) 90%-AN Sample Dealloyed 7h 6 9 12 15 18 21 24 0.0 0.2 0.4 0.6 Relative Frequency Pore Size (nm) 98%-AN Sample Dealloyed 3h Figure 4. Pore size distribution with corresponding Gauss fit for different samples as indicated. (a–d) CR samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively; (e–h) AN samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively. Figure 4. Pore size distribution with corresponding Gauss fit for different samples as indicated. (a–d) CR samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively; (e–h) AN samples with different thickness reductions of 0%, 60%, 90% and 98%, respectively. 6 of 7 6 of 7 Nanomaterials 2018, 8, 540 Na o ate ial 2018 8 FO Figure 5. FWHM of the Gauss function fitting curves of the pore size distribution of CR and AN samples as functions of thickness reduction. For the sample with a thickness reduction of 98% a dealloying time of 3 h was used and for the others a dealloying time of 7 h was used. 0 20 40 60 80 100 2 4 6 8 10 12 FWHM (nm) Thickness reduction (%) CR Sample AN Sample Figure 5. FWHM of the Gauss function fitting curves of the pore size distribution of CR and AN samples as functions of thickness reduction. 4. Conclusions 4. Conclusions We investigated the effects of pre-dealloying treatment by cold rolling and annealing the original alloy on the microstructure of NPG produced by dealloying. It was found that plastic deformation and annealing prior to dealloying can lead to reduced ligament and pore sizes of NPG. Moreover, post-deformation annealing can facilitate the formation of continuous, homogeneous, and refined NPG structures. The minima of pore and ligament sizes (both ~8 nm) with the most uniform distribution were obtained in the AN sample with a thickness reduction of 60% and a dealloying time of 7 h. Our study indicated the important role of pre-dealloying treatments of the original alloy (namely plastic deformation and annealing) in the formation and optimization of the NPG microstructure produced by dealloying. We investigated the effects of pre-dealloying treatment by cold rolling and annealing the original alloy on the microstructure of NPG produced by dealloying. It was found that plastic deformation and annealing prior to dealloying can lead to reduced ligament and pore sizes of NPG. Moreover, post-deformation annealing can facilitate the formation of continuous, homogeneous, and reﬁned NPG structures. The minima of pore and ligament sizes (both ~8 nm) with the most uniform distribution were obtained in the AN sample with a thickness reduction of 60% and a dealloying time of 7 h. Our study indicated the important role of pre-dealloying treatments of the original alloy (namely plastic deformation and annealing) in the formation and optimization of the NPG microstructure produced by dealloying. Author Contributions: Q.M. and R.X. conceived and designed the experiments; H.H., Y.M. and F.C. performed the experiments; H.H., Q.M., J.L and Y.L. analyzed the data; H.H. and Q.M. wrote the paper. Author Contributions: Q.M. and R.X. conceived and designed the experiments; H.H., Y.M. and F.C. performed the experiments; H.H., Q.M., J.L and Y.L. analyzed the data; H.H. and Q.M. wrote the paper. Funding: This work was financially supported by the National Natural Science Foundation of China (grants 51371128, 51401148) and the Fundamental Research Funds for the Central Universities of China (grant 2042017kf0190). J.Y.L. is supported by Research and Innovation Initiatives of Wuhan Polytechnic University (2016y07) Science and Technology Research Project of Hubei Provincial Education Department (B2017070) Funding: This work was ﬁnancially supported by the National Natural Science Foundation of China (grants 51371128, 51401148) and the Fundamental Research Funds for the Central Universities of China (grant 2042017kf0190). J.Y.L. 4. Conclusions 4. Conclusions is supported by Research and Innovation Initiatives of Wuhan Polytechnic University (2016y07), Science and Technology Research Project of Hubei Provincial Education Department (B2017070). (2016y07), Science and Technology Research Project of Hubei Pro C fli f I Th h d l fli f i Conﬂicts of Interest: The authors declare no conﬂict of interest. 2016y07), Science and Technology Research Project of Hubei Provincial Education Department (B2017070) C fli t f I t t Th th d l fli t f i t t Conﬂicts of Interest: The authors declare no conﬂict of interest. 3. Results and Discussion For the sample with a thickness reduction of 98% a dealloying time of 3 h was used and for the others a dealloying time of 7 h was used. 0 20 40 60 80 100 2 4 6 8 10 12 FWHM (nm) Thickness reduction (%) CR Sample AN Sample Figure 5. FWHM of the Gauss function fitting curves of the pore size distribution of CR and AN samples as functions of thickness reduction. 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https://openalex.org/W2955618520	https://bmchealthservres.biomedcentral.com/track/pdf/10.1186/s12913-019-4281-0	English	null	Cost-utility analysis of palonosetron in the antiemetic regimen for cisplatin-containing highly emetogenic chemotherapy in Japan	BMC health services research	2,019	cc-by	8,182	© 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. Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 https://doi.org/10.1186/s12913-019-4281-0 Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 https://doi.org/10.1186/s12913-019-4281-0 Open Access Keywords: Pharmacoeconomics, Cost-effective, Chemotherapy, Palonosetron, Emesis Society of Clinical Oncology (JSCO) indicate that antiemetic therapy should be used according to the level of emetic risk [1–4]. CINV occurs not only on the day of chemotherapy, but also on subsequent days, and it is a major factor affecting patients’ quality of life (QoL) and continuity of chemotherapy [5]. Cost-utility analysis of palonosetron in the antiemetic regimen for cisplatin-containing highly emetogenic chemotherapy in Japan Munenobu Kashiwa1,2* and Ryo Matsushita3 Background Chemotherapy-induced nausea and vomiting (CINV) is a typical adverse reaction to anti-cancer agents. The frequency of CINV varies widely depending on the drugs used, and emetogenicity is classified as high (> 90%), mod- erate (30–90%), low (10–30%), and minimal (< 10%). The guidelines of scientific societies such as the American Society of Clinical Oncology, the National Comprehensive Cancer Network, the Multinational Association of Supportive Care in Cancer (MASCC), and the Japan A 5-hydroxytryptamine3 receptor antagonist (5-HT3RA) is now the standard therapy for preventing CINV, due to its ability to block emesis caused by the stimulation of 5-HT3 receptors on vagal afferents. The first-generation 5- HT3RAs, ondansetron and granisetron, dramatically chan- ged the management of CINV in patients receiving highly or moderately emetogenic chemotherapy. Regimens that include cisplatin, which is widely used in the treatment of * Correspondence: munenobuk@stu.kanazawa-u.ac.jp * Correspondence: munenobuk@stu.kanazawa-u.ac.jp 1Division of Pharmacy, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan 2Department of Pharmacy, First Towakai Hospital, Takatsuki, Japan Full list of author information is available at the end of the article * Correspondence: munenobuk@stu.kanazawa-u.ac.jp 1Division of Pharmacy, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan 2Department of Pharmacy, First Towakai Hospital, Takatsuki, Japan Full list of author information is available at the end of the article Abstract Background: An antiemetic triplet regimen of 5-hydrotryptamine-3 receptor antagonist, dexamethasone, and aprepitant is the standard prophylaxis with highly emetogenic chemotherapy (HEC). A randomized phase III trial comparing palonosetron (PALO) versus granisetron (GRA) in the triplet antiemetic regimen (The TRIPLE study) showed the superiority of PALO over GRA for delayed-phase vomiting in patients receiving cisplatin-based HEC. However, economic efficiency evaluations including quality of life have not been done. The present study was a cost-utility analysis of PALO within the Japanese medical insurance system. Methods: The data source was the results of the TRIPLE study. A decision tree was constructed to assess the incremental cost-effectiveness ratio (ICER) using quality-adjusted life years (QALYs) and the medical service fees and the drug price for 2018 from the perspective of the payer. A one-way sensitivity analysis and a probabilistic sensitivity analysis (PSA) were performed to assess the robustness of the model. A threshold analysis was performed to determine the cost-effective price of PALO. Results: In the base case, the estimated incremental effect of PALO addition was 0.000645 QALYs, the estimated incremental cost was 10,455 JPY (93.21 USD), and the ICER was 16,204,591 JPY QALY (144,465 USD/QALY). In the PSA, the probability of superior cost-effectiveness was 3.64%. In the threshold analysis, the acceptable price of PALO was estimated to be 7,743 JPY (69.03 USD). Conclusions: If willingness-to-pay is taken as 5,000,000 JPY/QALY (44,575 USD/QALY), the antiemetic regimen using PALO for cisplatin-containing HEC was not cost-effective at this time. The cost of drugs, with the arrival of inexpensive generic drugs, will make a major contribution to its cost-effectiveness. Keywords: Pharmacoeconomics, Cost-effective, Chemotherapy, Palonosetron, Emesis Keywords: Pharmacoeconomics, Cost-effective, Chemotherapy, Palonosetron, Emesis Correspondence: munenobuk@stu.kanazawa-u.ac.jp 1Division of Pharmacy, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan 2Department of Pharmacy, First Towakai Hospital, Takatsuki, Japan Full list of author information is available at the end of the article The PROTECT study of a doublet regimen of 5-HT3RA and DEX found that, compared to granisetron (GRA), the efficacy of PALO was non-inferior for acute-phase CINV and superior for delayed-phase CINV [6]. However, since this study did not include aprepi- tant (APR), the results could not subsequently be directly extrapolated to everyday clinical practice. The usefulness of PALO in combination with APR and DEX was investigated in a randomized phase III trial (The TRIPLE study) [7]. This study compared PALO and GRA in an antiemetic triplet regimen of 5-HT3RA, APR, and DEX. In the TRIPLE study, the primary endpoint was complete response (CR; no vomiting/nausea and no rescue medica- tion) within 120 h after cisplatin-containing HEC. Eligible patients were randomly assigned to double-blind anti- emetic treatment with either GRA (1 mg) or PALO (0.75 mg). Although the primary endpoint was not achieved and the superiority of PALO was not shown in this clinical trial (P = 0.0539), PALO showed superiority to GRA in the CR rate in the delayed phase. Based on the results of this study, the JSCO Clinical Practice Guidelines for Antiem- esis in Oncology recommend the use of PALO with HEC containing 50 mg/m2 or more cisplatin. Guidelines in other countries also recommend PALO with moderately emetogenic chemotherapy [1–3]. However, a meta- analysis by Kolesar et al. reported insufficient grounds to recommend PALO over other 5-HT3RAs [8], and recent guidelines have positioned PALO at the same level as other 5-HT3RAs [1–3]. The positioning of PALO in clin- ical practice is thus different from other countries in the Japanese guidelines. Japan has a national health insurance (NHI) system and depending on the age, patients have to pay 10–30% of medical expenses. Lower out-of-pocket costs are particularly beneficial for the elderly, but the increase in medical expenditures in the national budget as a result of the aging population is a serious problem. The increased economic burden is a concern for PALO, which is a more expensive antiemetic than other 5HT3RAs. As to concerns about the economics of PALO, results of analyses in the USA and China reported that PALO Methods Modeling A decision tree that showed the acute phase and the delayed phase was constructed according to prior reports [12–14] to estimate and compare the health outcomes and cost of prophylactic antiemetic therapy using PALO (Fig. 1). The clinical outcome was defined as CR with no emesis and no rescue antiemetic therapy, and incomplete response (IR) was defined as some em- esis or some use of rescue antiemetic therapy. CR was subdivided into two mutually exclusive health outcomes: complete protection (CP), which was defined as no em- esis, no rescue antiemetic therapy, and no significant nausea; and complete response at best (CRB), which included those who achieved CR but not CP. For the purposes of this study, it was assumed that chemother- apy was administered on an outpatient basis, and the analysis period was defined as 5 days from administra- tion of chemotherapy. The positioning of PALO in clin- ical practice is thus different from other countries in the Japanese guidelines. Japan has a national health insurance (NHI) system and depending on the age, patients have to pay 10–30% of medical expenses. Lower out-of-pocket costs are particularly beneficial for the elderly, but the increase in medical expenditures in the national budget as a result of the aging population is a serious problem. The has poor cost-effectiveness [9, 10]. In Japan, Shimizu et al. reported that treatment with PALO is expensive based on retrospectively investigating the costs of patients in the TRIPLE study [11]. Although the cost per vomiting control was measured, there has been no economic evaluation including QoL. In order to com- pare with medical technologies, analyses including QoL are required. Cost-utility analysis is a measure used for this in the economic evaluation of medical technologies. If such an evaluation is completed within a clinical trial, it is desirable from the perspective of internal validity, but there may be a problem with the generalizability of the result. To the best of our knowledge, no study has directly compared the cost-effectiveness of the preven- tion of CINV associated with HEC with triple therapy combining DEX and APR with either a first-generation 5-HT3RA or palonosetron. The present study was a cost-utility analysis based on the Japanese health care system to define the cost-effectiveness of PALO com- pared to GRA as the standard prophylaxis in patients who received cisplatin-containing HEC. lung, stomach, and head and neck cancers, meet the criteria for highly emetogenic chemotherapy (HEC), and the JSCO Clinical Practice Guidelines for Antiemesis In Oncology recommend an antiemetic triplet regimen of 5-HT3RA, neurokinin 1 receptor antagonist, and dexamethasone (DEX) during HEC. Of these three agents, the 5-HT3RA palonosetron (PALO) was approved by the US Food and Drug Administration in addition to existing agents in 2003, and it was also approved for use in Japan in 2010. PALO has a plasma elimination half-life of approximately 40 h, which is longer than that of existing 5-HT3RAs, and it has approximately 100 times the affinity for 5-HT3R receptors. It is thus regarded as a second-generation 5-HT3RA that inhibits not only acute-phase, but also delayed-phase nausea and vomiting. Page 2 of 10 Page 2 of 10 Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 lung, stomach, and head and neck cancers, meet the criteria for highly emetogenic chemotherapy (HEC), and the JSCO Clinical Practice Guidelines for Antiemesis In Oncology recommend an antiemetic triplet regimen of 5-HT3RA, neurokinin 1 receptor antagonist, and dexamethasone (DEX) during HEC. Of these three agents, the 5-HT3RA palonosetron (PALO) was approved by the US Food and Drug Administration in addition to existing agents in 2003, and it was also approved for use in Japan in 2010. PALO has a plasma elimination half-life of approximately 40 h, which is longer than that of existing 5-HT3RAs, and it has approximately 100 times the affinity for 5-HT3R receptors. It is thus regarded as a second-generation 5-HT3RA that inhibits not only acute-phase, but also delayed-phase nausea and vomiting. The PROTECT study of a doublet regimen of 5-HT3RA and DEX found that, compared to granisetron (GRA), the efficacy of PALO was non-inferior for acute-phase CINV and superior for delayed-phase CINV [6]. However, since this study did not include aprepi- tant (APR), the results could not subsequently be directly extrapolated to everyday clinical practice. The usefulness of PALO in combination with APR and DEX was investigated in a randomized phase III trial (The TRIPLE study) [7]. This study compared PALO and GRA in an antiemetic triplet regimen of 5-HT3RA, APR, and DEX. In the TRIPLE study, the primary endpoint was complete response (CR; no vomiting/nausea and no rescue medica- tion) within 120 h after cisplatin-containing HEC. Eligible patients were randomly assigned to double-blind anti- emetic treatment with either GRA (1 mg) or PALO (0.75 mg). Although the primary endpoint was not achieved and the superiority of PALO was not shown in this clinical trial (P = 0.0539), PALO showed superiority to GRA in the CR rate in the delayed phase. Based on the results of this study, the JSCO Clinical Practice Guidelines for Antiem- esis in Oncology recommend the use of PALO with HEC containing 50 mg/m2 or more cisplatin. Guidelines in other countries also recommend PALO with moderately emetogenic chemotherapy [1–3]. However, a meta- analysis by Kolesar et al. reported insufficient grounds to recommend PALO over other 5-HT3RAs [8], and recent guidelines have positioned PALO at the same level as other 5-HT3RAs [1–3]. Clinical data h d The data source was the results of a phase III study with Japanese participants (The TRIPLE study [7]. In this study, patients aged 20 years over were included if they had solid cancer and were receiving HEC containing 50 mg/m2 or more of cisplatin, Eastern Cooperative Oncol- ogy Group performance status of 0–2, and no organ dysfunction during the 8 days prior to enrolment. The male:female ratio of subjects was 3:1, and 70% or more had exceeded 70 mg/m2 of cisplatin. The tumor site was respiratory in approximately 50% of cases, followed by esophageal, gastric, head and neck, and others. A total As to concerns about the economics of PALO, results of analyses in the USA and China reported that PALO Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 3 of 10 Fig. 1 Decision tree for cost-effectiveness analysis. PALO: palonosetron; GRA: granisetron g. 1 Decision tree for cost-effectiveness analysis. PALO: palonosetron; GRA: granisetron study were 59.1% (244/413 patients) for the GRA group and 67.2% (272/414 patients) for the PALO group. According to the rate of CR or complete control of acute and delayed CINV, transition probabilities were calculated by multiplying the probability of each state (CP, CRB, and IR) using the data from the TRIPLE study results (Table 1). of 842 patients were assigned to either the PALO regi- men or the GRA regimen, of which the results from 414 patients in the PALO regimen and 413 in the GRA regi- men were used for the analysis. The degree of nausea and vomiting and the use of rescue antiemetic therapy over the 120-h period following administration of the anti-cancer agent were examined from the patients’ symptom diaries. The primary outcome was CR over the whole period (0–120 h). For the secondary end point, CR was evaluated in the acute phase (0–24 h) and the delayed phase (> 24–120 h). The CR rates in the TRIPLE Cost Cost analysis was performed from the healthcare payer perspective, and direct medical costs associated with CINV prevention and the additional medical fees in- curred by CINV were estimated (Table 2). In Japan, pa- tients are covered by NHI system, and the copayment of a patient is 10–30% of the total medical cost according to his/her age. The Ministry of Health, Labour, and Wel- fare determines prescribing drug prices and expenses for medical treatment and care and registers them in the NHI standard list to which national insurance is applic- able. All costs in this study were assigned from the NHI Drug Price Standard listed in 2018 [15] and the IR in the acute or the delayed phase was taken to be the administration of additional antiemetic medication and fluid replacement as rescue medication. The drug cost of additional rescue treatment for CINV for each health state in the TRIPLE study was reported by Shimizu et al. [11]. In the trial, 5-HT3RAs were not administered additionally. In clinical practice, 5-HT3RAs are widely used for rescue treatment [17]. In this analysis, rescue treatment was set according to clinical practice and other previous studies instead of the cost result of the trial. Treatment strategy Prophylactic antiemetic triplet regimens of a 5-HT3RA, DEX, and APR (125 mg on day 1 and 80 mg/day on days Table 1 Health state probabilities used in the model, based on the TRIPLE study Health state outcome by phase PALO regimen N = 414 (%) GRA regimen N = 413 (%) Acute Phase (day 1) Delayed Phase (days 2–5) Complete protection Complete protection 58.7 50.4 Complete response at best 1.8 2.9 Incomplete response 29.6 36.9 Complete response at best Complete protection 1.1 1.0 Complete response at best 0.0 0.1 Incomplete response 0.6 0.7 Incomplete response Complete protection 5.3 4.6 Complete response at best 0.2 0.3 Incomplete response 2.7 3.4 Table 1 Health state probabilities used in the model, based on the TRIPLE study l h b h Table 1 Health state probabilities used in the model, based on the TRIPLE study Page 4 of 10 Page 4 of 10 Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 2–3) were analyzed. The regimens comprised 12 mg DEX on Day 1 and 8 mg/day on Days 2–4, administered intravenously; PALO or GRA administered intravenously on Day 1 together with DEX for less than 15 min, at least 30 min prior to cisplatin administration; and the dose of APR administered at least 60 min prior to cis- platin administration on Day 1, and the doses of APR administered before breakfast on Days 2 and 3. 2–3) were analyzed. The regimens comprised 12 mg DEX on Day 1 and 8 mg/day on Days 2–4, administered intravenously; PALO or GRA administered intravenously on Day 1 together with DEX for less than 15 min, at least 30 min prior to cisplatin administration; and the dose of APR administered at least 60 min prior to cis- platin administration on Day 1, and the doses of APR administered before breakfast on Days 2 and 3. Reimbursement Schedule of Social Insurance [16]. For rescue treatment, the costs associated with follow-up visits, the cost of test fees (biochemical tests [over 10 items], C-reactive protein, venous blood sampling), the cost of medications (prescription, preparation, and base dispensing fee if prepared at a hospital), and the cost of the intravenous drip infusion were included in the calcu- lation. The cost associated with the administration of chemotherapy other than the antiemetic therapy would be the same in both groups, and this cost was not in- cluded in the calculation. Utility y The utility value of the health state of CINV in Japanese has not been reported in clinical trials and there is no publicly available information. Sun et al. report a QoL evaluation using a visual analog scale according to the presence or absence of CINV by chemotherapy [20]. In- cluding this report, according to previous CINV reports [13, 14, 21, 22], utility values were applied to the three health states. The following utility values were defined: 0.9 for CP with no significant nausea or vomiting; 0.2 for IR with nausea and vomiting; and 0.7 for CRB with- out vomiting or use of rescue medication. The health state in the acute period was set as 1 day (24 h), and the health condition of the delay period was set as 4 days (96 h). The sum of the 5-day Quality-adjusted life years (QALYs) was calculated using the following formula: QALYs = ([utility value (acute phase) × 1 d] + [utility value (delayed phase) × 4 d])/365 d. Since information available for estimating the utility value of CINV is in- sufficient, a sensitivity analysis was performed to exam- ine the influence of these changes on the results. Cost In the delayed phase, the patient was re-assessed at a medical institution, with examination, prescription, and infusion performed by a doctor and pre- scriptions made up in the hospital by a pharmacist. The three drugs used in rescue therapy, which were prescribed with reference to the cost-effectiveness studies of DEX and PALO in prophylactic antiemetic therapy by Oshima et al. [18] and Yamanishi et al. [19], were 1 tablet of meto- clopramide 3 times a day for 5 days, one 4-mg tablet of dexamethasone twice a day for 5 days, and one tablet a day of the 5-HT3RA ramosetron for 5 days. However, the 5-HT3RA was not used after PALO administration. In addition, a transfusion of 500 mL of BFLUID® once a day for 2 days was given as intravenous feeding. Since there were no clinically relevant differences in the overall incidence of adverse events [7], in the present analysis, the costs relating to adverse reactions to the antiemetic treat- ment were not included in the totals. At the time of analysis, costs were calculated using the 2018 drug prices and medical fees and converted into dollars using the most recent annual exchange rate published by the OECD (1 USD = 112.17 JPY) [23]. dopamine receptor antagonists, steroids, or 5-HT3RAs are useful as additional agents for breakthrough nausea or vomiting. Further, for additional medication, the guide- lines recommend changing the 5-HT3RA to a different one from the 5-HT3RA used for prophylactic administra- tion. Accordingly, ramosetron, a tablet that is highly useful in clinical settings because it breaks down in the mouth and has a mid-range price, was selected as the additional 5-HT3RA. In the acute phase, additional medication was administered orally. In the delayed phase, the patient was re-assessed at a medical institution, with examination, prescription, and infusion performed by a doctor and pre- scriptions made up in the hospital by a pharmacist. The three drugs used in rescue therapy, which were prescribed with reference to the cost-effectiveness studies of DEX and PALO in prophylactic antiemetic therapy by Oshima et al. [18] and Yamanishi et al. [19], were 1 tablet of meto- clopramide 3 times a day for 5 days, one 4-mg tablet of dexamethasone twice a day for 5 days, and one tablet a day of the 5-HT3RA ramosetron for 5 days. However, the 5-HT3RA was not used after PALO administration. Cost-effectiveness analysis Cost-effectiveness analysis This study was carried out in compliance with the Guideline for Economic Evaluation of Healthcare Tech- nologies in Japan [24] and the Consolidated Health Eco- nomic Evaluation Reporting Standards Statement [25]. Cost-effectiveness was calculated from the costs incurred in antiemetic therapy and QALYs for 5 days following anticancer agent administration as a health outcome. The incremental cost-effectiveness ratio (ICER) of the base case was calculated. In this study, discounts were not applied during the 5-day short-term observation period. The threshold of willingness-to-pay (WTP) used 5, 000,000 JPY (44,575 USD/QALY) defined by Shiroiwa et al. [26]. TreeAge Pro 2016 (TreeAge Software, Inc., Williamstown, MA, USA) was used for the analysis. Sensitivity analysis y y For this study, a clinical decision analysis simulation model was constructed, and a hypothetical situation was defined with variables such as transition probabilities, utilities, and costs. One-way and probabilistic sensitivity analyses were carried out to assess the uncertainty and robustness of this model by evaluating the effects of differing model parameters. In the one-way sensitivity analysis, the efficacy ratio of antiemetic therapy was examined in the range of the 95% confidence interval of the effectiveness in the TRIPLE study. Changes were examined with the utility value and cost in the range ± 30%, although the cost of GRA was examined up to the cheapest price of the generic drug, and the cost of PALO is unlikely to increase with future drug price revision, and only the lower 50% limit was considered because only the possibility of a price decrease was considered. A probabilistic sensitivity analysis (PSA) was carried out to examine changes in data that included uncertainty of the base case and to investigate the robustness of the results, and the effects on the ICER were examined. In PSA, a 10,000-sample Monte Carlo simulation was performed with transition probability and utility value set as having beta distributions and cost as having a gamma distribu- tion. The probability of the ICER being less than the WTP value was determined from the cost-effectiveness acceptability curve (CEAC). Additionally, a threshold ana- lysis was performed to determine the cost-effectiveness price of PALO for WTP of 5,000,000 JPY. Cost The JSCO Clinical Practice Guidelines for Antiemesis in Oncology state that Table 2 Study parameter and ranges in one-way sensitivity analysis Table 2 Study parameter and ranges in one-way sensitivity analysis Pramater Values Range Ref CINV related health state probabilities Complete response in acute phase in PALO regimen 0.918 95% CI [7] Complete response in delayed phase in PALO regimen 0.672 95% CI [7] Complete response in acute phase in GRA regimen 0.918 95% CI [7] Complete response in delayed phase in GRA regimen 0.591 95% CI [7] Costs JPY (USD) JPY (USD) Palonosetron 0.75 mg (IV) 14,972 (133.5) 7,486-14,972 (66.7-133.5) [15] Granisetoron 1 mg (IV) 1,273 (11.3) 590 -1,273 (5.26-11.3) [15] Aprepitantcapusule set (125 mg /80 mg /80 mg) (PO) 11,638.2 (103.8) [15] Dexametazone 2 mg (IV) 99 (0.883) [15] Dexametazone 4 mg (PO) 34 (0.303) [15] Blood testb 1,580 (14.1) [16] Internal medicine for rescue medication for PALO regimen 432 (3.85) ± 30% [15, 18, 19] Internal medicine for rescue medication for GRA regimen 5,956.5 (53.1) ± 30% [15, 18, 19] Infusion therapy for rescue medication 1,374 (12.3) ± 30% [15, 16, 18, 19] Total cost per acute CINV in PALO regimen 432 (3.85) Total cost per delayed CINV in PALO regimen 2,396 (21.36) Total cost per acute CINV in GRA regimen 5,956.5 (53.10) Total cost per delayed CINV in GRA regimen 10,221 (91.12) Utility values Complete protection 0.9 ± 30% [13, 14, 20–22] Complete response at best 0.7 ± 30% [13, 14, 20–22] Incomplete response 0.2 ± 30% [13, 14, 20–22] CINV Chemotherapy induced nausea and vomitingn, PALO Palonosetron, GRA Granisetron, CI Confidence interval, JPY Japanese yen, IV Intravenous, PO Oral. Exchange rate, 1 USD = 112.17 JPY Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 5 of 10 Page 5 of 10 dopamine receptor antagonists, steroids, or 5-HT3RAs are useful as additional agents for breakthrough nausea or vomiting. Further, for additional medication, the guide- lines recommend changing the 5-HT3RA to a different one from the 5-HT3RA used for prophylactic administra- tion. Accordingly, ramosetron, a tablet that is highly useful in clinical settings because it breaks down in the mouth and has a mid-range price, was selected as the additional 5-HT3RA. In the acute phase, additional medication was administered orally. Cost In addition, a transfusion of 500 mL of BFLUID® once a day for 2 days was given as intravenous feeding. Since there were no clinically relevant differences in the overall incidence of adverse events [7], in the present analysis, the costs relating to adverse reactions to the antiemetic treat- ment were not included in the totals. At the time of analysis, costs were calculated using the 2018 drug prices and medical fees and converted into dollars using the most recent annual exchange rate published by the OECD (1 USD = 112.17 JPY) [23]. Base case results Expected costs for drug expenses of antiemesis treat- ment and utility values were estimated. The costs of antiemetic therapy per course were calculated to be 27, 406 JPY (244.33 USD) with the PALO regimen and 13, 707 JPY (122.20 USD) with the GRA regimen. The med- ical expenses were 1,580 JPY (14.09 USD) for blood Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 6 of 10 Page 6 of 10 Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 combination with APR and DEX compared to that of GRA with APR and DEX for cisplatin-containing HEC. The results showed the incremental effect per person to be 0.0006452 QALYs, and the incremental cost was 10, 455 JPY (93.21 USD). The base case ICER was approxi- mately 16,200,000 JPY QALY (144,000 USD/QALY). No consensus exists regarding the threshold for acceptable cost per QALY ratios in Japan’s health policy. Therefore, the common WTP threshold of 5,000,000 JPY/QALY from a previous study was adopted. Based on this standard, it was concluded that prophylaxis with PALO was not cost- effective for HEC in Japan. This conclusion was consid- ered robust based on the results of the sensitivity analyses. In the one-way sensitivity analyses, the clinical trial results were examined on the basis of the 95% confidence inter- val. The results indicate that the CR rate in the delayed phase had the greatest effect on the ICER. The onset of delayed-phase CINV was thus attributable to the patients’ decreased QoL and increases in costs associated with rescue therapy, such as medical consultation fees, test fees, and drug costs. In addition, a 50% reduction in the drug price of PALO reduced the ICER to approximately 46,000, 000 JPY/QALY (41,000 USD/QALY), bringing it to below the WTP value. Using the results of the threshold analysis, the acceptable price of PALO was estimated to be 7743 JPY (69.03 USD). This price is 51.7% of the current price of 14,972 JPY. Therefore, a price reduction is necessary for PALO to be considered cost-effective by commonly applied thresholds. The cost of drugs, with the arrival of inexpensive generic drugs, will make a major contribution to the cost-effectiveness of the therapy. In the PSA, with the WTP at 5,000,000 JPY, the probability of the PALO regimen being judged to be cost-effective was only 3.64%. Base case results Even taking the diversity of diagnostic patterns and the individual differences of patients into account, antiemetic therapy using PALO was shown to be less cost-effective. The JSCO Clinical Practice Guidelines for Antiemesis in Oncology recommend the use of PALO with HEC containing 50 mg/m2 or more cisplatin from the point of view of efficacy. However, similar to the report of Shimizu et al., the results of the present study indicate that there is still concern about the cost-effectiveness of antiemetic therapy using PALO at this time. The results of the present study can be compared to th lt f i t di i th t i A it h testing, 590 JPY (5.25 USD) for pharmacy costs, and 1, 374 JPY (12.25 USD) for supplementary nutrition infu- sion a single time with both regimens. The costs of oral agents for rescue medication were calculated to be 432 JPY (3.85 USD) with the PALO regimen and 5,957 JPY (53.1 USD) with the GRA regimen. The base case results over 5 days after cycle 1 of chemotherapy are shown in Table 3. For the PALO regimen, the incremental effect per person was 0.0006452 QALY, the incremental cost was 10,455 USD (93.21 USD), and the ICER was 16,204, 591 JPY QALY (144,465 USD/QALY). The economic re- sults with the two strategies for each cost category are shown in Table 4. Although the cost for rescue medica- tion in the PALO regimen was low, the total cost was higher than in the GRA regimen. testing, 590 JPY (5.25 USD) for pharmacy costs, and 1, 374 JPY (12.25 USD) for supplementary nutrition infu- sion a single time with both regimens. The costs of oral agents for rescue medication were calculated to be 432 JPY (3.85 USD) with the PALO regimen and 5,957 JPY (53.1 USD) with the GRA regimen. The base case results over 5 days after cycle 1 of chemotherapy are shown in Table 3. For the PALO regimen, the incremental effect per person was 0.0006452 QALY, the incremental cost was 10,455 USD (93.21 USD), and the ICER was 16,204, 591 JPY QALY (144,465 USD/QALY). The economic re- sults with the two strategies for each cost category are shown in Table 4. Although the cost for rescue medica- tion in the PALO regimen was low, the total cost was higher than in the GRA regimen. Sensitivity analysis The results of the one-way sensitivity analysis are shown in a tornado diagram (Fig. 2). This diagram shows the effects of uncertainly on the ICER for each parameter. The largest effect of PALO on the increase in the ICER was on the CR rate in the delayed phase in the PALO regimen and the CR rate in the delayed phase in the GRA regimen, and the greatest ICER was approximately 27,120,000 JPY/QALY (242,000 USD/QALY) and 26,460, 000 JPY/QALY (236,000 USD/QALY). With a reduction of 50% in the drug price for PALO, the ICER decreased to approximately 4,600,000 JPY/QALY (41,000 USD/ QALY). Maximums in the ICER due to other parameters were less than 20,000,000 JPY/QALY (178,300 USD). An incremental cost-effectiveness plane and a CEAC were drawn from the results of the PSA. Many points in this analysis existed in the northeastern quadrant (i.e., more effective and more expensive) and existed above the diagonal line showing ICER of 5,000,000 JPY per QALY (Fig. 3). At a WTP threshold of 5,000,000 JPY, the PALO regimen had an acceptability of 3.64% (Fig. 4). The results of the threshold analysis are shown in Fig. 5. The estimated threshold value of PALO was 7743 JPY (69.03 USD) when PALO in a triplet antiemetic regimen was compared with GRA in the base case. Discussion The results of the present study can be compared to the results of prior studies in other countries. Avritscher et al. [9] compared the cost-effectiveness in the US med- ical care system of the triplet regimen of PALO, APR, and DEX with a control of combined ondansetron and The present study used the results of the TRIPLE study to perform a cost-utility analysis within the Japanese health care system from the health care payer perspective. This is the first report in which the ICER was calculated with QALYs of the antiemetic triplet regimen of PALO in Table 3 Base-case results Strategy Cost JPY (USD) Incrmental Cost JPY (USD) QALY Incremental QALY ICER JPY/QALY (USD/QALY) PALO regimen 30,348 (270.55) 10,455 (93.21) 0.009598 0.000645208 16,204,591 (144,465) GRA regimen 19,893 (177.35) 0.008952 JPY Japanese yen, QALY Quality-adjusted life year, ICER Incremental cost-effectiveness ratio. Exchange rate, 1 USD = 112.17 JPY Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 7 of 10 Table 4 Economic results of the two strategies per each cost category Strategy Cost category JPY (USD) Total cost Rescue medication Medical fee Prophyraxis PALO Regimen 30,348 (270.55) 617 (5.50) 949 (8.46) 27,406 (244.33) GRA Regimen 19,893 (177.35) 3,290 (29.33) 1,183 (10.55) 13,707 (122.20) JPY Japanese yen. Exchange rate, 1 USD = 112.17 JPY effective and less expensive [28]. The present study com- pared a triplet regimen of APR, DEX, and PALO against a control of APR, DEX, and GRA in the Japanese medical care system. The data source was Japanese patients under- going cisplatin-containing HEC from the TRIPLE study. Thus, the present study examined different types of cancer and chemotherapy from Avritscher et al. [9] and differs from Du et al. [10] in the doublet regimen of APR, and it is the first to report the cost-utility that evaluated QoL of PALO in comparison to GRA in triplet regimens contain- ing APR for cisplatin-containing HEC. The fact that calcu- lations were made using the unit price from each country, the differences between countries in drug prices and insurance systems, and the differences in the analysis models are reflected in the results of these studies, but all of them indicate that the cost-effectiveness of antiemetic therapy using PALO is not favorable. Discussion The drug price for PALO in Japan is over five times that of GRA, and it may be assumed that the same results were obtained, since there is the same or greater price difference in the USA and China. With the dramatic rises in drug prices in the past several decades, the use of new treatments should be DEX in breast cancer patients who had undergone anthracycline and cyclophosphamide treatment using a Markov model. The results showed that the ICER for the triplet regimen including PALO was 115,490 USD (approx. 13,000,000 JPY). Du et al. [10] compared the cost-effectiveness in the Chinese medical care system of a doublet regimen of PALO and DEX against a control of ondansetron or GRA and DEX using a decision tree, and they reported the ICER of PALO to be 167,914 USD (approx. 19,000,000 JPY). Cawston et al. reported the cost-effectiveness of a combination drug (NEPA: netupi- tant plus palonosetron), comparing the combination with APR and PALO using a Markov model in the UK medical care system. They reported that NEPA was cost- effective for preventing CINV associated with highly or moderately emetogenic chemotherapy in the UK [27]. Restelli et al. reported the cost-effectiveness of NEPA, comparing the combination with NK1-RA (APR or fosaprepitant) and 5-HT3RA (PALO or ondansetron) in patients receiving highly or moderately emetogenic chemotherapy using a Markov model in the Italian med- ical care system. The results showed that NEPA was more DEX in breast cancer patients who had undergone anthracycline and cyclophosphamide treatment using a Markov model. The results showed that the ICER for the triplet regimen including PALO was 115,490 USD (approx. 13,000,000 JPY). Du et al. [10] compared the cost-effectiveness in the Chinese medical care system of a doublet regimen of PALO and DEX against a control of ondansetron or GRA and DEX using a decision tree, and they reported the ICER of PALO to be 167,914 USD (approx. 19,000,000 JPY). Cawston et al. reported the cost-effectiveness of a combination drug (NEPA: netupi- tant plus palonosetron), comparing the combination with APR and PALO using a Markov model in the UK medical care system. They reported that NEPA was cost- effective for preventing CINV associated with highly or moderately emetogenic chemotherapy in the UK [27]. Restelli et al. Discussion reported the cost-effectiveness of NEPA, comparing the combination with NK1-RA (APR or fosaprepitant) and 5-HT3RA (PALO or ondansetron) in patients receiving highly or moderately emetogenic chemotherapy using a Markov model in the Italian med- ical care system. The results showed that NEPA was more Fig. 2 Tornado diagram for one-way sensitivity analyses. WTP: Willingness to pay; ICER: incremental cost-effectiveness ratio; JPY: Japanese yen; QALY: quality-adjusted life year; CR: complete response; PALO: palonosetron; GRA: granisetron; CP: complete protection; IR: incomplete response; PO: oral; IV: ntravenous Fig. 2 Tornado diagram for one-way sensitivity analyses. WTP: Willingness to pay; ICER: incremental cost-effectiveness ratio; JPY: Japanese yen; QALY: quality-adjusted life year; CR: complete response; PALO: palonosetron; GRA: granisetron; CP: complete protection; IR: incomplete response; PO: oral; IV: ntravenous Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 8 of 10 Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 8 of 10 Fig. 3 Incremental cost-effectiveness plane for probabilistic sensitivity analyses. WTP: Willingness-to-pay; JPY: Japanese yen does not reflect differences in the costs of antiemetic ther- apy, calculation on the basis of fee for service should be performed. Shimizu et al. investigated the rescue treatment cost and total hospitalization cost of the TRIPLE study, but the results, such as shorter hospital stay and lower readmis- sion rate due to CINV suppression, were not evaluated. These results are needed to evaluate differences in anti- emetic outcomes in DPC systems. Because of the lack of data and evidence, the analysis was not able to examine in- patients. However, there is no evidence for these in Japan and studies with these results are awaited. Once the results are available, the model can incorporate the cost of hospitalization for DPC and analyze inpatient chemother- apy as well as outpatient chemotherapy. The third limita- tion is that the present study used only data from first-time chemotherapy. tailored to those patients who are likely to benefit. For expensive PALO used for HEC as well, it is necessary to optimize prophylactic antiemetic therapy according to in- dividual patient risk. The limitations of the present study are as follows. First, the utility values in the analysis model conformed to prior studies, but they were based on data measured in other countries. Discussion It is highly likely that differences will be seen be- tween the health care systems of different countries and re- gions, but since it is very difficult to evaluate the difference in utility values between Japan and other countries, the util- ity value patterns were assumed to be the same in Japan and other countries. The results of the one-way sensitivity analysis indicated that changes in the utility values did not greatly impact the results. The second limitation is that the present study analyzed outpatient chemotherapy on the basis of the TRIPLE study, but many hospitals that carry out inpatient chemotherapy have adopted a diagnosis procedure combination (DPC) payment system. Since DPC This study showed the ICER of a triplet regimen of PALO, APR, and DEX for prevention of CINV in patients receiving cisplatin-containing HEC on the basis Fig. 4 Cost-effectiveness acceptability curve for probabilistic sensitivity analyses. PALO: palonosetron; GRA: granisetron; JPY: Japanese yen Kashiwa and Matsushita BMC Health Services Research (2019) 19:438 Page 9 of 10 Fig. 5 Results of the threshold analysis for the cost of palonosetron. PALO: palonosetron; GRA: granisetron; JPY: Japanese yen Fig. 5 Results of the threshold analysis for the cost of palonosetron. PALO: palonosetron; GRA: granisetron; JPY: Japanese of the Japanese medical insurance system. In the future, it will be necessary to search for other cost-effective anti- emetics such as olanzapine, and the result of this study is useful. Optimizing pricing of expensive medicines based on cost-effectiveness is an important financial issue in Japan. Based on the present findings, the Japanese govern- ment, as for other clinical interventions, may need to adjust the price of PALO in antiemetic therapy. Received: 19 February 2019 Accepted: 19 June 2019 5-HT3RA: 5-hydrotryptamine-3 receptor antagonist; APR: Aprepitant; CEAC: Cost-effectiveness acceptability curve; CINV: Chemotherapy-induced nausea and vomiting; CP: Complete protection; CR: Complete response; CRB: Complete response at best; DEX: Dexamethasone; DPC: Diagnosis procedure combination; GRA: Granisetron; HEC: Highly emetogenic chemotherapy; ICER: Incremental cost-effectiveness ratio; IR: Incomplete response; JPY: Japanese yen; JSCO: Japan Society of Clinical Oncology; NHI: National health insurance; PALO: Palonosetron; PSA: Probabilistic sensitivity analysis; QALYs: Quality-adjusted life years; QoL: Quality of life; USD: US dollar; WTP: Willingness -to-pay Consent for publication Not applicable. Consent for publication Not applicable. Acknowledgements Not applicable. 4. Takeuchi H, Saeki T, Aiba K, Tamura K, Aogi K, Eguchi K, et al. Japanese Society of Clinical Oncology clinical practice guidelines 2010 for antiemesis in oncology: executive summary. Int J Clin Oncol. Springer Japan. 2015;21:1–12. Author details 1 f 1Division of Pharmacy, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. 2Department of 1Division of Pharmacy, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. 2Department of y p p Pharmacy, First Towakai Hospital, Takatsuki, Japan. 3Division of y p p Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan. Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan. Competing interests The use of PALO instead of GRA for prevention of CINV in patients receiving HEC through the Japanese health insurance system is not cost-effective at this time. The cost of drugs, with the arrival of inexpensive generic drugs, will make a major contribution to its cost-effectiveness. The authors declare that they have no competing interests. Abbreviations h d Received: 19 February 2019 Accepted: 19 June 2019 Availability of data and materials y The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. References 1. Hesketh PJ, Kris MG, Basch E, Bohlke K, Barbour SY, Clark-Snow RA, et al. Antiemetics: American Society of Clinical Oncology clinical practice guideline update. JCO. 2017;35:3240–61. guideline update. JCO. 2017;35:3240–61. 2. Berger MJ, Ettinger DS, Aston J, Barbour S, Bergsbaken J, Bierman PJ, et al. NCCN guidelines insights: Antiemesis, version 2.2017. J Natl Compr Canc Netw. Harborside Press, LLC. 2017;15:883–93. 3. 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https://openalex.org/W2899542989	https://europepmc.org/articles/pmc6232309?pdf=render	English	null	Uterine Microbiota of Dairy Cows With Clinical and Subclinical Endometritis	Frontiers in microbiology	2,018	cc-by	9,425	Edited by: Edited by: Suhelen Egan, University of New South Wales, Australia Reviewed by: Vinicius Machado, Texas Tech University, United States Fabio S. Lima, University of Illinois at Urbana-Champaign, United States Correspondence: Yao-Hong Zhu zhu_yaohong@hotmail.com Correspondence: Yao-Hong Zhu zhu_yaohong@hotmail.com Specialty section: This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology Received: 29 May 2018 Accepted: 22 October 2018 Published: 06 November 2018 Citation: Wang M-L, Liu M-C, Xu J, An L-G, Wang J-F and Zhu Y-H (2018) Uterine Microbiota of Dairy Cows With Clinical and Subclinical Endometritis. Front. Microbiol. 9:2691. doi: 10.3389/fmicb.2018.02691 Specialty section: This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology Uterine Microbiota of Dairy Cows With Clinical and Subclinical Endometritis Meng-Ling Wang, Ming-Chao Liu, Jin Xu, Li-Gang An, Jiu-Feng Wang and Yao-Hong Zhu* Meng-Ling Wang, Ming-Chao Liu, Jin Xu, Li-Gang An, Jiu-Feng Wang and Yao-Hong Zhu* Department of Veterinary Clinical Sciences, College of Veterinary Medicine, China Agricultural University, Beijing, China The objective of this study was to characterize the uterine microbiota of dairy cows with clinical and subclinical endometritis and to identify the potential bacterial genera as well as their interactions associated with uterine disease. Uterine ﬂush samples (n = 27) were collected from 13 healthy, 5 subclinical endometritic (SE), and 9 clinical endometritic (CE) cows at 30 days postpartum. Microbial DNA from uterine ﬂush samples was subjected to sequencing of the 16S rRNA gene on the Illumina MiSeq platform. The uterine microbiota of healthy, SE, and CE cows had similarly complex microbial diversity, and shared 293 of 445 operational taxonomic units. However, endometritic and healthy cows could be discriminated by the relative abundance of bacterial genera. In CE cows, the uterine microbiota was characterized by increased abundance of Fusobacterium and unique presence of Trueperella and Peptoniphilus. For SE cows, known intrauterine pathogens were almost absent and the uterine microbiota was characterized by enrichment of Lactobacillus and Acinetobacter. Analysis of correlations between bacterial genera showed that the uterine microbiota exhibited two co-occurrence groups (i.e., the Lactococcus and the Fusobacterium COGs), indicating that the synergistic effect by co-occurred bacteria may be an important aspect of pathogenesis. Our ﬁndings support that common uterine pathogens are not associated with subclinical endometritis at 30 days postpartum and indicate the need of investigating the role of commensal bacteria such as Lactobacillus, and Acinetobacter in the inﬂammatory process of uterine endometrium. Keywords: uterus, microbiota, uterine ﬂush, cow, endometritis ORIGINAL RESEARCH published: 06 November 2018 doi: 10.3389/fmicb.2018.02691 INTRODUCTION Received: 29 May 2018 Accepted: 22 October 2018 Published: 06 November 2018 Endometritis is one of the most important causes of infertility in dairy cows, resulting in high economic losses in the dairy industry (Sheldon et al., 2009; Wagener et al., 2017). Endometritis is a superﬁcial inﬂammation of the endometrium without systemic signs (Sheldon et al., 2006). Clinical endometritis is deﬁned as the presence of purulent or mucopurulent vaginal discharge at 21 or more days postpartum, accompanied by a prominent leukocyte inﬁltration into the uterine lumen. Subclinical endometritis is characterized by an increased proportion of polymorphonuclear neutrophils (PMN) cells in the endometrium, with the absence of signs of clinical endometritis (Kasimanickam et al., 2004). Indeed, a broad diversity of bacteria, including potential pathogens, can be observed in the uterus of 80–100% of dairy cows during the ﬁrst 2 weeks postpartum Endometritis is one of the most important causes of infertility in dairy cows, resulting in high economic losses in the dairy industry (Sheldon et al., 2009; Wagener et al., 2017). Endometritis is a superﬁcial inﬂammation of the endometrium without systemic signs (Sheldon et al., 2006). Clinical endometritis is deﬁned as the presence of purulent or mucopurulent vaginal discharge at 21 or more days postpartum, accompanied by a prominent leukocyte inﬁltration into the uterine lumen. Subclinical endometritis is characterized by an increased proportion of polymorphonuclear neutrophils (PMN) cells in the endometrium, with the absence of signs of clinical endometritis (Kasimanickam et al., 2004). Indeed, a broad diversity of bacteria, including potential pathogens, can be observed in the uterus of 80–100% of dairy cows during the ﬁrst 2 weeks postpartum Citation: Depending on the balance between the immune response and uterine infection, about 25–40% of cows develop metritis within ﬁrst 3 weeks postpartum (Markusfeld, 1987; Drillich et al., 2001); subsequently, 15–20% of cows develop clinical endometritis, and 30% develop subclinical endometritis beyond 3 weeks postpartum (LeBlanc et al., 2002; Gilbert et al., 2005; Cheong et al., 2011). Postpartum endometritis has a negative eﬀect on reproductive performance as it delayed resumption of ovarian cycles, prolonged postpartum luteal phases, increased days to ﬁrst service and days open, and decreased the conception rate (Kasimanickam et al., 2004; Ribeiro et al., 2013). Studies using culture-dependent methods have identiﬁed several uterine pathogens associated with endometritis, including Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella species (Dohmen et al., 1995; Williams et al., 2005; Carneiro et al., 2016). Members of the genera Bacillus, Streptococcus, and Enterococcus, in addition to coagulase-negative Staphylococci, are among the most frequently isolated intrauterine bacteria and have been described as potential or opportunistic pathogens (Wagener et al., 2014, 2015; Carneiro et al., 2016). Recently developed culture- independent molecular approaches based on sequencing have expanded our current knowledge of the uterine microbiome in cows with metritis, pyometra, and endometritis (Santos and Bicalho, 2012; Jeon et al., 2015; Knudsen et al., 2015, 2016; Bicalho et al., 2017a,b). T. pyogenes was the most important bacteriological risk factor for clinical endometritis, but not for subclinical endometritis (Prunner et al., 2014b). Bacterial growth density on the agar plates increased the risk for subclinical endometritis (Prunner et al., 2014a). Knowledge regarding the uterine microbiota in subclinical endometritic (SE) cows, mainly gleans from studies based on routine microbial isolation and culture techniques. The results from culture-based studies indicated that uterine infections with known pathogens play a minor role in SE cows (Sens and Heuwieser, 2013; Madoz et al., 2014; Prunner et al., 2014a,b). Uterine ﬂush samples were collected from cows at 30 days postpartum. Uterus was ﬂushed with saline, using a pipette (Santos and Bicalho, 2012). Brieﬂy, each cow was restrained and the perineum area was disinfected with 70% ethanol. The infusion pipette covered with a protective plastic sheath was introduced into the cervix; the sheath was subsequently ruptured and the clean pipette tip was manipulated through the cervix into the uterus. The pipette has a deﬂated balloon in the tip. Once inside the uterus, the balloon was inﬂated to prevent vaginal or cervix contamination. Citation: A total of 30 mL of sterile saline was infused into the uterus, agitated gently, and a sample of the ﬂuid aspirated. Recovered ﬂuid was transferred to two polypropylene centrifuge tubes and placed on ice for transport to the laboratory within 4 h. One tube of each uterine ﬂush sample was stored at −80◦C for DNA extraction, the other tube was taken for cytological examination. Brieﬂy, the uterine ﬂush samples were centrifuged at 750 × g for 10 min. After discarding the supernatant, the remaining pellets were re-suspended and smeared onto microscope slides. The slides were ﬁxed and stained with DiﬀQuick. A total amount of 300 cells (endometrial epithelial cells and PMNs) were counted under a microscope by ×400 magniﬁcation to determine the proportion of PMN. A proportion of 18% PMN was set as the threshold for the In this study, we explored the uterine microbiota from the uterine ﬂush samples of healthy, SE and clinical endometritic (CE) cows in an attempt to identify bacterial genera that were associated with endometritis via 16S rRNA gene proﬁling by high-throughput sequencing. Furthermore, we performed the co- occurrence network analysis to identity potential interactions between genera in the uterine microbiota of dairy cows. The data generated through this work might ultimately facilitate the development of eﬃcient disease prevention and intervention strategies. Citation: Wang M-L, Liu M-C, Xu J, An L-G, Wang J-F and Zhu Y-H (2018) Uterine Microbiota of Dairy Cows With Clinical and Subclinical Endometritis. Front. Microbiol. 9:2691. doi: 10.3389/fmicb.2018.02691 November 2018 \| Volume 9 \| Article 2691 1 Frontiers in Microbiology \| www.frontiersin.org Endometritis-Associated Bacteria in Uteri Wang et al. The study was conducted on a commercial dairy farm in Beijing, China. The herd consisted of 800 milking Holstein dairy cows with an average milk production of 9,527 kg per lactation. A total of 38 cows were enrolled in the study. During the sample collection period, nine cows were excluded because of a systemic antibiotic treatment. Reasons for these antibiotic treatments were mastitis (n = 5), metritis (n = 2), pyometra (n = 1), vaginal lacerations (n = 1). Another two cows were excluded because of the poor quality of cytological smears. Therefore, the complete data set of 27 cows was used for statistical analyses. The average parity was 3.3, and an average body condition score (BCS) was 3.1 (see Supplementary Table S1). No diﬀerences were found among groups in parity and BCS. On day 30 postpartum, all cows underwent a vaginal inspection, rectal palpation of the uterus, endometrial cytological examination, and their overall condition was recorded. The BCS was evaluated on a scale from 1 to 5. The cows were selected for the study dependent on the health status of the uterus. Vaginal discharge was scored as previously described (Williams et al., 2005): score 0 with clear or translucent mucus; score 1 with mucus containing ﬂecks of white or oﬀ-white pus; score 2 with less than 50% white or oﬀ-white mucopurulent material in the mucus; and score 3 with more than 50% purulent material, usually white or yellow, but occasionally sanguineous in the mucus. Cows exhibiting mucopurulent or worse (purulent or foul) vaginal discharge without signs of systemic illness as well as the presence of purulent material within the uterine lumen were classiﬁed as having clinical endometritis (n = 9). In the absence of purulent vaginal discharge, cows with the proportion of PMN ≥18% by cytological examination were classiﬁed as having subclinical endometritis (n = 5). Cows with a clear or translucent vaginal discharge that was not fetid or mucopurulent and with the proportion of PMN < 18% by cytological examination were classiﬁed as healthy (n = 13). (Sheldon et al., 2009). MATERIALS AND METHODS Experimental Design and Sampling DNA Extraction and Sequencing Uterine ﬂush samples collected from 27 cows were prepared for DNA extraction and sequencing of the 16S rRNA gene. An aliquot of 2 mL was centrifuged for 30 min at 15,000 × g at 4◦C. The supernatant was discarded, the pellets were suspended in 200 µL of phosphate-buﬀered saline (PBS) to concentrate microbial cells. The PBS suspension was used to isolate bacterial genomic DNA using a QIAamp DNA minikit (Qiagen, Valencia, CA, United States) according to the manufacturer’s protocol, with a minor modiﬁcation: before AL buﬀer was added, samples were incubated with 400 mg of lysozyme for 12 h at 56◦C to maximize bacterial DNA extraction. The purity and concentration of genomic DNA were determined using a spectrophotometer (Nanodrop 1000; Thermo Scientiﬁc, Waltham, MA, United States). Genomic DNA was ampliﬁed by PCR with primers that target the V3 and V4 hypervariable regions of the 16S rRNA gene. The forward primer sequence was 338F (5′- ACTCCTACGGGAGGCAGCAG-3′), and the reverse primer sequence was 806R (5′-GGACTACHVGGGTWTCTAAT-3′). An eight-base sequence unique to each sample preceded the primers for sample identiﬁcation using a HotStarTaq Plus master mix kit (Qiagen) according to a custom Illumina preparation protocol. Amplicons were excised from 1.5% agarose gels and puriﬁed using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) according to the manufacturer’s protocol and quantiﬁed using ST ﬂuorometer (Promega, Madison, WI, United States). A composite sample library for sequencing was created by combining equimolar ratios of amplicons from the individual samples. The composite sample library was cleaned using an UltraClean-htp 96-well PCR cleanup kit (Mo Bio Laboratories, Carlsbad, CA, United States). Pooled amplicons were paired-end sequenced (PE 2 × 250) on an Illumina MiSeq platform according to standard protocols. Taxonomic classiﬁcation of the representative sequence for each OTU was performed using the Ribosomal Database Project classiﬁer2 (Release 11.1) with a cutoﬀof 80% homology against the Silva Gold reference database3(Release 128). OTUs were grouped at diﬀerent levels of classiﬁcation (phylum, class, order, family, and genus), at each level, unclassiﬁed OTUs were grouped together by the highest available resolution. A heat map was generated with average linkage hierarchical clustering of Bray– Curtis distance based on the relative abundances of genera per animal. The correlations between the 28 most abundant genera were calculated using pairwise Spearman’s rank based on relative abundance in the R stats package. Experimental Design and Sampling Experimental Design and Sampling All animal procedures were performed in accordance with the approved guidelines and regulations, and the ethical approval of the Animal Ethics Committee of the China Agricultural University (CAU20140728-2). November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 2 Endometritis-Associated Bacteria in Uteri Wang et al. diagnosis of subclinical endometritis in cows with clear vaginal discharge (Kasimanickam et al., 2004). (version 6.1.544). All singleton OTUs were removed in an attempt to discard the majority of chimera sequences. The OTUs that reached at a 97% similarity level were used for alpha diversity, Good’s coverage, Venn diagram, rarefaction, and rank abundance curve analysis using Mothur (version 1.31.2). Sequences were subsampled to the lowest number of sequences found in all samples (9,666 reads) to evaluate alpha diversity. Alpha diversity was assessed by Shannon index, and the number of observed OTUs. The principal coordinate analysis (PCoA) based on Bray– Curtis distance was performed using OTUs from each sample and plotted by the vegan package in R. 1http://drive5.com/usearch/manual/uchime_algo.html Statistical Analysis y Statistical analysis was carried out using R Statistical Language and GraphPad Prism (version 7.0) software. All continuous variables, such as relative abundance of bacteria, Shannon index, and the number of observed OTUs were analyzed using the Kruskal–Wallis analysis of variance on ranks, followed by Dunn’s test to adjust for multiple comparisons. Permutational multivariate analysis of variance (PERMANOVA) was performed using the vegan package in R. Correlations were calculated by Spearman’s rank correlation in the R stats package. The linear discriminant analysis (LDA) eﬀect size (LEfSe)4 method was used to identify indicator bacteria diﬀerentiating the uterine microbiota between healthy and endometritic cows, which emphasizes both statistical signiﬁcance and biological relevance. LEfSe uses the Kruskal–Wallis rank sum test with a normalized relative abundance matrix to detect diﬀerentially abundant features between groups and performs LDA to estimate the eﬀect size of each feature (Segata et al., 2011). A signiﬁcance level (alpha) of 0.05 and an eﬀect size threshold of 3 were used for all DNA Extraction and Sequencing The correlation matrix was visualized and clustered in R using the Made4 package and Heatplot function, and hierarchical Ward-linkage clustering was used to deﬁne genus co-occurrence groups (COGs) (Biagi et al., 2016). The correlations were visualized in network interface with Cytoscape software (Shannon et al., 2003). The nodes represented genera, and the size of each node is proportional to the average relative abundance. The edges between nodes represented signiﬁcant (P < 0.05) correlations, and the thickness of edge is proportional to the correlation strength. Bacterial Diversity of the Uterine Microbiota The relative abundance of Fusobacterium increased with increasing vaginal discharge score (Spearman’s rs = 0.51, P = 0.006) (Figure 4A). Likewise, the relative abundance of Trueperella increased with increasing vaginal discharge score (Spearman’s rs = 0.48; P = 0.017) (Figure 4B). We also compared the relative abundances of the 28 most abundant genera in the uterine microbiota using Kruskal–Wallis rank-sum test (Figure 4 and Supplementary Table S3). Compared with healthy cows, the relative abundance of Fusobacterium increased in CE cows, the abundance of Lactococcus, Bacillus, Solibacillus, Pseudomonas, and Arthrobacter decreased (P < 0.05). Lactobacillus and Acinetobacter were more abundant in SE cows than in healthy cows (P < 0.05). The abundance of Fusobacterium and Trueperella decreased in SE cows, compared with those in CE cows (P < 0.05). No diﬀerences in the abundance of Lactococcus, Bacillus, Solibacillus, Pseudomonas, and Arthrobacter were observed between the healthy and SE cows. Bacterial Diversity of the Uterine Microbiota To identify genera associated with endometritis, LEfSe was performed using the 28 most abundant bacterial genera in the uterine microbiota of healthy and endometritic cows. Compared with healthy cows, Fusobacterium, Trueperella, and Peptoniphilus were discriminately enriched (LDA sores > 3.5) in CE cows (Figure 3A and Supplementary Table S2). In SE cows, Lactobacillus, and Acinetobacter were discriminately increased (LDA scores > 3), compared with healthy cows (Figure 3B and Supplementary Table S3). To proﬁle the bovine uterine microbiota, we performed 16S rRNA sequencing for 27 uterine ﬂush samples using an Illumina MiSeq platform. A total of 468,846 raw sequence reads were obtained. Following quality trimming and chimera checking, 392,246 high quality reads remained, accounting for 83.7% of the valid reads. The rarefaction curves (Supplementary Figure S1 and Figure 1A) showed that this sequencing depth was suﬃcient to cover the overall bacterial diversity. The value of Good’s coverage of each cow were greater than 99%, indicating that this sequencing method can characterize the true composition of uterine microbiota. The rank-abundance curves showed that a few species accounted for more than 1% abundance in the uterine microbiota (Figure 1B). Species diversity was measured as Shannon index, species richness was calculated as the number of OTUs. No diﬀerences were found among groups in species diversity and species richness (Figures 1C,D). Venn diagram showed that 293 of the 445 total OTUs were shared among groups (Figure 1E). These 293 shared OTUs dominated the uterine microbiota, represented 99.68%, 99.39%, and 98.67% of the total OTUs abundance in the healthy, SE, and CE cows, respectively. The uterine microbiota of healthy, SE, and CE cows had similar level of microbial diversity and shared most bacterial species (Figures 1C–E). The PCoA analysis showed that the samples from CE cows could be separated from healthy cows, although two CE samples were clustered with the healthy group (Figure 1F). The PERMANOVA analysis of the uterine samples showed signiﬁcant diﬀerences in community composition between the healthy and CE groups (P = 0.004, R2 = 0.182), and no signiﬁcant diﬀerence in community composition between the healthy and SE groups (P = 0.167, R2 = 0.097). To further explore the relationship between speciﬁc bacteria and clinical endometritis, we stratiﬁed cows by vaginal discharge score as a clinical sign and performed a Spearman’s rank correlation tests on the relative abundances of the 28 most abundant bacterial genera. Sequence Analysis The sequence data were deposited in the NCBI Sequence Read Archive database (accession number SRP102408). Raw fastq ﬁles were demultiplexed and quality-ﬁltered using the Quantitative Insights Into Microbial Ecology (QIIME) (Caporaso et al., 2010) with the following criteria: (i) The 250 bp reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window, discarding the truncated reads that were shorter than 50 bp; (ii) there were exact barcode matching and a maximum of two nucleotide mismatches to primer sequences; (iii) no ambiguous bases; and (iv) only sequences that overlap longer than 10 bp were assembled according to their overlap sequence. Reads that could not be assembled were discarded. Chimeras were checked and excluded using the Uchime algorithm (version 4.2.401 ). The resulting high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% identity level using Usearch 6.1 methodology November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 3 Endometritis-Associated Bacteria in Uteri Wang et al. SE cows (Supplementary Table S4). In CE cows, the Lactococcus, Bacillus, Fusobacterium, ratAN060301.norank, and Solibacillus were the top ﬁve abundant genera. The genus-level heat map analysis showed an obvious shift in the uterine microbiota of CE cows, with an increase in the genera Fusobacterium, Parvimonas, Porphyromonas, Peptoniphilus, Helcococcus, Trueperella, and ratAN060301.norank (OTU154, uncultured Porphyromonas species in the family ratAN060301, order Bacteroidales), compared with healthy and SE cows. indicators discussed. All tests for signiﬁcance were two-sided, and a signiﬁcance level of 0.05 was considered statistically signiﬁcant in this study. Interactions Between Bacterial Genera in the Uterine Microbiota A total of 17 phyla were identiﬁed in the uterine microbiota of all samples. Taxonomic assignment showed that the Firmicutes (76.7%), Proteobacteria (8.1%), Actinobacteria (5.9%), Bacteroidetes (4.6%), Fusobacteria (4.3%), and Tenericutes (0.2%) were the six most abundant phyla in the uterus of all dairy cows, accounting for 99.8% of the total abundance. In total, 206 genera were identiﬁed across all the samples. A heat map of the 40 most abundant genera (at 0.1% or greater abundance within either group) is shown in Figure 2, accounting for 99.66%, 99.48%, and 98.61% of the total genera abundance in the healthy, SE, and CE groups, respectively. Lactococcus, Bacillus, Solibacillus, Pseudomonas, and Arthrobacter were the ﬁve most abundant genera with little variations in the uterus of healthy and We evaluated the correlations between the 28 most abundant bacterial genera, and performed hierarchical Ward-linkage clustering of the correlations to deﬁne co-occurrence group (COG) (Figure 5A). The distribution of COGs diﬀered signiﬁcantly among groups, determined by PERMANOVA analysis using the Bray–Curtis dissimilarity (Fusobacterium COG F = 2.31, P = 0.003; Lactococcus COG F = 2.39, P = 0.002). The Lactococcus COG represented the majority of the uterine microbiota in terms of high predominate abundance, accounted for 94.1%, 91.1%, and 66.1% in the healthy, SE, and CE groups, respectively. The Fusobacterium COG (accounted for 28.39%) was exclusively enriched in the November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 4 Endometritis-Associated Bacteria in Uteri Wang et al. E 1 \| Structural comparison of the uterine microbiota. The rarefaction curves (A), rank abundance curves (B), Shannon index (C), and the number of nal taxonomic units (OTUs) (D) were used to estimate alpha diversity of the uterine microbiota in healthy, SE, and CE cows. Symbols represent data from al cows, data shown as median with 95% CI. (E) Venn diagram illustrating the common and exclusive OTUs in the uterine microbiota of the three groups. cipal coordinate analysis based on the relative abundance of OTUs with Bray–Curtis distances, showing the differences between each individual cow. healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. FIGURE 1 \| Structural comparison of the uterine microbiota. DISCUSSION and ratN060301C.norank were positively correlated (P < 0.05) with Peptoniphilus. Helcococcus was also positively correlated (P < 0.05) with ratN060301C.norank and Porphyromonas. Bacteroides was also positively correlated (P = 0.002) with Ruminococcaceae uncultured. Fusobacterium showed a positive correlation with Parvimonas (P = 0.003). Within the Lactococcus COG, Arthrobacter was positively correlated with Lactobacillus (Spearman’s rs = 0.56, P = 0.018), and with Psychrobacter (Spearman’s rs = 0.80, P = 0.00004). Arthrobacter, Psychrobacter, Bacillus, Solibacillus, and Pseudomonas were positively correlated with many bacteria including Psychrobacter, Carnobacterium, Exiguobacterium, and Brochothrix. In the present study, we investigated the disease-related alterations as well as bacteria interactions in the uterine microbiota of CE and SE cows. Previous studies have described the diversity and complexity of the bacterial community in the postpartum uterus of dairy cows (Santos and Bicalho, 2012; Jeon et al., 2015; Knudsen et al., 2015, 2016; Bicalho et al., 2017a,b). Here, we performed co-occurrence analysis to obtain a comprehensive understanding of the complex bacterial interactions in bovine uterus. In the present study, we investigated the disease-related alterations as well as bacteria interactions in the uterine microbiota of CE and SE cows. Previous studies have described the diversity and complexity of the bacterial community in the postpartum uterus of dairy cows (Santos and Bicalho, 2012; Jeon et al., 2015; Knudsen et al., 2015, 2016; Bicalho et al., 2017a,b). Here, we performed co-occurrence analysis to obtain a comprehensive understanding of the complex bacterial interactions in bovine uterus. We demonstrated that Fusobacterium, Trueperella, and Peptoniphilus were associated with clinical endometritis, and Fusobacterium and Trueperella were positively correlating with purulent vaginal discharge. Notably, CE-associated genera Fusobacterium, Trueperella, and Peptoniphilus, along with other pathogens such as Porphyromonas, Parvimonas, Bacteroides, and Helcococcus were found to belong to the Fusobacterium COG, and had positive correlations. Therefore, it is likely that Fusobacterium acts synergistically with Trueperella, Porphyromonas, Parvimonas and other bacteria, to cause dysbiosis in the uterine microbiota of CE cows. F. necrophorum, and T. pyogenes have been recognized as the major uterine pathogens associated with metritis, endometritis and purulent vaginal discharge (Dohmen et al., 1995; Williams et al., Negative correlations were found between genera from the Lactococcus COG and the Fusobacterium COG (Figure 5C and Supplementary Table S6). The abundance of Fusobacterium was negatively correlated with the abundance of Arthrobacter (Spearman’s rs = −0.53, P = 0.028), and with the abundance with Psychrobacter (Spearman’s rs = −0.64, P = 0.0003). Interactions Between Bacterial Genera in the Uterine Microbiota The rarefaction curves (A), rank abundance curves (B), Shannon index (C), and the number of operational taxonomic units (OTUs) (D) were used to estimate alpha diversity of the uterine microbiota in healthy, SE, and CE cows. Symbols represent data from individual cows, data shown as median with 95% CI. (E) Venn diagram illustrating the common and exclusive OTUs in the uterine microbiota of the three groups. (F) Principal coordinate analysis based on the relative abundance of OTUs with Bray–Curtis distances, showing the differences between each individual cow. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. uterine microbiota of CE cows (Figure 5B). Fusobacterium, Porphyromonas, Trueperella, Helcococcus, and Peptoniphilus represented 10.58%, 3.05%, 2.24%, 1.55%, and 0.66% of the total bacterial population in CE cows, while little abundance was found in the uterine microbiota of healthy and SE cows (representing less than 0.2% of the total bacterial population) (Supplementary Table S4). At species level, sequences from F. necrophorum (OTU430), T. pyogenes (OTU99), Helcococcus ovis (OTU311) and Peptoniphilus indolicus (OTU295) were identiﬁed in this study (Supplementary Table S5). A number of positive correlations were found among genera from the same COG (Figure 5C and Supplementary Table S6). Major uterine pathogens such as Fusobacterium, Trueperella, Bacteroides, and Porphyromonas along with other uterine pathogens such as Peptoniphilus and Helcococcus belonged to the Fusobacterium COG, and had positive correlations. Fusobacterium showed a positive correlation with Trueperella (Spearman’s rs = 0.43, P = 0.026), which was positively correlated (P < 0.05) with Peptoniphilus, Helcococcus, ratN060301C.norank, and Bacteroides. Helcococcus November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 5 Wang et al. Endometritis-Associated Bacteria in Uteri FIGURE 2 \| Heat map with average linkage clustering based on Bray–Curtis distance showing the relative abundances of the top 40 genera in each cow. The relative abundance of each genus is indicated by a gradient of color from blue (low abundance) to red (high abundance). Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. FIGURE 2 \| Heat map with average linkage clustering based on Bray–Curtis distance showing the relative abundances of the top 40 genera in each cow. The relative abundance of each genus is indicated by a gradient of color from blue (low abundance) to red (high abundance). Interactions Between Bacterial Genera in the Uterine Microbiota Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION Psychrobacter, Bacillus, Solibacillus, and Pseudomonas were negatively (P < 0.05) correlated with Trueperella. Genera with negative correlations with Fusobacterium and/or Trueperella, comprising Arthrobacter, Psychrobacter, Bacillus, Solibacillus, and Pseudomonas, were positively correlated (P < 0.05) with many other bacteria including Psychrobacter, Carnobacterium, Exiguobacterium, and Brochothrix. November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 6 Endometritis-Associated Bacteria in Uteri Wang et al. FIGURE 3 \| The linear discriminant analysis (LDA) effect size plots showing the differences in the uterine microbiota between healthy cows and CE cows (A), and between healthy cows and SE cows (B). The histogram shows the LDA effect size computed for features at genus level. Healthy-enriched genera are indicated with positive LDA scores (green), and genera enriched in CE or SE cows are indicated with negative LDA scores (red). Only genera meeting a signiﬁcant level of 0.05 and an effect size threshold of 3 are plotted. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. FIGURE 3 \| The linear discriminant analysis (LDA) effect size plots showing the differences in the uterine microbiota between healthy cows and CE cows (A), and between healthy cows and SE cows (B). The histogram shows the LDA effect size computed for features at genus level. Healthy-enriched genera are indicated with positive LDA scores (green), and genera enriched in CE or SE cows are indicated with negative LDA scores (red). Only genera meeting a signiﬁcant level of 0.05 and FIGURE 3 \| The linear discriminant analysis (LDA) effect size plots showing the differences in the uterine microbiota between healthy cows and CE cows (A), and between healthy cows and SE cows (B). The histogram shows the LDA effect size computed for features at genus level. Healthy-enriched genera are indicated with positive LDA scores (green), and genera enriched in CE or SE cows are indicated with negative LDA scores (red). Only genera meeting a signiﬁcant level of 0.05 and an effect size threshold of 3 are plotted. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. 2005; Bicalho et al., 2012; Prunner et al., 2014b). Frontiers in Microbiology \| www.frontiersin.org DISCUSSION Cows were assigned to four groups according to vaginal discharge score: 0, clear or translucent mucus; 1, clear discharge with ﬂecks of pus; 2, mucopurulent, not fetid discharge; 3, purulent or fetid discharge. r, Spearman’s coefﬁcient. (C) Analysis of the relative abundance of Lactococcus, Bacillus, Solibacillus, Pseudomonas, and Arthrobacter in healthy, SE and CE cows by Kruskal–Wallis rank-sum test. Symbols represent data from individual cows, data shown as median with 95% CI. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. ∗P < 0.05, ∗∗P < 0.01. FIGURE 5 \| Co-occurrence groups (COGs) assignment depended on heat plot (A) showing Spearman correlations between genera, clustered by Ward-linkage hierarchical clustering. (B) The average cumulative abundance of genera in each COG for healthy, SE and CE cows is also plotted. Colors are indicative of the two identiﬁed COGs: Lactococcus COG (blue) and Fusobacterium COG (yellow). The proportion of genera not considered in the COG assignment is colored gray. (C) The network plot showing correlations between bacterial genera in the uterine microbiota of all cows (C). The nodes represent the genera; the circle size of each node is proportional to its average relative abundance in all samples. Blue and yellow nodes indicate genera belong to Lactococcus COG and Fusobacterium COG, respectively. The edges between nodes represent signiﬁcant (P < 0.05) correlations between genera, the thickness of edge is proportional to the correlation strength (see Supplementary Table S5). Green and red edges indicate positive and negative correlations, respectively. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. FIGURE 5 \| Co-occurrence groups (COGs) assignment depended on heat plot (A) showing Spearman correlations between genera, clustered by Ward-linkage hierarchical clustering. (B) The average cumulative abundance of genera in each COG for healthy, SE and CE cows is also plotted. Colors are indicative of the two identiﬁed COGs: Lactococcus COG (blue) and Fusobacterium COG (yellow). The proportion of genera not considered in the COG assignment is colored gray. (C) The network plot showing correlations between bacterial genera in the uterine microbiota of all cows (C). The nodes represent the genera; the circle size of each node is proportional to its average relative abundance in all samples. Blue and yellow nodes indicate genera belong to Lactococcus COG and Fusobacterium COG, respectively. DISCUSSION Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. ∗P < 0.05, ∗∗P < 0.01. FIGURE 5 \| Co-occurrence groups (COGs) assignment depended on heat plot (A) showing Spearman correlations between genera, clustered by Ward-linkage ierarchical clustering. (B) The average cumulative abundance of genera in each COG for healthy, SE and CE cows is also plotted. Colors are indicative of the two dentiﬁed COGs: Lactococcus COG (blue) and Fusobacterium COG (yellow). The proportion of genera not considered in the COG assignment is colored gray. C) The network plot showing correlations between bacterial genera in the uterine microbiota of all cows (C). The nodes represent the genera; the circle size of each ode is proportional to its average relative abundance in all samples. Blue and yellow nodes indicate genera belong to Lactococcus COG and Fusobacterium COG, espectively. The edges between nodes represent signiﬁcant (P < 0.05) correlations between genera, the thickness of edge is proportional to the correlation strength see Supplementary Table S5). Green and red edges indicate positive and negative correlations, respectively. Healthy, healthy cows, n = 13; SE, subclinical ndometritic cows, n = 5; CE, clinical endometritic cows, n = 9. dometritis at 21 days postpartum was not associated with pyogenes (Prunner et al., 2014a). Early culture-based study also major pathogens play a minor role in SE cows compared with CE cows. The establishment of uterine infections depends on FIGURE 4 \| The relative abundance distribution of Fusobacterium (A), Trueperella (B) among groups, and correlations between vaginal discharge scores. Cows were assigned to four groups according to vaginal discharge score: 0, clear or translucent mucus; 1, clear discharge with ﬂecks of pus; 2, mucopurulent, not fetid discharge; 3, purulent or fetid discharge. r, Spearman’s coefﬁcient. (C) Analysis of the relative abundance of Lactococcus, Bacillus, Solibacillus, Pseudomonas, and Arthrobacter in healthy, SE and CE cows by Kruskal–Wallis rank-sum test. Symbols represent data from individual cows, data shown as median with 95% CI. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. ∗P < 0.05, ∗∗P < 0.01. FIGURE 4 \| The relative abundance distribution of Fusobacterium (A), Trueperella (B) among groups, and correlations between vaginal discharge scores. DISCUSSION It has been proposed that Trueperella and the Gram-negative anaerobes Fusobacterium, Bacteroides, and Porphyromonas act synergistically to cause metritis and endometritis in the uterus (Bonnett et al., 1991; Bicalho et al., 2012; Prunner et al., 2014b). T. pyogenes causes cytolysis in the endometrium by secreting pyolysin (Amos et al., 2014). F. necrophorum produces leukotoxins (Nagaraja et al., 2005), Bacteroides produces short-chain fatty acids (Rotstein, 1993), and Porphyromonas levii produces an immunoglobulin protease that inhibits phagocytosis (Lobb et al., 1999). It is widely believed that T. pyogenes support F. necrophorum growth and colonization by producing an unknown growth factor (Dadarwal et al., 2017). A synergy between F. necrophorum and Porphyromonas levii has been hypothetically suggested for their co-localization in the lamina propria of the uterus (Karstrup et al., 2017a). It is therefore plausible that uterine pathogens might assist each other in avoiding uterine defense mechanisms and interact to facilitate colonization of the endometrium. Similar cooperative interactions between pathogens were also observed in cows with metritis or purulent vaginal discharged (Bicalho et al., 2012, 2017a,b; Jeon et al., 2015, 2017). Pathogenic bacteria (such as Trueperella spp., Fusobacterium spp.) were also present in the uterus of virgin heifers and of pregnant cows (Karstrup et al., 2017b,c; Moore et al., 2017). Collectively, the co-occurrence of uterine pathogens could be considered of major importance in the development of uterine infection. The cooperative interspecies signaling and mechanism behind synergisms need to be elucidated. Contrast with clinical endometritis, cows with subclinical endometritis harbored a small proportion of the Fusobacterium COG, constituting only 1.5% of the total number of sequences. Fusobacterium, Trueperella were rarely detected in samples from SE cows. Early culture-based study demonstrated that subclinical November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 7 Endometritis-Associated Bacteria in Uteri Wang et al. FIGURE 4 \| The relative abundance distribution of Fusobacterium (A), Trueperella (B) among groups, and correlations between vaginal discharge scores. Cows were assigned to four groups according to vaginal discharge score: 0, clear or translucent mucus; 1, clear discharge with ﬂecks of pus; 2, mucopurulent, not fetid ischarge; 3, purulent or fetid discharge. r, Spearman’s coefﬁcient. (C) Analysis of the relative abundance of Lactococcus, Bacillus, Solibacillus, Pseudomonas, and Arthrobacter in healthy, SE and CE cows by Kruskal–Wallis rank-sum test. Symbols represent data from individual cows, data shown as median with 95% CI. DISCUSSION The edges between nodes represent signiﬁcant (P < 0.05) correlations between genera, the thickness of edge is proportional to the correlation strength (see Supplementary Table S5). Green and red edges indicate positive and negative correlations, respectively. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; CE, clinical endometritic cows, n = 9. endometritis at 21 days postpartum was not associated with T. pyogenes (Prunner et al., 2014a). Early culture-based study also found that T. pyogenes were frequently isolated from CE cows, and no bacteria were isolated from SE cows (Madoz et al., 2014). Our observations by sequencing of 16S rRNA gene corroborate previous observations, supporting that uterine infections with major pathogens play a minor role in SE cows compared with CE cows. The establishment of uterine infections depends on the pathogenicity of invading bacteria and the local immune state. Many factors inﬂuence bacteria pathogenicity, including bacterial load, various strains, bacterial virulence factors, and interactions between species etc. (Dadarwal et al., 2017). PMN November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 8 Endometritis-Associated Bacteria in Uteri Wang et al. inﬁltration into the uterine lumen as an indicator of subclinical endometritis is associated with increased endometrial mRNA expression of pro-inﬂammatory mediators, including cytokines antimicrobial peptides, acute phase proteins and prostaglandins (Fischer et al., 2010; Brodzki et al., 2015). Brodzki et al. (2015) hypothesized that high levels of IL-10 in SE cows contribute to a weakened local immune response in the endometrium, leading to persistent uterine inﬂammation in the postpartum period (Brodzki et al., 2015). Metabolic imbalances also increase the risk of subclinical endometritis, particularly a negative energy balance, which interferes with an adequate immune response (Wagener et al., 2017). Elevated concentrations of non-esteriﬁed fatty acids and beta-hydroxybutyric acid, and a poor BCS increase the risk for subclinical endometritis (Galvão et al., 2010; Heidarpour et al., 2012). promote group survival under nutritionally challenging conditions (Zelezniak et al., 2015). The diﬀerent uterine bacterial composition between healthy and diseased cows likely reﬂect the diﬀering nutritional and physiological conditions. Identifying the source of critical nutrients that support pathogenic overgrowth will be crucial to increasing our understanding of disease pathogenesis and could potentially assist in developing novel treatment strategies (Zengler and Zaramela, 2018). Such competitive and cooperative interactions and mechanisms of establishing infection warrants further detailed studies. ACKNOWLEDGMENTS We would like to thank Dr. David Robinette for revising and editing the manuscript. DISCUSSION Investigation the uterine microbiota in uterine ﬂush samples from postpartum dairy cows by high throughput sequencing of 16S rRNA gene showed that major uterine pathogens such as Fusobacterium, Trueperella, and Peptoniphilus were enriched in the uterine microbiota of CE cows, but were almost absent in the uterine microbiota of SE cows. Our results demonstrated that known uterine pathogens had co-existence relationships with other bacteria, such as Porphyromonas, Bacteroides, Helcococcus, and Parvimonas, suggesting that their synergistic eﬀects may be crucial contributors in uterine infection. Our ﬁndings support that major uterine pathogens are not associated with subclinical endometritis at 30 days postpartum and indicate the need of investigating the role of commensal bacteria such as Lactobacillus, and Acinetobacter in the inﬂammatory process of uterine endometrium. The uterine microbiota of SE cows was characterized by enrichment of Lactobacillus and Acinetobacter. Intravaginal administration of certain strains of Lactobacillus reduced the incidence of uterine diseases in treated cows with enhanced secretory immunoglobulin A production in the vaginal mucus (Deng et al., 2015). Lactobacillus species isolated from the bovine uterus, such as L. amylovorus and L. ruminis, stimulate an immune response without cytotoxic eﬀects (Gärtner et al., 2015). Lactobacillus rhamnosus GR-1 reduces E. coli-induced release of pro-inﬂammatory cytokines in primary bovine endometrial epithelial cells in vitro (Liu et al., 2016). Acinetobacter spp. are observed in the environment (e.g., soil and water) and present in the microbiota of healthy human skin, cattle udder skin, and cattle gut (Yeoman et al., 2018). A. baumannii is the most important species, since it causes serious infections in human (De Amorim and Nascimento, 2017). Acinetobacter strains isolated from samples of milk and milk derivatives could be opportunistic pathogens (Gurung et al., 2013). Acinetobacter strains can be isolated from intrauterine samples collected from repeat breeder cows (Pothmann et al., 2015). Due to the limited information of 16S rRNA sequencing method, it is diﬃcult to deﬁne pathogenic strains or harmless commensals associated with subclinical endometritis. In this study, whether these bacterial changes are a cause or a consequence of uterine inﬂammation is uncertain. Our ﬁndings increase the current knowledge of the uterine microbiota in SE cows and provide a basis for future detailed in vitro studies to decipher the aﬀect of these bacteria on immune response in uterine endometrium. AUTHOR CONTRIBUTIONS M-LW, M-CL, Y-HZ, and J-FW conceived and designed the experiments. M-LW, M-CL, JX, and L-GA performed the experiments. M-LW performed sequencing analysis and wrote the manuscript. FUNDING The present work was funded by the National Key R&D Program of China (Project No. 2017YFD0502200), the Program for the Beijing Dairy Industry Innovation Team (BAIC06-2018), and the National Natural Science Foundation of China (Project Nos. 31873034, 31672613, and 31472242). The present work was funded by the National Key R&D Program of China (Project No. 2017YFD0502200), the Program for the Beijing Dairy Industry Innovation Team (BAIC06-2018), and the National Natural Science Foundation of China (Project Nos. 31873034, 31672613, and 31472242). The Fusobacterium COG had negatively correlations with the Lactococcus COG and dramatically more abundant in CE cows, implying that there might be local colonization resistance between the Fusobacterium COG and Lactococcus COG, linked to the microbiota dysbiosis in CE cows. Accordingly, the overgrowth of the Lactococcus COG may result in a decrease of the Fusobacterium COG. Therefore, competition between members of the Fusobacterium COG and Lactococcus COG might restrict the overgrowth of potential pathogens. The composition and function of microbial communities is thought to be largely shaped by interspecies competition for the available resources (Zengler and Zaramela, 2018). The cooperative interactions among co-occurring bacteria, such as metabolite exchanges, could REFERENCES D., Chae, M. H., Jang, G. C., Jung, S. C., et al. (2013). Prevalence and antimicrobial susceptibility of Acinetobacter from raw bulk tank milk in Korea. J. Dairy Sci. 96, 1997–2002. doi: 10.3168/jds.2012-5965 Bonnett, B. N., Martin, S. W., Gannon, V. P., Miller, R. B., and Etherington, W. G. (1991). Endometrial biopsy in Holstein-Friesian dairy cows. III. Bacteriological analysis and correlations with histological ﬁndings. Can. J. Vet. Res. 55, 168–173. Heidarpour, M., Mohri, M., Fallah-Rad, A. H., Dehghan Shahreza, F., and Mohammadi, M. (2012). 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Association between virulence factors of Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium pyogenes and uterine diseases of dairy cows. Vet. Microbiol. 157, 125–131. doi: 10.1016/j.vetmic.2011.11.034 Gurung, M., Nam, H. M., Tamang, M. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02691/full#supplementary-material November 2018 \| Volume 9 \| Article 2691 Frontiers in Microbiology \| www.frontiersin.org 9 Wang et al. Endometritis-Associated Bacteria in Uteri FIGURE S1 \| Rarefaction curves of each individual cow. Healthy, healthy cows, n = 13; SE, subclinical endometritic cows, n = 5; and CE, clinical endometritic cows, n = 9. TABLE S4 \| Relative abundance of the 28 most abundant genera detected in the uterine of postpartum healthy and endometritic dairy cows at 30 days postpartum. TABLE S4 \| Relative abundance of the 28 most abundant genera detected in the uterine of postpartum healthy and endometritic dairy cows at 30 days postpartum. TABLE S1 \| The basic information of cows included in this study. TABLE S5 \| Relative abundance of 28 most abundant species detected in the uterine of postpartum healthy and endometritic dairy cows at 30 days postpartum. TABLE S5 \| Relative abundance of 28 most abundant species detected in the uterine of postpartum healthy and endometritic dairy cows at 30 days postpartum. TABLE S2 \| Key features are statistically different between the uterine microbiota of healthy and clinical endometritic cows. TABLE S6 \| Correlations among the 28 most abundant genera detected in the uterus of 27 cows at 30 days post parturition. Only signiﬁcant (P < 0.05) Spearman coefﬁcients of correlation are presented. TABLE S3 \| Key features are statistically different between the uterine microbiota of healthy and subclinical endometritic cows. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution 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. Frontiers in Microbiology \| www.frontiersin.org REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Santos, T. M. A., and Bicalho, R. C. (2012). Diversity and succession of bacterial communities in the uterine ﬂuid of postpartum metritic, endometritic and healthy dairy cows. PLoS One 7:e53048. doi: 10.1371/journal.pone.0053048 Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W. S., et al. (2011). Metagenomic biomarker discovery and explanation. 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https://openalex.org/W2598719676	https://europepmc.org/articles/pmc5441401?pdf=render	English	null	Whole exome sequencing of a patient with suspected mitochondrial myopathy reveals novel compound heterozygous variants in <i><scp>RYR</scp>1</i>	Molecular genetics & genomic medicine	2,017	cc-by	4,654	Results Whole exome sequencing revealed two novel compound heterozygous variants in RYR1 (c.7060_7062del, p.Val2354del and c.4485_4500del, p.Tyr1495X). Conclusion doi: 10.1002/mgg3.280 doi: 10.1002/mgg3.280 Review of her clinical, pathologic, and genetic ﬁndings pointed to a diagnosis of a congenital myopathy with ﬁber-type disproportion. in clinical presentation, and varying modes of inheritance, determining the genetic diagnosis has become increasingly challenging, even with the advent of next generation sequencing in clinical practice (B€onnemann et al. 2014). Pathogenic variants in the ryanodine receptor 1 (RYR1, MIM# 180901) gene have been implicated in a number of ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Correspondence Paldeep S. Atwal, Assistant Professor of Medical Genetics and Medicine, Department of Clinical Genomics, Mayo Clinic, 4500 San Pablo Road South, Jacksonville, FL 32224. Tel: +1 904 953 6466; Fax: 904-953-6056; E-mail: Atwal.paldeep@mayo.edu Funding Information The authors would like to thank the Mayo Clinic Center for Individualized Medicine for supporting this research. Received: 7 December 2016; Revised: 18 January 2017; Accepted: 8 February 2017 Received: 7 December 2016; Revised: 18 January 2017; Accepted: 8 February 2017 Whole exome sequencing of a patient with suspected mitochondrial myopathy reveals novel compound heterozygous variants in RYR1 Patrick R. Blackburn1,2 , Duygu Selcen3, Jennifer M. Gass1 , Jessica L. Jackson4, Margot A. Cousin5,6 , Nicole J. Boczek5,6 , Eric W. Klee5,6,7,8, Elliot L. Dimberg9, Kathleen D. Kennelly9 & Paldeep S. Atwal1,4 1Center for Individualized Medicine, Mayo Clinic, Jacksonville, Florida 2Department of Health Sciences Research, Mayo Clinic, Jacksonville, Florida 3Department of Neurology, Mayo Clinic, Rochester, Minnesota 4Department of Clinical Genomics, Mayo Clinic, Jacksonville, Florida 5Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota 6Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota 7Department of Clinical Genomics, Mayo Clinic, Rochester, Minnesota 8Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 9Department of Neurology, Mayo Clinic, Jacksonville, Florida Keywords CFTD, congenital ﬁber-type disproportion, congenital myopathy, malignant hyperthermia, ryanodine receptor 1, RYR1 Methods We present a patient with global developmental delay, hypotonia, myopathy, joint hypermobility, and multiple other systemic complaints that were noted early in life. Later she was found to have multiple bone deformities involving her spine, with severe scoliosis that was corrected surgically. She was also diag- nosed with ophthalmoplegia, chronic hypercapnic respiratory failure, and hypertension. At 22 years of age she presented to the genetics clinic with a diagnosis of mitochondrial myopathy and underwent whole exome sequencing (WES). Background Pathogenic variants in ryanodine receptor 1 (RYR1, MIM# 180901) are the cause of congenital myopathy with ﬁber-type disproportion, malignant hyper- thermia susceptibility type 1, central core disease of muscle, multiminicore dis- ease and other congenital myopathies. Clinical description The patient is a 22-year-old African American female with a complicated past medical history since birth involving myopathy, weakness, joint hypermobility, and multiple other systemic complaints that were attributed initially to a mitochondrial myopathy. The patient was born to term after an uncomplicated pregnancy with no known expo- sures. The patient had a normal birth weight of 6 lbs 11 oz and her Apgar scores were 7 at 1 min and 8 at 5 min. She was unresponsive after birth and was given oxygen via hood for 10 days. After 3 days, she was transferred to the neona- tal intensive care unit (NICU) and was diagnosed with hypotonia and cerebral palsy. She had failure to thrive with trouble sucking and swallowing and at 10 months of age had a J-tube placed. She remained tube-dependent until she was 3 years old. She received physical, occupational, and speech therapy for global developmental delay. The patient never crawled, and she walked initially using a walker while working with a physical therapist at around 2 years of age. She also had speech delay and did not start speaking until she was 2 years old. As a young child, she was diagnosed with external oph- thalmoplegia with little upward eye movement, limited lateral gaze, and mild ptosis (Recent photographs are shown in Fig. 1A–D). She is reported to have had a mus- cle biopsy when she was 3 years old that showed evidence of oxidative phosphorylase deﬁciency and was given a diagnosis of a mitochondrial disorder. The patient was placed on CoQ10, carnitine, levocarnitine, and other sup- plements, which did not help her symptoms. Pathogenic variants in RYR1, TPM3 (MIM# 191030), TPM2 (MIM# 190990), ACTA1 (MIM# 102610), SEPN1 (MIM# 606210), LMNA (MIM# 150330), and MYH7 (MIM# 160760) genes can cause CFTD, with TPM3 being the most common cause of this form of myopathy (Clarke et al. 2010; North et al. 2014). Clarke et al. (2010) described four families with the same pattern of recessive RYR1 variants including one frameshift or trun- cating variant together with a missense change who had CFTD. The pathology and clinical phenotypes of patients with CFTD can be present in core myopathy, and it can be difﬁcult to distinguish between these disorders (Clarke et al. 2010). Introduction The congenital myopathies and muscular dystrophies are clinically and genetically heterogeneous disorders (B€onne- mann et al. 2014). Because of the overlap in disease phe- notype across many of these disorders, marked variability 295 Exome Sequencing Reveals Two Novel RYR1 Variants P. R. Blackburn et al. and in-frame deletion variants in RYR1 and further pathological characterization revealed this to be CFTD. While the patient described in this report has a pheno- type consistent with autosomal recessive RYR1-related congenital myopathy, it highlights the importance of this condition in the differential diagnosis for mitochondrial myopathy. different disorders including congenital myopathy with ﬁber-type disproportion (CFTD, MIM# 255310). The RYR1 gene contains 106 exons that encode a homotetrameric calcium channel that controls communi- cation between transverse-tubules and the sarcoplasmic reticulum in skeletal muscle by regulating cytosolic Ca2+ levels and excitation–contraction coupling (Jungbluth 2007; Hernandez-Ochoa et al. 2015). RYR1 is required for normal development of muscle ﬁbers, skin, and heart during embryogenesis and pathogenic variation is thought to result in altered properties of RYR1 and changes in cal- cium homeostasis that can lead to a number of pathologi- cal states. RYR1 variants associated with susceptibility to malignant hyperthermia and central core disease are pri- marily dominant missense variants, and very few small deletions or duplications have been described (Klein et al. 2012). These variants produce hypersensitive channels (prone to activation by muscle ﬁber depolarization) as in malignant hyperthermia or leaky (Ca2+ dysregulation and depletion of Ca2+ from the sarcoplasmic reticulum) RYR1 channels as in classic central core disease (Hernandez- Ochoa et al. 2015). Autosomal recessive RYR1-related myopathies on the other hand, often result from a com- pound heterozygous missense variant in combination with a nonsense, splice-site, or frameshift variant (Klein et al. 2012). These variants can cause excitation-contrac- tion uncoupling in the severe recessive from of central core disease or loss of normal RYR1 expression as in mul- timinicore disease (Hernandez-Ochoa et al. 2015). As whole exome and whole genome sequencing are increas- ingly utilized, RYR1 variants are being identiﬁed more frequently and underlie a signiﬁcant proportion of neuro- muscular disease cases. ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Electromyography ﬁndings Nerve conduction studies of the left upper and lower limbs revealed a borderline ulnar sensory response peak latency. Concentric needle examination of selected left upper and lower limb muscles demonstrated rapid recruitment of short duration, low amplitude, complex motor unit potentials, indicating a diffuse myopathy without abnormal spontaneous activity. Echocardiogram ﬁndings A three-generation family pedigree was obtained (Fig. 2). No family members are known to have similar phenotype. One ﬁrst cousin has rhabdomyolysis and muscle disease; he is currently undergoing genetic testing. The paternal grandmother has an eye abnormality. An echocardiogram (ECG) was done to determine the eti- ology of tachycardia, hypertension, and suspected postural tachycardia (POTS). There was no evidence of POTS and left ventricular function was normal with a calculated ejection fraction of 69%. Her blood pressure is well con- trolled and the tachycardia is likely related to the severe deconditioning and restrictive pulmonary pattern seen in the patient. Neuromuscular pathology A biopsy of the right vastus lateralis muscle was per- formed when the patient was 21 years of age. The mus- cle ﬁbers varied pathologically from 5 to 100 micrometers in diameter. Fibers smaller than 25 micrometers occurred both singly and in small groups with up to ﬁve ﬁbers per group. There was a mild increase of internal nuclei. Rare ﬁbers were regenerating (Fig. 3A). No necrotic ﬁbers were observed. There was a mild focal increase in perimysial ﬁbrous connective tis- sue. In an NADH dehydrogenase reacted section, a few ﬁbers displayed irregularly circumscribed decreases of enzyme activity (Fig. 3B). Type 1 ﬁbers had a signiﬁ- cantly smaller mean diameter than type 2 ﬁbers, and all atrophic ﬁbers were histochemically type 1 (Fig. 3C). Based on these ﬁndings, the patient was given a diagno- sis of CFTD. Clinical description It is also known that cores become more prominent with age, and rebiopsy of patients later in life can lead to different pathological ﬁndings (Clarke et al. 2010). She also had multiple bone deformities involving her back, with mandibular abnormalities and severe scoliosis that required her to use a wheelchair. Secondary to her sco- liosis and chest wall deformities she developed restrictive lung disease, obstructive and central apnea, frequent pneu- monia, episodes of acute respiratory failure requiring mechanical ventilation, and hypertension. She also had epi- sodes of tachycardia and postural orthostatic tachycardia syndrome (POTS) was suspected. She had maxillary sur- gery due to facial dysmorphism and teeth misalignment (Fig. 1E). She also had dysplastic changes involving the pel- vis and acetabula for which she underwent a bilateral femoral osteotomy (Fig. 1F). The patient had rods and screw ﬁxation in the upper thoracic and lower lumbar spine In this report, we describe a patient who was diagnosed with a mitochondrial myopathy early in life. After a pro- tracted diagnostic odyssey, whole exome sequencing (WES) revealed novel compound heterozygous frameshift 296 ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. P. R. Blackburn et al. Exome Sequencing Reveals Two Novel RYR1 Variants Figure 1. Patient photographs and skeletal survey. Photographs show mild ptosis and slight facial dysmorphism (A, B). Kyphoscoliosis is evident (C, D). Patient had maxillary surgery due to facial dysmorphism and teeth misalignment. Bilateral femoral osteotomy was performed. Pronounced scoliosis of the thoracolumbar spine with postsurgical changes of the posterior rods and bilateral pedicle screws traversing the thoracic and lumbar spine are evident. Skeletal abnormalities were secondary to congenital myopathy. Figure 1. Patient photographs and skeletal survey. Photographs show mild ptosis and slight facial dysmorphism (A, B). Kyphoscoliosis is evident (C, D). Patient had maxillary surgery due to facial dysmorphism and teeth misalignment. Bilateral femoral osteotomy was performed. Pronounced scoliosis of the thoracolumbar spine with postsurgical changes of the posterior rods and bilateral pedicle screws traversing the thoracic and lumbar spine are evident. Skeletal abnormalities were secondary to congenital myopathy. at age 11 for her scoliosis (Fig. 1G,H). Since high school, she has not been able to ﬂex her neck and, due to weakness, must hold her head upright while she walks. Neuromuscular pathology Laboratory ﬁndings Laboratory studies including a mucopolysaccharide urine screen, urine organic acids, lactic acid, creatine kinase, acyl- carnitine proﬁle, and plasma amino acids were unrevealing. 297 ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. P. R. Blackburn et al. Exome Sequencing Reveals Two Novel RYR1 Variants Figure 2. Family Pedigree. A three-generation family pedigree showing the proband (arrow) and relatives. Note that each parent carries a different RYR1 variant. Both the proband’s mother and a maternal aunt have ﬁbromyalgia and a maternal cousin has an undiagnosed myopathy with rhabdomyolysis. Figure 3. Histologic ﬁndings. Note a few ﬁbers with internal nuclei and a regenerating ﬁber (arrow) (A); scattered ﬁbers display irregularly circumscribed attenuations of oxidative enzyme activity (B); type 1 ﬁbers have a smaller mean diameter than type 2 ﬁbers and all atrophic ﬁbers are type 1 (C). Section (A) is stained with hematoxylin and eosin, section (B) with NADH dehydrogenase, and section (C) is reacted for ATPase at pH = 4.3. Bars = 20 lm in (A), 50 lm in (B), and 100 lm in (C). q g Figure 2. Family Pedigree. A three-generation family pedigree showing the proband (arrow) Figure 2. Family Pedigree. A three-generation family pedigree showing the proband (arrow) and relatives. Note that each parent carries a different RYR1 variant. Both the proband’s mother and a maternal aunt have ﬁbromyalgia and a maternal cousin has an undiagnosed myopathy with rhabdomyolysis. Figure 2. Family Pedigree. A three-generation family pedigree showing the proband (arrow) and relatives. Note that each parent carries a different RYR1 variant. Both the proband’s mother and a maternal aunt have ﬁbromyalgia and a maternal cousin has an undiagnosed myopathy with rhabdomyolysis. Figure 3. Histologic ﬁndings. Note a few ﬁbers with internal nuclei and a regenerating ﬁber (arrow) (A); scattered ﬁbers display irregularly circumscribed attenuations of oxidative enzyme activity (B); type 1 ﬁbers have a smaller mean diameter than type 2 ﬁbers and all atrophic ﬁbers are type 1 (C). Section (A) is stained with hematoxylin and eosin, section (B) with NADH dehydrogenase, and section (C) is reacted for ATPase at pH = 4.3. Bars = 20 lm in (A), 50 lm in (B), and 100 lm in (C). Figure 3. Histologic ﬁndings. Laboratory ﬁndings Note a few ﬁbers with internal nuclei and a regenerating ﬁber (arrow) (A); scattered ﬁbers display irregularly circumscribed attenuations of oxidative enzyme activity (B); type 1 ﬁbers have a smaller mean diameter than type 2 ﬁbers and all atrophic ﬁbers are type 1 (C). Section (A) is stained with hematoxylin and eosin, section (B) with NADH dehydrogenase, and section (C) is reacted for ATPase at pH = 4.3. Bars = 20 lm in (A), 50 lm in (B), and 100 lm in (C). analyzed for sequence variants using a proprietary analysis tool (Xome Analyzer, GeneDx, Gaithersburg, MD, USA). Sanger sequencing was used to conﬁrm all potentially pathogenic variants identiﬁed in this individual and in the parental samples. Sequence alterations were reported according to the Human Genome Variation Society (HGVS) nomenclature guidelines. The exome was covered to a mean depth of 91x, with a quality threshold of 95.6%. Ethical compliance The patient and her parents consented for sample collec- tion and subsequent analysis under a protocol approved by the institutional review board of the Mayo Clinic. Consent was obtained from the patient to publish photographs. The clinical analysis for the proband included evaluation of variants that were identiﬁed to be de novo, compound heterozygous, homozygous, heterozygous and X-linked and prioritization of variants was based on the family structure and reported phenotype. Analysis in this case speciﬁcally included review of variants in genes asso- ciated with myopathy, mitochondrial disease, oxidative phophorylation deﬁciency, skeletal dysplasia, scoliosis, anemia, asthma, dysphagia, dyspnea, exercise intolerance, fatigue, facial dysmorphism, failure to thrive in infancy, hypotonia, global developmental delay, growth delay, Discussion As next generation sequencing becomes increasingly uti- lized in the clinic, more and more cases of RYR1-related congenital myopathy are being uncovered. RYR1 congenital myopathies are highly clinically variable and have a broad phenotypic spectrum with overlap with numerous other neuromuscular disorders with diverse molecular etiologies. These disorders can have neonatal or early childhood onset and the clinical manifestations may include neonatal hypo- tonia, delayed motor development, muscle weakness with feeding difﬁculty and failure to thrive in some cases (Beggs and Agrawal 2013). Scoliosis and secondary complications including respiratory impairment can also occur (Beggs and Agrawal 2013). The spectrum of RYR1-related myopa- thies is also expanding with the recent characterization of polyhydramnios and fetal akinesia leading to arthrogrypo- sis multiplex congenita, also known as lethal multiple pterygium syndrome (Kariminejad et al. 2016). Whole exome sequencing and next generation sequencing panels are extremely useful in unraveling the cause of complex neuromuscular disease and identifying the variants involved in a more efﬁcient and cost-effective manner as demonstrated in this case. ( ) ( j , ; ) Previous sequence and deletion/duplication analysis of the mitochondrial genome and 139 nuclear genes associ- ated with mitochondrial disorders (GeneDx) for this indi- vidual identiﬁed several heterozygous VUSs including a p.Leu46Phe homoplasmic VUS in mitochondrially encoded cytochrome C oxidase subunit II (MT-CO2, MIM# 516040) (NC_012920.1: m.7721C>T, NC_012920. 1: c.136C>T, p.COX2: Leu46Phe), a p.Met5Thr heterozy- gous VUS in seryl-tRNA ligase (SARS2, MIM# 612804) (Chr19(GRCh37): g.39421363A>G, NM_001145901.1: c.14T>C, NP_001139373.1: p.Met5Thr), a p.Thr139Arg heterozygous VUS in acylglycerol kinase (AGK, MIM# 610345) (Chr7(GRCh37): g.141313971C>G, NM_018238. 3: c.416C>G, NP_060708.1: p.Thr139Arg), a p.Ser447Thr heterozygous likely benign variant in translocase of inner mitochondrial membrane 44 homolog (TIMM44, MIM# 605058) (Chr19(GRCh37): g.7992091C>G, NM_006351.3: c.1340G>C, NP_006342.2: p.Ser447Thr, and a duplication of exons 1-4 in polyribonucleotide nucleotidyltransferase 1 (PNPT1, MIM# 610316) (Table 1). Through the integration of the clinical ﬁndings including muscle biopsy, genetic results, and other assessments, we were able to determine the correct underlying gene defect and pathological diagnosis of CFTD in this individual after a lengthy diagnostic odyssey. The two variants seen in this individual (p.Val2354del and p.Tyr1495X) have not been reported previously, but given her clinical presentation and pathologic diagnosis, they are likely to be pathogenic. Pathogenic variants in RYR1 are typically associated with core myopathies (Amburgey et al. 2013). However recent evidence suggests that up to 50% of recessive RYR1-related myopathies may exhibit non-core pathology (Amburgey et al. 2013). Results Whole exome sequencing (XomeDx, GeneDx) revealed a likely pathogenic 3 bp deletion (Chr19(GRCh37): g.38990307_38990309del, NM_000540.2: c.7060_7062del, NP_000531.2: p.Val2354del) in exon 44 of RYR1 resulting in the in frame deletion of a highly conserved valine residue within the intracellular calcium release channel domain and was inherited from the patient’s mother (Table 1). A second pathogenic deletion (Chr19(GRCh37): g.38969105_38969120del, NM_000540.2: c.4485_4500del, NP_000531.2: p.Tyr1495X) in RYR1 leads to a premature stop in exon 31 (SPIa/RYanodine Receptor SPRY domain) and was detected in the patient’s father (Table 1). A patient with a c.4485G>A missense variant that results in an identical premature stop codon (p.Tyr1495X) in RYR1 has been previously described in Klein et al. in an individ- ual who presented >10 years of age with proximal weak- ness, scoliosis, and muscle biopsy that revealed type 1 predominance in both cores and minicores (Klein et al. 2012). This individual also had two other missense variants of uncertain signiﬁcance (VUS) in RYR1 including c.1453A>G; p.Met485Val and c.325C>T; p.Arg109Trp. Nei- ther of our patient’s variants have been seen in approxi- mately 6500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project nor in the 60,706 unrelated individuals in the ExAC database (Table 1) (Project NGES, 2012; Lek et al. 2016). Exome Sequencing Reveals Two Novel RYR1 Variants headache, hypertension, joint hypermobility, malnutrition, muscle weakness, obstructive sleep apnea, central apnea, oligohydramnios, ophthalmoplegia, ptosis, orthostatic tachycardia, and tachycardia. Comparison of the mitochondrial and nuclear genome panel and the whole exome sequencing results showed the SARS2 variant to be paternally inherited and the AGK variant to be maternally inherited. No other potentially pathogenic variants were observed in the SARS2 gene at 100% coverage of the coding region of this gene or the AGK gene at 96.3% coverage. Due to the lack of pheno- typic overlap with these disorders, the RYR1 variants identiﬁed are likely responsible for the patient’s pheno- type (Table 1). Whole exome sequencing methodology Genomic DNA was extracted from blood from the pro- band, mother, and father. The Agilent Clinical Research Exome capture kit was used for exome enrichment and sequencing was done on an Illumina HiSeq 2000 that gen- erates 100 bp paired-end reads. Bi-directional sequence was assembled, aligned to reference gene sequences based on human genome build GRCh37/UCSC hg19, and 298 ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. P. R. Blackburn et al. Exome Sequencing Reveals Two Novel RYR1 Variants Discussion Multiminicore disease, centronuclear myopa- thy, and CFTD are inherited in a recessive manner (Amburgey et al. 2013). Most patients with RYR1-related myopathies present in infancy or early childhood and loss- of-function variants that result in reductions of RYR1 pro- tein levels correlate with a more severe clinical presentation 299 ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Table 1. Whole exome sequence and comprehensive mitochondrial nuclear gene panel results. Table showing the location of variants found in the proband including cDNA change, protein change, in silico prediction algorithm results, zygosity, mode of inheritance, association with disease, ACMG variant classiﬁcation, population frequency (ExAC and ESP), rs number, and ClinVar accession number. Test Gene RefSeq Accession Number cDNA change Protein Change SIFT PolyPhen-2 Mutation Taster2 Zygosity Inheritance Mode of Inheritance OMIM ACMG classiﬁcation ExAC Frequency ESP Frequency DbSNP ClinVar Accession GeneDx XomeDx/ Whole Exome Sequence Analysis RYR1 NM_000540.2 c.7060_7062del p.Val2354del N/A N/A N/A Heterozygous Maternal AD/AR Central core disease MIM: 117000; Neuromuscular disease, congenital, with uniform type 1 ﬁber MIM: 117000; King-Denborough syndrome MIM: 145600; Minicore myopathy with external ophthalmoplegia MIM: 255320; {Malignant hyperthermia susceptibility 1} MIM: 145600 Likely Pathogenic Variant N/R N/R N/R GeneDx XomeDx/ Whole Exome Sequence Analysis RYR1 NM_000540.2 c.4485_4500del p.Tyr1495X N/A N/A N/A Heterozygous Paternal AD/AR Central core disease MIM: 117000; Neuromuscular disease, congenital, with uniform type 1 ﬁber MIM: 117000; King-Denborough syndrome MIM: 145600; Minicore myopathy with external ophthalmoplegia MIM: 255320; {Malignant hyperthermia susceptibility 1} MIM: 145600 Pathogenic Variant N/R N/R N/R GeneDx Comprehensive Mitochondrial Nuclear Gene Panel MT-CO2 NC_012920.1 c.136C>T p.Leu46Phe Tolerated Probably Damaging N/A Homoplasmic N/A MT Cytochrome c oxidase deﬁciency MIM: 220110 N/R VUS N/A N/A N/R 300 ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodic Exome Sequencing Reveals Two Novel RYR1 Variants P. R. Blackburn Exome Sequencing Reveals Two Novel RYR1 Variants P. R. Blackburn et al. N/R ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. 300 P. R. Blackburn et al. Exome Sequencing Reveals Two Novel RYR1 Variants Table 1. Continued. ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. References Amburgey, K., A. Bailey, J. H. Hwang, M. A. Tarnopolsky, C. G. B€onnemann, L. Medne, et al. 2013. Genotype-phenotype correlations in recessive RYR1-related myopathies. Orphanet J. Rare Dis. 8:117. The p.Tyr1495X truncating variant uncovered in our patient likely undergoes nonsense mediated decay or results in a severely truncated non-functional protein. The presence of CFTD (a non-core pathology) found in this patient is in line with these previously observed asso- ciations. The p.Val2354del (3 bp in-frame deletion) observed in our patient likely results in reduced RYR1 function, but additional studies will need to be done to conﬁrm this. Several missense variants have been observed in neighboring residues adjacent to p.Val2354 in individuals with malignant hyperthermia and a 3 bp in- frame deletion of a single residue (p.Glu2347) has been observed in two unrelated malignant hyperthermia-sus- ceptible families (Sambuughin et al. 2001). The p.Glu2347 in-frame deletion in these families was evalu- ated using in vitro skeletal-muscle-contracture testing and found to produce a large electrically evoked contraction tension, suggesting that this small deletion leads to a sig- niﬁcant alteration in skeletal-muscle calcium regulation (Sambuughin et al. 2001). Based on the proximity of the patient’s variant to other previously reported variants associated with malignant hyperthermia, we recom- mended that precautions be taken for the patient and her mother to avoid this anesthetic-drug-induced hyperme- tabolic syndrome (Sambuughin et al. 2001). Beggs, A. H., and P. B. Agrawal. 2013. Multiminicore disease. In: R. A. Pagon, M. P. Adam, H. H. Ardinger, et al., eds. GeneReviews [Internet]. University of Washington, Seattle, WA; 1993–2017. Available from: https://www.ncbi.nlm.nih. gov/books/NBK1290/ (Accessed 2013 Jan 24). gov/books/NBK1290/ (Accessed 2013 Jan 24). B€onnemann, C. G., C. H. Wang, S. Quijano-Roy, N. Deconinck, E. Bertini, A. Ferreiro, et al. 2014. Diagnostic approach to the congenital muscular dystrophies. Neuromuscul. Disord. 24:289–311. Neuromuscul. Disord. 24:289–311. Clarke, N. F., L. B. Waddell, S. T. Cooper, M. Perry, R. L. L. Smith, A. J. Kornberg, et al. 2010. Recessive mutations in RYR1 are a common cause of congenital ﬁber type disproportion. Hum. Mutat. 31:E1544–E1550. Dowling, J. J., S. Arbogast, J. Hur, D. D. Nelson, A. McEvoy, T. Waugh, et al. 2012. Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy. Brain 135:1115–1127. Hernandez-Ochoa, E. O., S. J. P. Pratt, R. M. Lovering, and M. F. Schneider. 2015. Critical role of intracellular RyR1 calcium release channels in skeletal muscle function and disease. Front. Physiol. 1:420. Jungbluth, H. Acknowledgments The authors would like to thank the patient and her fam- ily for their willingness to participate in our study. Sambuughin, N., S. McWilliams, A. De Bantel, K. Sivakumar, and T. E. Nelson. 2001. Single-amino-acid deletion in the RYR1 gene, associated with malignant hyperthermia susceptibility and unusual contraction phenotype. Am. J. Hum. Genet. 69:204–208. Discussion Test Gene RefSeq Accession Number cDNA change Protein Change SIFT PolyPhen-2 Mutation Taster2 Zygosity Inheritance Mode of Inheritance OMIM ACMG classiﬁcation ExAC Frequency ESP Frequency DbSNP ClinVar Accession GeneDx Comprehensive Mitochondrial Nuclear Gene Panel SARS2 NM_ 001145901.1 c.14T>C p.Met5Thr Tolerated Benign Polymorphism Heterozygous Paternal AR Hyperuricemia, pulmonary hypertension, renal failure, and alkalosis, HUPRA Syndrome MIM: 613845 VUS 0.00000008 N/R rs538446780 N/R GeneDx Comprehensive Mitochondrial Nuclear Gene Panel AGK NM_018238.3 c.416C>G p.Thr139Arg Tolerated Benign Polymorphism Heterozygous Maternal AR Autosomal recessive cataract 38 MIM: 614691; Sengers syndrome MIM: 212350 VUS 0.00000448 EA: G = 0.00% – AA: G = 0.43% rs144706178 N/R GeneDx Comprehensive Mitochondrial Nuclear Gene Panel TIMM44 NM_006351.3 c.1340G>C p.Ser447Thr Tolerated Benign Disease causing Heterozygous N/A N/R N/R VUS 0.00001612 EA: G = 0.00% – AA: G = 1.79% rs7257461 N/R GeneDx Comprehensive Mitochondrial Nuclear Gene Panel PNPT1 NM_033109.4 N/A N/A N/A N/A N/A Het N/A AR Combined oxidative phosphorylation deﬁciency 13 MIM: 614932; autosomal recessive deafness 70 MIM: 614934 VUS N/R N/R N/R N/A, not available; N/R, not reported; VUS, variant of uncertain signiﬁcance. Genetics & Genomic Medicine published by Wiley Periodicals, Inc. N/A, not available; N/R, not reported; VUS, variant of uncertain signiﬁcance. 301 Exome Sequencing Reveals Two Novel RYR1 Variants P. R. Blackburn et al. in patients (Dowling et al. 2012; Amburgey et al. 2013). Non-central core pathology also seems to correlate with reduced RYR1 protein expression (Amburgey et al. 2013). References 2007. Multi-minicore disease. Orphanet J. Rare Dis. 2:31. Despite RYR1-related congenital myopathy being slowly progressive or static in most cases, some patients may expe- rience decreased respiratory function or even respiratory failure, signiﬁcant cardiac impairment, and increasing axial and proximal muscle weakness, as was the case in the patient described in this report. Currently only supportive care for respiratory symptoms, physical therapy for muscle weakness and contractures, and orthopedic correction are used to manage the disorder, but new treatment options may be on the horizon. The patient described in this report was recently enrolled in a phase I and phase II efﬁcacy study of N-acetylcysteine for the treatment of RYR1-conge- nital myopathy at the National Institutes of Health in Bethesda, MD. Kariminejad, A., S. Ghaderi-Sohi, H. H-N Nedai, V. Varasteh, A.-R. Moslemi, and H. Tajsharghi. 2016. Lethal multiple pterygium syndrome, the extreme end of the RYR1 spectrum. BMC Musculoskelet. Disord. 17:109. Klein, A., S. Lillis, I. Munteanu, M. Scoto, H. Zhou, R. Quinlivan, et al. 2012. Clinical and genetic ﬁndings in a large cohort of patients with ryanodine receptor 1 gene- associated myopathies. Hum. Mutat. 33:981–988. Lek, M., K. J. Karczewski, E. V. Minikel, K. E. Samocha, E. Banks, T. Fennell, et al. 2016. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536:285–291. North, K. N., C. H. Wang, N. Clarke, H. Jungbluth, M. Vainzof, J. J. Dowling, et al. 2014. Approach to the diagnosis of congenital myopathies. Neuromuscul. Disord. 24:97–116. Project NGES. 2012. Exome variant server. NHLBI GO Exome Sequencing Project (ESP), Seattle, WA. Conﬂicts of Interest The authors have no conﬂicts of interest. 302 ª 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.
https://openalex.org/W3029165146	https://publikationen.bibliothek.kit.edu/1000120605/80097446	English	null	Surface-Energy-Balance Closure over Land: A Review	Boundary-layer meteorology/Boundary - layer meteorology	2,020	cc-by	18,862	* Matthias Mauder matthias.mauder@kit.edu Abstract Quantitative knowledge of the surface energy balance is essential for the prediction of weather and climate. However, a multitude of studies from around the world indicate that the turbulent heat fluxes are generally underestimated using eddy-covariance measure- ments, and hence, the energy balance is not closed. This energy-balance-closure problem, which has been heavily covered in the literature for more than 25 years, is the topic of the present review, in which we provide an overview of the potential reason for the lack of closure. We demonstrate the effects of the diurnal cycle on the energy balance closure, and address questions with regard to the partitioning of the energy balance residual between the sensible and the latent fluxes, and whether the magnitude of the flux underestimation can be predicted based on other variables typically measured at micrometeorological stations. Remaining open questions are discussed and potential avenues for future research on this topic are laid out. Integrated studies, combining multi-tower experiments and scale-cross- ing, spatially-resolving lidar and airborne measurements with high-resolution large-eddy simulations, are considered to be of critical importance for enhancing our understanding of the underlying transport processes in the atmospheric boundary layer. Keywords Eddy covariance · Energy balance closure · Large-eddy simulation · Secondary circulations · Surface energy balance Matthias Mauder1 · Thomas Foken2 · Joan Cuxart3 Matthias Mauder1 · Thomas Foken2 · Joan Cuxart3 Received: 25 November 2019 / Accepted: 2 May 2020 © The Author(s) 2020 Received: 25 November 2019 / Accepted: 2 May 2020 © The Author(s) 2020 Boundary-Layer Meteorology https://doi.org/10.1007/s10546-020-00529-6 Boundary-Layer Meteorology https://doi.org/10.1007/s10546-020-00529-6 Boundary-Layer Meteorology y y y https://doi.org/10.1007/s10546-020-00529-6 RESEARCH ARTICLE 1 Institute of Meteorology and Climate Research, Karlsruhe Institute of Technology, 82467 Garmisch‑ Partenkirchen, Germany 3 Department of Physics, University of the Balearic Islands, 07122 Palma, Mallorca, Spain 1.1 The Surface Energy Balance and Its Relevance The surface energy balance (SEB) is an essential cornerstone of any theoretical description of the Earth’s climate system. It can be assessed locally on the ecosystem scale if the turbu- lent heat fluxes, the soil heat flux and net radiation are measured independently, 2 Bayreuth Center of Ecology and Environmental Research, University of Bayreuth, 95440 Bayreuth, Germany 3 Department of Physics, University of the Balearic Islands, 07122 Palma, Mallorca, Spain 3 Department of Physics, University of the Balearic Islands, 07122 Palma, Mallorca, Spain 123456789) 1 3 23456789) 1 3 M. Mauder et al. (1) H + 휆E + G + Imb = Rn, H + 휆E + G + Imb = Rn, (1) where H is the sensible heat flux, λE is the latent heat flux, both of which are most directly measured using the eddy-covariance (EC) technique, G is the soil heat flux at the surface, which can be quantified by a combination of heat-flux plates, soil temperature, and soil water content sensors (energy storage in the canopy can potentially also be included here), and Rn is net radiation, which is ideally measured by a high-quality four-component net radiometer. The imbalance (Imb) is considered as the total contribution of neglected effects and uncertainties. All terms on the left-hand side of Eq. 1 are defined positive for transfer- ring energy away from the surface, while Rn is traditionally defined positive in the daytime.i i This budget equation is to be ideally applied for an interface of infinitesimal depth between the atmosphere and the surface. However, constraints on how the different terms of the budget are measured make it more convenient to define the energy budget for a vol- ume surrounding the interface. There is a correspondence between the evolution equation of the temperature of this volume and the SEB equation, as shown for instance in Cuxart et al. (2015). Hence, the term Imb includes at least, (1) the tendency and advection terms, respectively, related to surface heterogeneities and non-stationarity; (2) natural and anthro- pogenic thermal processes, including heat storage, plant metabolism or urban-induced ther- mal processes; (3) the vertical divergences of the turbulent heat fluxes or the net radiation within the volume of interest, and (4) the uncertainties related to the experimental display. 1.1 The Surface Energy Balance and Its Relevance Therefore, strictly speaking, closure of the SEB can only be expected under the assump- tion of a horizontally uniform two-dimensional exchange surface without a canopy, while the fluxes are perpendicular to this surface. These conditions are assumed to be fulfilled in commonly used numerical land-surface models. In spite of these shortcomings, accu- rate field measurements of the SEB are essential for the calibration and validation of land- surface models. Nevertheless, when comparing observed and modelled energy fluxes, the uncertainty of the observed values has to be considered, since models have a closed SEB in contrast to the observations. It is important that these models provide a realistic representation of the underlying pro- cesses, especially for seasonal predictions and climate simulations, where land–atmosphere feedbacks play a critical role (Arneth et al. 2012; Green et al. 2017). In order to address the discrepancies in energy balance closure between models and observations, already Sell- ers et al. (1989) included a correction factor for the evaporation fraction of the turbulent fluxes in his simple biosphere model. Kracher et al. (2009) investigated how different land- surface models compare to field measurements of the energy balance components. They found that if the model uses the surface temperature to calculate all terms of the energy balance iteratively the resulting fluxes agree quite well with the EC data corrected under the assumption that the Bowen ratio is preserved. Some land-surface models compute explicitly H and λE at the surface and use Rn pro- vided by the radiation scheme, obtaining G as the residual of those three terms. In the case of realistic values for 휆E, H and Rn when compared to observations, this approach implies that the residual is added to G, resulting in too high values of this flux and exces- sive heat transport into the ground (as shown in Cuxart et al. 2015 for the ECMWF model). Similarly, some remote-sensing applications estimate H, Rn and G, and obtain λE as the residual, also very often overestimating the value of evapotranspiration (see the review of Liou and Kar 2014). Similar problems occur when EC measurements are used for the validation of biological models, e.g. to calculate the surface conductance of water, which also implicitly assume a closed energy balance (Wohlfahrt et al. 2009). 1.2 Historical Development and Description of the Problem The first indications of a non-closed energy balance became obvious in the comparison of EC measurements with Bowen-ratio measurements; the latter close the energy balance by definition. Similar results were found in experiments in Australia in 1981 (Leuning et al. 1982) and in June of the years 1982, 1983, and 1984 in Germany (Koitzsch et al. 1988). In both experiments net radiometers were used that were highly accurate for the 1980s, the Funk (1959) and the Sonntag (1975) devices, respectively (see also Sect. 2.1). In both stud- ies problems of the EC method were postulated. Desjardins (1985) aimed to measure pri- marily CO2 fluxes and found an underestimation in the turbulent energy fluxes in compari- son to the available energy. During an experiment at Kursk (Russia) in 1988 an unclosed energy balance was found, initially not explicitly published (Tsvang et al. 1991), but then analyzed by Foken (1990) and later by Panin et al. (1998). Nevertheless, an investigation of the SEB closure was formulated as the aim of a field experiment near Tartu (Estonia) in 1990 (Foken et al. 1993) and the follow-up experiment at Kursk 1991 (Panin et al. 1998). G. N. Panin was also involved in the First International Satellite Land Surface Climatol- ogy Project (ISLSCP) Field Experiment (FIFE) in Kansas, USA in 1989, and found an unclosed energy balance there too (Kanemasu et al. 1992), while a closed energy balance was reported in other published FIFE studies. Further pioneering work on the SEB closure problem was conducted by Blanford et al. (1991) and Bernhofer (1992a), who were the first to explain the flux underestimation with large-scale non-turbulent transport mechanisms. About ten years later, the problem was addressed through ecological networks (Aubinet et al. 2000; Wilson et al. 2002) and a first correction was proposed for general flux meas- urements (Twine et al. 2000). At the beginning of the 1990s, T. Foken submitted an overview article on the problem, but this was rejected because reviewers doubted the accurate calibration of the instruments. An updated version was later published (Foken 1998). A first international workshop addressing the problem was held in Grenoble in 1994 (Foken and Oncley 1995). During this workshop, the idea of the first concerted field experiment to address the problem was born, which then actually took place in California in 2000, in the framework of the Energy- Balance Experiment (EBEX-2000) (Oncley et al. 2007). 1.1 The Surface Energy Balance and Its Relevance Another important question in biological 1 3 Surface‑Energy‑Balance Closure over Land: A Review and ecological research is whether trace gas fluxes measured by eddy correlation are equally affected by a related systematic error (Wang 2009; Foken et al. 2011). A study on a large number of FLUXNET sites suggests that there is a link between the energy imbal- ance and CO2 fluxes (Wilson et al. 2002). For a given value of the photosynthetically active radiation, the magnitude of CO2 uptake was less when the energy imbalance was greater. Similarly, respiration (estimated by nocturnal CO2 release to the atmosphere) was signifi- cantly less when the energy imbalance was greater.i The journal Boundary-Layer Meteorology has played a prominent role in the scientific discussion of the SEB closure problem over the last 25 years. It has provided a platform for highly innovative, pioneering and ground-breaking work, particularly related to atmos- pheric transport processes and EC methodology: see Table 1 for highly cited articles. 1.3 Analysis of Energy Balance Closure at Multiple Sites Studies on the energy balance closure are particularly valuable when datasets from multi- ple sites are analyzed, in order to generalize the problem and to extract common underlying reasons. At a single arbitrary site, a large variety of errors can cause non-closure of the energy balance. Hence, a single-site SEB closure analysis may provide insights on how to optimize the instrumental set-up. However, the phenomenon of a general systematic behav- iour becomes only apparent in an analysis of data from many sites with different character- istics, e.g., with respect to instrumentation, canopy structure, and atmospheric conditions. For example, Panin et al. (1998) investigated several sites in North America and Europe, and found a relation with surface inhomogeneity around the EC measurements. Moreover, Wilson et al. (2002) found a clear relationship between SEB closure and friction velocity u∗ for 22 FLUXNET (Baldocchi et al. 2001) sites. An investigation on the SEB closure of eight ChinaFLUX (Li et al. 2005) sites discusses certain factors that contribute to the imbalance of energy, such as systematic errors associated with the mismatch of sampling areas, systematic instrument bias, neglected energy sinks, low and high frequency losses of turbulent fluxes and advection of heat and water vapour. A dependence of SEB clo- sure on atmospheric stability, in particular the flux Richardson number, was found by Stoy et al. (2006) for a successional chronosequence in the south-eastern United States. A simi- lar response of the SEB closure to u∗ and atmospheric stability is described by Barr et al. (2006) for several Canadian sites and by Hendricks-Franssen et al. (2010) for several Euro- pean FLUXNET sites. Based on multi-site analyses, surface heterogeneity on the landscape scale beyond the actual flux footprint has been found to play an important role (Mauder et al. 2007c; Panin and Bernhofer 2008), because this scale is relevant to the formation of secondary circula- tions, as shown, e.g., for the LITFASS-2003 (Lindenberg Inhomogeneous Terrain—Fluxes between Atmosphere and Surface: a Long-term Study) multi-site experiment (Foken et al. 2010). This hypothesis is supported by fluxes derived from area-averaging measurement techniques, i.e. scintillometers and aircraft, which measure larger heat fluxes than EC tow- ers in the same area (Meijninger et al. 2006; Xu et al. 2017a). The relationship with the friction velocity u∗ and landscape-scale heterogeneity is confirmed by the most compre- hensive analysis of SEB closure for 173 FLUXNET sites around the world (Stoy et al. 1 It should not be overlooked that this topic has even influenced popular culture through the publication of the song “Energy Balance Closure Blues”, which was written and composed by Henk de Bruin and inter- preted by Klaus Blume (https://soundcloud.com/klaus-blume/energy-balance-closure-blues). 1.2 Historical Development and Description of the Problem Based on these results and many 3 1 M. Mauder et al. other studies, the first peer-reviewed review article on the SEB closure problem was pub- lished (Foken 2008), stressing the importance of secondary circulations.1 1.3 Analysis of Energy Balance Closure at Multiple Sites 2013). In addition, it was demonstrated that an accurate measurement of the storage term is important for closing the surface energy balance for tall vegetation canopies, particularly at half-hourly time scales (Leuning et al. 2012), under the condition that all sources of meas- urement and data processing errors in the EC system are minimized (Xu et al. 2019). 1 1 3 3 Surface‑Energy‑Balance Closure over Land: A Review Instrument error Systematic error of soil heat flux Systematic error of radiation measurement Systematic error of sonic anemometers Systematic error of humidity measurement Mismatch of footprints Data processing error Systematic error of correction algorithms Uncertainty due to averaging operator Additional sources of energy Canopy heat storage Bio-chemical energy storage (photosynthesis) Potential energy of water sub-mesoscale transport/secondary circulations Horizontal flux divergence Vertical/horizontal advection Fig. 1 Overview and classification of hypothesis for the underlying reasons of the SEB closure problem Fig. 2 Schematic view on the measurement area of the differ- ent parts of the energy balance. Modified after Foken (2017), first published in 2003, © Springer Nature Fig. 2 Schematic view on the measurement area of the differ- ent parts of the energy balance. Modified after Foken (2017), first published in 2003, © Springer Nature Fig. 2 Schematic view on the measurement area of the differ- ent parts of the energy balance. Modified after Foken (2017), first published in 2003, © Springer Nature 1.4 Overarching Research Question Based on the extensive amount of literature available on the SEB closure problem, we structure our review based on the following overarching research questions: • What are the reasons/underlying processes for the non-closure?f • What are the reasons/underlying processes for the non-closure?f • Is the closure problem different in daytime and nighttime conditions?fl f • How should the residual be partitioned (including effects on other scalar fluxes)? f • How can one predict the magnitude of the energy balance residual? 2.1 Instrumental Errors Instrumental errors must be discussed in the context of the measurement area that is represented by the sensors, ranging from horizontal scales of 0.1 m for the ground heat flux, 10 m for the net radiation, and up to several 100 m for the turbulent fluxes (Schmid and Oke 1990; Schmid 1997; Foken 2008), as illustrated in Fig. 2. To over- come this problem, the surface characteristics below the net radiometer should be similar to the surface in the flux footprint of the EC system, and the ground heat flux should be measured under the same condition; more than one sensor is highly recom- mended. In addition, the EC footprint should be determined for each averaging inter- val, since it varies with wind speed, stratification and boundary-layer depth. However, this effect should not lead to a general systematic underclosure but rather can be con- sidered as a random error for most sites or only a small bias (Richardson et al. 2012). 2 Hypotheses for the Underlying Reason The hypotheses for the underlying reasons of the SEB closure problem can be grouped in four major topics: instrumental errors, data processing errors, usually neglected additional terms of the SEB equation, and sub-mesoscale transport processes, which contradict the theoretical assumptions of tower-based EC measurements, and therefore are inherently not captured (Fig. 1). 3 3 M. Mauder et al. 2.1.2 Hygrometers There was a significant change in the application of optical hygrometers around the year 2000; before 2000, most of the instruments were institutional fabrications or from small companies. Foken et al. (1995) gave an overview of the developments of ultraviolet and infrared hygrometers. If the instruments were carefully calibrated, no significant influence on the energy balance closure and bias to recent sensors can be expected. However, the fre- quently-used Lyman-alpha hygrometers were not very stable. This issue can be controlled by an additional humidity measurement and re-calibration. For field calibrations of these ultraviolet sensors, a method with changing pathlength was developed that can be applied under nearly constant absolute humidity for Lyman-alpha hygrometers (Foken et al. 1998) or under constant pressure conditions for Krypton hygrometers (Foken and Falke 2012) because Krypton hygrometers are affected by an oxygen cross-sensitivity (Tanner and Campbell 1985). A prototype of the latter system was applied during EBEX-2000. Because ultraviolet hygrometers are nearly no longer in use, the calibration techniques are not applied any more. Nevertheless, ultraviolet hygrometers have the advantage that they are very sensitive and therefore only require a very short pathlength on the order of 0.01 m, while the pathlength of an infrared hygrometer is typically on the order of 0.1 m.i The first comparisons of the Krypton hygrometer with the infrared open-path hygrom- eter LI-7500, which is currently considered the reference sensor, were conducted during EBEX-2000 (Mauder et al. 2007d). Since then, infrared hygrometers have almost com- pletely replaced the ultraviolet hygrometers because they have a number of advantages, such as superior long-term stability and the additional measurement of CO2 concentration. However, a larger pathlength is needed for infrared hygrometers in comparison with ultra- violet hygrometers, which leads to spectral losses. Closed-path hygrometers are afflicted with additional low-pass filtering effects due to tube dampening (Haslwanter et al. 2009). These spectral losses are corrected effectively as a function of relative humidity (Ibrom et al. 2007; Fratini et al. 2012) as long as the error is not too large. Therefore, closed-path gas sampling systems need to be carefully designed and maintained (Aubinet et al. 2016; Metzger et al. 2016). 2.1.1 Sonic Anemometers Sonic anemometers are the core instrument for EC measurements. Therefore, research- ers intensively investigated their response characteristics to determine whether these instruments explain a larger part of the SEB closure problem. Based on the results of intercomparison experiments, error estimates were assigned to several types of sonic anemometers, which had been classified into two groups during the 1994 SEB clo- sure workshop in Grenoble (Foken and Oncley 1995; Mauder et al. 2006). However, no general systematic biases were found here for the standard deviation of the verti- cal velocity, which would translate into a bias of energy fluxes. In parallel, several researchers found a larger systematic error for certain wind-tunnel experiments on the order of 10% of the flux, which is comparable to the magnitude of imbalances of many sites (van der Molen et al. 2004; Nakai et al. 2006). The term angle-of-attack correc- tion was coined for the proposed adjustment method. Nevertheless, doubts were raised whether these corrections determined under quasi-laminar wind-tunnel conditions are transferable to real-world turbulence (Högström and Smedman 2004), because the extent of wakes behind an obstacle is much shorter for conditions of high turbulence intensity (Wyngaard 1981; Rodríguez et al. 2015). This topic was also addressed as part of the EBEX-2000 campaign by a comprehen- sive intercomparison experiment, with the result that there were some characteristic differences between different sonic anemometers, but none of them was sufficiently large and systematic to explain the magnitude of the energy balance residual (Mauder et al. 2007d). To date, the discussion in the literature about possible systematic errors of flux measurements using sonic anemometers is still ongoing (Kochendorfer et al. 2012; Horst et al. 2015; Frank et al. 2016), indicating that the error might be on the order of 3–5% for the energy fluxes. New experimental designs have recently been explored to employ additional independent reference estimates for sonic anemometers, particularly under turbulent conditions, such as large-eddy simulation (LES, Huq et al. 2017), spectral ratios in the inertial sub-range (Peña et al. 2019), and high-resolution Doppler lidar (Mauder et al. 2020). While the absolute accuracy of sonic anemom- eters is still not fully quantified, it was at least established that the precision of various types of modern sonic anemometers is very good regarding general flux measurements (Mauder and Zeeman 2018). 1 3 Surface‑Energy‑Balance Closure over Land: A Review 2.1.4 Ground‑Heat‑Flux Measurements In the beginning of the investigations on the lack of energy balance closure, the ground heat flux was discussed as a potential reason for the lack of closure because of its large relative errors (Culf et al. 2004). A frequently-found asymmetry of the closure between the morning and afternoon hours supported this assumption. Kukharets and Tsvang (1999) and Kukharets et al. (2000) investigated possible errors mainly of the heat storage in the upper soil layer, with a special focus on the time shift between the air temperature and the surface temperature changes, and changes of the soil heat capacity due to a changing soil water content (Peters-Lidard et al. 1998).fl Liebethal et al. (2005) conducted a sensitivity analysis on different ground-heat-flux measurement techniques and concluded that heat-flux plates should be installed at a depth of about 0.2 m and the heat storage should be calculated from a high-resolution tempera- ture profile with associated soil moisture measurements. Furthermore, Liebethal and Foken (2007) compared different parametrization approaches for the ground heat flux with the results that a simplified measurement and force-restore method best approximates the measurements. In contrast to these mostly calorimetric approaches, Heusinkveld et al. (2004) developed an harmonic approach for homogeneous soils. Both approaches can reduce the residual of the energy balance, the harmonic approach with a larger effect, but it cannot solve the general problem (Jacobs et al. 2008). Nevertheless, for some sites over flat and homogeneous terrain, the energy balance was nearly closed when the ground heat flux was carefully calculated (Kabat et al. 1997; Heusinkveld et al. 2004; Mauder et al. 2007c). For weather conditions not evolving drastically, the integration of the ground heat flux over a full diurnal cycle should give a value close to zero. Thus, omitting G and integrating Eq. 1 over one or several days might help to characterize the imbalance term apart from the potential errors related to G. 2.1.3 Net Radiometers The reason that the non-closed surface energy balance was not found already in the 1970s and 1980s was the lack of accurate net radiometers, which was shown by Halldin and Lin- droth (1992). They compared different net radiometers with the Schulze-Däke instrument with polyethylene (Lupolen®) dome (Däke 1972) that was one of the best instruments at that time (the Sonntag (1975) instrument was similarly constructed). It was found that the Funk (1959) net radiometer underestimated net radiation only by a few percent but the widely used Fritschen (1963) net radiometer underestimated net radiation in the same order as the residual of the energy balance. Therefore, the SEB closure problem was only found in experiments where the Fritschen instrument was not used. During the EBEX-2000 experiment several types of instruments were compared, among them also the Schulze-Däke and the Q7 Fritschen type net radiometers (Kohsiek et al. 2007). The Schulze-Däke agreed well with the widely used Kipp&Zonen CNR1, which is now mostly replaced by the ventilated CNR4 with improved accuracy. Again, the Q7 radiometer significantly underestimated net radiation. A similar underestimation was also 1 3 3 M. Mauder et al. found for the NR-lite instrument (Brotzge and Duchon 2000). In general, careful and fre- quent cleaning of the sensors was found to be essential for high-quality data. Nevertheless, the relative uncertainty of net radiation sensors can still be considerable, i.e. 10% already under favourable conditions and larger percentages at night when absolute radiative fluxes are small. However, this should normally not lead to a systematic error. Since the beginning of the 1990s, much progress was made in radiation measurements (Ohmura et al. 1998), and the measurement of shortwave radiation (pyranometer) is now often separated from longwave radiation (pyrgeometer). The latest state of the art is docu- mented in a recent ISO guideline (ISO 2018). 2.2.1 Averaging Time It is a fundamental assumption for EC measurements that the mean vertical wind compo- nent over the averaging period should be zero. In the past, different methods were applied to achieve this condition: a running mean filter (McMillen 1988), linear detrending (Rannik and Vesala 1999), or block averaging, often over 30 or 60 min, (Kaimal and Finnigan 1994; Finnigan et al. 2003; Wang 2010). It is important to note that all of these averaging opera- tors act as high-pass filters. Sometimes, a clear spectral gap is found between the frequencies of atmospheric motions, which however often appears not to be as evident, especially above 1 3 Surface‑Energy‑Balance Closure over Land: A Review rough surfaces and in unstable stratification. Some scientists prefer using block averaging by calculating the arithmetic mean over a certain time period, in order to avoid potential conflicts with the Reynolds decomposition rules (Aubinet et al. 2012). Nevertheless, if low-frequency contributions beyond the averaging interval are significant, and if no appropriate high-pass filtering correction is applied, this may produce an underestimation of the total flux. rough surfaces and in unstable stratification. Some scientists prefer using block averaging by calculating the arithmetic mean over a certain time period, in order to avoid potential conflicts with the Reynolds decomposition rules (Aubinet et al. 2012). Nevertheless, if low-frequency contributions beyond the averaging interval are significant, and if no appropriate high-pass filtering correction is applied, this may produce an underestimation of the total flux. il Wavelet methods are useful to determining the relevant scales in the covariances, allow- ing determination whether a spectral gap is present (Terradellas et al. 2001). The so-called ogive method (Desjardins et al. 1989; Oncley et al. 1990) is often employed to check the low- frequency part of the cospectra for significant flux contributions. This was also done by Foken et al. (2006) to investigate the SEB closure problem with the conclusion that an extended aver- aging time would lead to larger fluxes in some cases. l Finnigan et al. (2003) proposed a long-term integration over several days to close the energy balance. This was also shown for another dataset by Mauder et al. (2006) with the result that most of the missing energy would be added to the sensible heat flux. 2.2.2 Flux Corrections An ideal EC system would be able to measure the vertical wind fluctuations and the scalar to be transported at a single point at the same instant and in units that do not change with changes of atmospheric pressure. In reality, these conditions are not completely fulfilled and therefore a number of so-called flux corrections, which are sometimes merely unit conver- sions, are required (Foken et al. 2012). The basis for accurate flux estimates is a consensus about a suite of required corrections, which has been laid out in the Handbook of Micromete- orology (Lee et al. 2004). Nevertheless, different choices of methods to correct for the same effect and different implementations in a certain software package can lead to small discrepan- cies in the resulting fluxes (Fratini and Mauder 2014).ffl l The effect of different flux corrections on the SEB closure was investigated by Mauder et al. (2006) for the LITFASS-2003 field campaign, which was carried out over a heterogene- ous but flat area in central Europe. In addition, Mauder et al. (2007d) also compared different EC processing software from different institutions and countries as part of EBEX-2000. These studies conclude that it is important to apply the appropriate corrections in the right way, but the potential discrepancies in correction algorithms cannot explain the observed general lack of SEB closure. 2.2.1 Averaging Time The problem of this method is that the averaging time needs to be on the order of days, and within this period no significant changes of the weather condition should occur, in order to fulfil the sta- tionarity requirement. The study of Mauder et al. (2006) was later extended to other sites of the same field campaign (Charuchittipan et al. 2014) with the result that SEB closure does not improve in the same way for different land-use classes. The observed remaining lack of clo- sure even for very long averaging times can be explained because large-scale eddies often do not propagate with the mean wind; hence they violate Taylor’s frozen turbulence hypothesis, and therefore inherently cannot be fully captured by single-tower measurements (Mahrt 2010). 2.3 Additional, Normally Neglected, Terms There is a series of additional energy balance terms, which are often, but not always, insig- nificant compared to the error margins of flux measurements (Eq. 1). In the framework of EBEX-2000, the following aspects were investigated (Oncley et al. 2007): 3 1 M. Mauder et al. • Heat storage in the canopy • Biochemical storage in the canopy due to photosynthesis and respiration • Vertical and horizontal flux divergence • Horizontal advection • Water pumping by the plants • Heat storage in the canopy • Biochemical storage in the canopy due to photosynthesis and respiration • Vertical and horizontal flux divergence • Horizontal advection • Water pumping by the plants • Heat storage in the canopy • Biochemical storage in the canopy due to photosynthesis and respiration • Vertical and horizontal flux divergence • Horizontal advection • Water pumping by the plants Accounting for all these additional terms did indeed improve the energy balance closure for EBEX-2000, although they were often difficult to estimate with the available meas- urements. Nevertheless, despite this effort, the EBEX dataset still shows an imbalance on the order of 10% of the available energy that could not be explained for this rather low- vegetation measurement site (cotton). The values in crops of the canopy heat storage and biochemical processes added are typically less than 10 W m−2 (Oke 2002). Water pumping by plants is driven by suction at the stomata in the leaves and osmotic pressure at the root level, and the involved energy being related to these processes at the cell level must be added. For tall vegetation sites, especially forests, it is clear that the above-ground heat storage can play an important role for the SEB closure. This has been demonstrated for a number of sites in different climate zones (Haverd et al. 2007; Moderow et al. 2009; Lindroth et al. 2010). Leuning et al. (2012) showed that including the above-ground heat storage can even lead to a satisfactory energy balance closure for several largely homogeneous forest sites in Australia, where advective fluxes are probably small. l The energy converted into biochemical storage through photosynthesis is typically only a few percent of the incoming shortwave radiation. In order to determine this term, gross primary productivity of the plants needs to be quantified and then converted into energetic units. Oncley et al. 2.3 Additional, Normally Neglected, Terms (2007) did this for EBEX-2000 and report a daily average of 8 W m−2 for a cotton canopy. Other estimates in the literature are, e.g., daytime values of 10–20 W m−2 for a maize crop and 5–10 W m−2 for a soybean crop under favourable con- ditions (Meyers and Hollinger 2004). Adding this photosynthesis term in the SEB closure equation normally improves its closure, but a major part of the imbalance remains in most cases. 1 3 2.4 Effect of the Diurnal Cycle on the SEB Figure 3a displays the evolution of the terms of the SEB equation obtained at a station on the island of Majorca for a clear-sky summer day with a very dry surface. The shadowed area between − 10 and 10 W m−2 indicates the range of values where the turbulent fluxes are below the limit of detection, and comprises most of the nocturnal values and some small values of the latent heat flux in the daytime that do not fulfill the quality criteria of Mauder et al. (2013). During daytime, when Rn is positive, this term is the largest and it is maximum in the central part of the day. Under these dry conditions, the evapotranspiration is small and most of the energy is used in transferring heat to the atmosphere by turbulence mixing (H) with a substantial part being transported into the ground (G). Since the soil is very dry, most of the heat storage takes place in the upper soil layers. The imbalance (Imb) is of the same order as G and very significant in the morning and evening transitions. i In the night-time, the radiative cooling of the surface is mostly compensated by heat transfer from the ground. The turbulence fluxes are very small, with some occur- rences of positive values of λE above the detection limit indicating evaporation from 1 3 Surface‑Energy‑Balance Closure over Land: A Review -100 0 100 200 300 400 500 0000 0200 0400 0600 0800 1000 1200 1400 1600 1800 2000 2200 Energy luxes (W m f -2) Time (UTC) Rn H λE G Imb (a) 0 50 100 150 200 250 0000 0200 0400 0600 0800 1000 1200 1400 1600 1800 2000 2200 Imb, advection (W m -2) Time (UTC) Imb 10 50 100 AS L7 (b) Fig. 3 a Surface energy budget for a dry summer day (21 July 2016) at the University of the Balearic Islands, Mallorca, 10 km away from the coast. The black, yellow and green lines join the 30-min values of net radiation (Rn), the ground heat flux at the surface (G) and the imbalance (Imb). The symbols indicate the 30-min values of the turbulent sensible (H, brown squares) and latent (λE, blue triangles) heat fluxes. The rose band between − 10 and 10 W m−2 signals the range corresponding to the detection limit of the turbulent fluxes. 2.4 Effect of the Diurnal Cycle on the SEB b The energy imbalance term (green line) compared to the estimated values of the thermal advection using land-surface temperature fields at different spatial resolutions from a remotely-pilot aircraft system and from satellite. The symbols indicate the advection estimates at scales around 10 m (yellow trian- gles), 50 m (red circles) and 100 m (blue squares). The crosses are the estimates from satellites Landsat 7 at 30-m resolution (vertical crosses) and ASTER at 90-m resolution (tilted cross) 0 50 100 150 200 250 0000 0200 0400 0600 0800 1000 1200 1400 1600 1800 2000 2200 Imb, advection (W m -2) Time (UTC) Imb 10 50 100 AS L7 (b) -100 0 100 200 300 400 500 0000 0200 0400 0600 0800 1000 1200 1400 1600 1800 2000 2200 Energy luxes (W m f -2) Time (UTC) Rn H λE G Imb (a) (b) Time (UTC) Fig. 3 a Surface energy budget for a dry summer day (21 July 2016) at the University of the Balearic Islands, Mallorca, 10 km away from the coast. The black, yellow and green lines join the 30-min values of net radiation (Rn), the ground heat flux at the surface (G) and the imbalance (Imb). The symbols indicate the 30-min values of the turbulent sensible (H, brown squares) and latent (λE, blue triangles) heat fluxes. The rose band between − 10 and 10 W m−2 signals the range corresponding to the detection limit of the turbulent fluxes. b The energy imbalance term (green line) compared to the estimated values of the thermal advection using land-surface temperature fields at different spatial resolutions from a remotely-pilot aircraft system and from satellite. The symbols indicate the advection estimates at scales around 10 m (yellow trian- gles), 50 m (red circles) and 100 m (blue squares). The crosses are the estimates from satellites Landsat 7 at 30-m resolution (vertical crosses) and ASTER at 90-m resolution (tilted cross) the surface. Small nocturnal values for the turbulent fluxes are common, as shown in Cuxart et al. (2015, Table 1), where two-year averages for an inland site in the Iberian Peninsula indicate nocturnal values below 20 W m−2 for H and below 15 W m−2 for λE, values comparable to those of the Imb term. 2.4 Effect of the Diurnal Cycle on the SEB Therefore, the usually neglected processes in the SEB, such as storage, soil and plant respiration, lateral transport or significant vertical divergences of radiative or turbulent heat fluxes may have nocturnal contributions of the same order of magnitude as the turbulent fluxes. The result is that these processes should not be neglected and eventu- ally the Imb term could be taken as the surrogate of their combined contribution. Simi- lar arguments may be used in the morning and evening transitions, when the turbulent fluxes are small and the processes at the surface are very relevant. In absolute numbers, however, the Imb term is much larger during daytime. For a better understanding of the processes causing this behaviour with the diurnal cycle, Fig. 3b compares the imbalance to advection estimates based on remotely-sensed land-surface temperature (LST) fields, following Garcia-Santos et al. (2019). In that work, LST was determined at 1-m resolution from a remotely-piloted aircraft system flying at a height of 200 m above the ground in the area surrounding the SEB station. The advection term is estimated for the volume of interest as (2) A = 휌cpzmU(ΔT∕Δx) (2) where ρ is the air density, cp is the heat capacity of air at constant pressure, U is the wind speed, zm is the measurement height of the atmospheric variables, while ΔT is the tempera- ture difference between two parcels separated a distance Δx along the wind direction. This advection term is estimated using the wind speed at the SEB station and the LST of the 3 M. Mauder et al. Table 1 Overview of the most cited papers in Boundary-layer Meteorology on the topic of SEB closure in chronological order; citation numbers are according to Web of Sci- ence (Clarivate Analytics), accessed on 18 March 2020 References Times cited Significance for SEB closure Etling and Brown (1993) 320 First description of the relationship between large-scale coherent structures in the boundary layer and the bias of single-tower measurements Kanda et al. (2004) 139 First systematic LES study dedicated to the SEB problem Oncley et al. (2007) 243 First concerted field campaign to address the SEB problem Mauder et al. (2007b) 154 First comprehensive comparison of EC sensors and processing software 1 3 Surface‑Energy‑Balance Closure over Land: A Review adjacent parcels for different resolutions obtained degrading the original high-resolution field. adjacent parcels for different resolutions obtained degrading the original high-resolution field. 2.4 Effect of the Diurnal Cycle on the SEB i The symbols in Fig. 3b indicate the average values of the advection estimated at scales of 10 m, 50 m and 100 m. In addition, the same computation is made from the LST fields of the available Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) and Landsat 7 for that day, which are respectively at resolutions of 90 and 30 m. It is seen that in the daytime the estimates at 50 and 100 m are comparable to the SEB imbalance, while those at 10 m are much larger and essentially describe small-scale hetero- geneities that are expected to be blended by turbulence mixing. These occur probably due to the high surface variability of the LST related to the patchiness of the vegetation cover at small scales in the summertime semi-arid conditions. At night, the advection estimates are significantly smaller than at daytime and there is no evidence that the hectometre scale is the one closest to the imbalance, indicating that other mechanisms besides advection may contribute significantly to the lack of closure. 2.5 Sub‑mesoscale Transport Processes and Secondary Circulations Roll vortices are a very common phenomenon in the daytime convective boundary layer. In the presence of such large-scale organized structures, single-tower measurement must be biased, because the associated vertical energy transport is inherently not captured (Etling and Brown 1993). Under strongly unstable conditions, hexagonal cell-like structures develop instead of rolls, and these also result in a bias of single-tower measurements, if they are not carried along the EC system within a given flux-averaging time by the mean flow (Segal and Arritt 1992; Etling and Brown 1993). Both phenomena generally occur in the convective boundary layer even over homogeneous surfaces as shown by many LES studies (Kanda et al. 2004; Huang et al. 2008; Patton et al. 2016; De Roo et al. 2018).l Over heterogeneous surfaces, additional flux-sampling errors are to be expected due to non-propagating circulations or standing cells, which are often linked to surface features, e.g., patches of different surface temperature (Blanford et al. 1991). Under such circum- stances, ergodicity, and as a result Taylor’s frozen turbulence hypothesis, is violated, which is an important assumption underlying the EC method. As a consequence, “energy trans- ported non-turbulently will not be sensed by EC systems and a bias towards lower energy fluxes will result” (Blanford et al. 1991). Only a spatial EC method, instead of the usual temporal EC method, will result in the correct flux estimates (Mahrt 1998).l l After applying Reynolds averaging, the total sensible heat flux in kinematic units can be written as (3) Hkin = wT = ̄w(T −T0) + wT, (3) where w is the vertical wind speed, T is the temperature, and T0 is the so-called base tem- perature (e.g. Webb et al. 1980). It is the temperature from which each parcel of air is warmed (or cooled) during the vertical transfer of the heat supplied (or removed) at the underlying surface (Webb 1982). The overbar denotes a temporal average and the prime denotes a perturbation from a time-averaged quantity. Following Webb et al. (1980) the mean vertical velocity ̄w can be estimated as (4) ̄w = wT∕̄T. ̄w = wT∕̄T. (4) 1 3 M. Mauder et al. Fig. 4 Schematic of the mechanism causing a systematic bias in tower-based EC measurements in the pres- ence of large-scale organized structures in the convective boundary layer. 2.5 Sub‑mesoscale Transport Processes and Secondary Circulations Warm (moist) air near the sur- face is transported upward by secondary circulations, which leads to a positive sensible (latent) heat flux that cannot be capture by single-tower measurements, if these structures are not carried along the EC sys- tem by the mean wind within the flux-averaging time. Virtual control volumes are indicated by a blue box around the EC tower, indicating how secondary circulations (bending grey arrows) lead to advection locally. Please note that the potential temperature θ is generally lower when it enters the control volume than when it leaves the control volume, as long as the surface is heated and the atmosphere is unstably stratified. Also note that a red upward arrow and a blue downward arrow both represent a positive transport of sensible heat, because they correspond to upward and downward vertical motion Fig. 4 Schematic of the mechanism causing a systematic bias in tower-based EC measurements in the pres- ence of large-scale organized structures in the convective boundary layer. Warm (moist) air near the sur- face is transported upward by secondary circulations, which leads to a positive sensible (latent) heat flux that cannot be capture by single-tower measurements, if these structures are not carried along the EC sys- tem by the mean wind within the flux-averaging time. Virtual control volumes are indicated by a blue box around the EC tower, indicating how secondary circulations (bending grey arrows) lead to advection locally. Please note that the potential temperature θ is generally lower when it enters the control volume than when it leaves the control volume, as long as the surface is heated and the atmosphere is unstably stratified. Also note that a red upward arrow and a blue downward arrow both represent a positive transport of sensible heat, because they correspond to upward and downward vertical motion This first term on the right-hand side of Eq. 3 is usually neglected for planar homo- geneous flows, since (T −T0) is mostly much smaller than ̄T . This leads to the conclu- sion that the transport of sensible heat can be described by the covariance only. 1 3 2.5 Sub‑mesoscale Transport Processes and Secondary Circulations However, accu- rate measurements of advection under field conditions remain a methodological challenge (Aubinet et al. 2000). Several researchers attempted to quantify advective heat fluxes, either by using single towers (Gay and Bernhofer 1991; Bernhofer 1992b; Lee 1998; Paw et al. 2000), or directly by using multiple towers (Oncley et al. 2007; Moderow et al. 2007, 2009). However, accu- rate measurements of advection under field conditions remain a methodological challenge (Aubinet et al. 2000). Moreover, a control volume approach was applied to LES, in order to explain the observed SEB closure at two contrasting sites in a highly heterogeneous setting (Eder et al. 2015a). According to earlier theoretical considerations (Finnigan et al. 2003; Wang 2010), the total surface flux of potential temperature in kinematic units, H0,kin, can be determined at a height zm as (6) H0,kin = w𝜃\|\|\|zm + zm ∫ 0 𝜕𝜃 𝜕t dz + zm ∫ 0 ( ̄u𝜕𝜃 𝜕x + ̄v𝜕𝜃 𝜕y ) dz + zm ∫ 0 ̄w𝜕𝜃 𝜕z dz + zm ∫ 0 ( 𝜕u𝜃 𝜕x + 𝜕v𝜃 𝜕y ) dz, (6) where the first term represents the usual EC measurement, the second term is the storage in the layer below (including above ground biomass), the third and fourth terms are horizontal and vertical advection respectively, and the fifth term is the horizontal flux divergence. An analogous equation can be written for the latent heat flux. 2.5 Sub‑mesoscale Transport Processes and Secondary Circulations How- ever, this simplification may not be justified in in some cases as the first term can be expanded by using a perturbation from spatial averages, resulting in (5) Hkin = wT =< ̄w >< T −T0 > + < ̄w∗̄T∗> +wT, (5) where angle brackets enclose a spatial average and a star indicates a local perturbation from a spatial average. The important second term on the right refers to the energy transported by standing eddies or spatially organized time-invariant convection cells, which is associ- ated with a dispersive flux (Raupach and Shaw 1982). In contrast, the first term of Eq. 5 can now indeed be neglected in most cases with the assumption ̄w = 0. Such theoretical considerations on the SEB closure problem inspired researchers to apply spatial EC methods in field experiments by calculating spatial fluxes from air- craft and multi-tower data, as soon as such an undertaking became feasible. Indeed, they found additional (sub-)mesoscale fluxes sufficiently large in magnitude to explain a major part of the imbalance (Mauder et al. 2007a, 2008b, 2010).l Large-eddy simulation studies show that the sum of the additional dispersive fluxes is likely to be positive under unstable conditions in the presence of sub-mesoscale sec- ondary circulations (Kanda et al. 2004), and hence the EC measurements systematically 1 3 Surface‑Energy‑Balance Closure over Land: A Review underestimate the total surface flux (Fig. 4). An analogous mechanism can be described for the latent heat flux as well (Huang et al. 2008). Hence, sub-mesoscale transport is the most prominent underlying hypothesis for studies on the SEB closure problem today.l underestimate the total surface flux (Fig. 4). An analogous mechanism can be described for the latent heat flux as well (Huang et al. 2008). Hence, sub-mesoscale transport is the most prominent underlying hypothesis for studies on the SEB closure problem today.l underestimate the total surface flux (Fig. 4). An analogous mechanism can be described for the latent heat flux as well (Huang et al. 2008). Hence, sub-mesoscale transport is the most prominent underlying hypothesis for studies on the SEB closure problem today. Several researchers attempted to quantify advective heat fluxes, either by using single towers (Gay and Bernhofer 1991; Bernhofer 1992b; Lee 1998; Paw et al. 2000), or directly by using multiple towers (Oncley et al. 2007; Moderow et al. 2007, 2009). 3.1 Idealized LES Studies Large-eddy simulation is a powerful tool for investigating the processes behind the energy balance closure problem quantitatively. It can help to explore the effect of large-scale coherent structures on single-tower measurements systematically quantitatively under con- trolled conditions, where the true flux is known. As an early example, Lohou et al. (2000) used LES to examine the impact of coherent structures on vertical fluxes. Their finding has an important consequence for traditional flux measurements based on the hypothesis of isotropic and homogeneous turbulence, since it can explain part of the underestimation of the surface fluxes often reported in the literature. l Subsequently, Kanda et al. (2004) showed that secondary circulations lead to local advection by the mean flow, which cannot be captured by single-tower measurements, and that this leads to an underestimation of the EC measurement on average. A few years later, the LES study of Inagaki et al. (2006) distinguished between turbulent organized struc- tures, which develop over homogenous surfaces in the convective boundary layer, and ther- mally-induced circulations, which are driven by differential heating. Moreover, LES was used to assess the representativity bias of an EC measurement and to calculate the number of towers needed to capture the total surface flux using spatial eddy covariance (Steinfeld et al. 2007). Using idealized LES over homogenous surfaces, Huang et al. (2008) found a relationship of the energy balance residual with u∗∕w∗ , where w∗ is the convective velocity scale (Dear- dorff 1970). A dependence on u∗ was also found in a year-long simulation by Schalkwijk et al. 1 3 3 M. Mauder et al. Fig. 5 Correlation between normalized horizontal flux divergence and normalized advection by the mean flow versus energy balance ratio for kilometre scale heterogeneity (top row) and hectometre scale heteroge- neity (bottom row). Modified after De Roo and Mauder (2018). Here, the energy balance ratio is defined as the ratio of the simulated eddy-covariance estimate at the measurement height divided by the surface flux Fig. 5 Correlation between normalized horizontal flux divergence and normalized advection by the mean flow versus energy balance ratio for kilometre scale heterogeneity (top row) and hectometre scale heteroge- neity (bottom row). Modified after De Roo and Mauder (2018). Here, the energy balance ratio is defined as the ratio of the simulated eddy-covariance estimate at the measurement height divided by the surface flux (2016). The LES study of Zhou et al. 1 3 3.1 Idealized LES Studies (2018) found, for homogeneous surface forcing, that the imbalance depends on atmospheric stability (and hence also on u∗ ). For heterogeneous sur- faces, it also depends additionally on the turbulent kinetic energy and the difference between the potential temperature at the measurement height and surface temperature.l Recently, De Roo and Mauder (2018) investigated the dependence of the flux underestima- tion on the scale heterogeneity and on the location within a heterogeneous landscape. They found that horizontal flux divergence and advection are anti-correlated for heterogeneous heat- ing with patch sizes on the order of kilometres, but advection dominates the overall effect. However, for heterogenous heating with patch sizes on the order of hectometres, horizontal flux divergence and advection are positively correlated, so that flux divergence amplifies the effect of advection by the mean flow. Moreover, areas with higher than average sensible heat fluxes generally have better closure (Fig. 5). 1 3 Surface‑Energy‑Balance Closure over Land: A Review Fig. 6 Diurnal variations in the energy balance components during EBEX-2000 on 7 August 2000. The fluxes were measured at 8.7 and 2.7 m above ground level. Ruw is the correlation coefficient between the u and w velocity components and Res is the energy balance residual (Zhang et al. 2010, (C) Springer Nature) 3.2.1 Multi‑tower Experiments EBEX-2000 was the first multi-tower experiment addressing the SEB closure problem. It comprised 10 sites on an area of 1600 × 800 m2, and at several of the sites more than one EC system was deployed in order to characterize the instrumental uncertainty with side-by-side comparisons and in order to quantify vertical flux divergence with measure- ments at different heights (Oncley et al. 2007). In addition, the horizontal advection of sensible and latent energy by the mean flow was quantified from this rich dataset, and the energy balance closure gap was reduced by approximately 50%, however with quite a large uncertainty. Inspired by the LES study of Steinfeld et al. (2007), Mauder et al. (2008b) conducted a multi-tower experiment with 25 towers distributed over an area of 4 × 4 km2. A simpli- fied spatio–temporal EC technique was employed, so that only one tower was required to have high-frequency turbulence measurements and the other 24 were equipped with slow- response sensors, which were used to determine a spatio–temporal mean temperature. The goal was to capture the flux due to large-scale organized structures, which was partly suc- cessful. This approach helped to improve energy balance closure, but it was not able to measure the entire exchange of energy between the surface and the atmosphere, probably because it still invokes assumptions during the derivation of the method, amongst others horizontal homogeneity, which was obviously not fulfilled (Mauder et al. 2010). i A similar idea was implemented by Engelmann and Bernhofer (2016) in an experiment using nine sonic anemometers in an array covering 10 × 10 m2. The resulting sensible heat flux increased by 9% when applying spatio–temporal instead of purely temporal averag- ing for the covariance calculation. Also other studies report that accounting for dispersive fluxes yields a better spatial ensemble (Christen and Vogt 2004; Poggi et al. 2004; Kloster- halfen et al. 2019) and highlights the limitations of Taylor’s frozen turbulence hypothesis (Tsvang et al. 1991).f Zhang et al. (2010) analyzed turbulence measurements at different heights during EBEX-2000, and they extracted two important findings. Firstly, they focused on an obvious sawtooth pattern in the latent heat flux. Despite a smooth forcing by Rn, large variations in λE were observed with magnitudes as high as 200 W m−2 (Fig. 6). These variations suggest that other external forcings beyond Rn affect evapotranspiration. Moreover, these variations 1 3 3 M. Mauder et al. 3.2.1 Multi‑tower Experiments (a) (c) (b) (d) Fig. 7 Cospectra for uw and wq as a function of normalized frequency for HF cases (a, b) and LF cases (c, d) as measured during EBEX-2000. Here, u∗ is the friction velocity and q∗= wq∕u∗. Modified after Zhang et al. (2010), (C) Springer Nature (c) (c) (d) (b) (d) Fig. 7 Cospectra for uw and wq as a function of normalized frequency for HF cases (a, b) and LF cases (c, d) as measured during EBEX-2000. Here, u∗ is the friction velocity and q∗= wq∕u∗. Modified after Zhang et al. (2010), (C) Springer Nature in λE are more significant at a height of 8.7 m than at 2.7 m and follow a similar pattern in the afternoon. This behaviour can be explained by turbulent organized structures penetrat- ing from above, thereby transporting latent heat away from the surface in those 30-min periods with very low λE. However, they are not fully captured by the EC system, because they are either at time scales larger than the 30-min averaging time or not propagating with the mean flow.i l This interpretation is supported by spectral analysis. When the authors classified the entire dataset into two groups, one with high latent heat fluxes (HF) and one with low latent heat fluxes (LF), they found striking differences in the spectra between the two heights, particularly also in the cospectra. The ensemble cospectra for the HF cases behave as expected in accordance with common cospectral models. However, for the LF cases, cospectra are depressed in the mid-frequency range. This effect is stronger for the meas- urements at 8.7 m than for those at 2.7 m (Fig. 7). The reduced latent heat fluxes of the LF group are primarily responsible for the concurrently increased residuals of the SEB closure (Fig. 6). It is intriguing that the energy balance was closed for this EBEX-2000 dataset when correcting for the phase shift between vertical velocity and the respective scalar, either temperature or humidity (Gao et al. 2017). Based on this result, the authors conclude that enlarged phase differences of large eddies are linked to entrainment or advec- tion occurrence, which leads to increased residuals of the surface energy balance. Eder et al. (2015b) reported similar sawtooth pattern as in Zhang et al. 1 3 3.2.1 Multi‑tower Experiments (2010), however it affected both latent and sensible heat fluxes equally at their site, which may be related to 1 3 Surface‑Energy‑Balance Closure over Land: A Review the much higher Bowen ratio there. Similar to Zhang et al. (2010), high heat fluxes (and small energy balance residual) were correlated with high momentum fluxes or friction velocity u∗ . An anti-correlation between u∗ and the energy balance residual was found at many other sites (Hendricks-Franssen et al. 2010; Stoy et al. 2013; Eder et al. 2015a). Also similar to Zhang et al. (2010), low heat fluxes (and large energy balance residuals) are cor- related to increased low-frequency motions in the horizontal velocity spectra. While Zhang et al. (2010) based their interpretation only on indirect evidence of large organized eddies, Eder et al. (2015b) actually measured the large organized eddies, either roll-like or cell- like, depending on the background wind speed, by using a triple Doppler lidar configura- tion, which further substantiates this explanation. 3.2.2 Airborne Measurements Airborne measurements represent one of the most direct means of obtaining spatially- resolved 3D turbulence data (Mahrt 1998). Therefore, such datasets illustrate the presence of secondary circulations in the surface layer. With the help of wavelet analysis, it is possi- ble to extract the associated flux at longer wavelengths (Mauder et al. 2007a). It was shown for a dataset of 20 flights, at 30-m height over a length 115 km above heterogeneous boreal forest, that the mesoscale flux is of similar magnitude to the lack of energy balance closure of nearby EC towers (Mauder et al. 2007a). Eder et al. (2014) analyzed the same dataset to investigate the Bowen ratio of the mesoscale fluxes. They found the mesoscale Bowen ratio to be similar to the small-scale turbulent Bowen ratio in most cases.l For a heterogeneous, but flat, region in Central Europe, Foken et al. (2010) found that the energy balance closure generally improved when using the airborne flux measurements instead of a tower-based composite, however, the uncertainty was quite large. Despite the advantages of airborne measurements, they also have challenges, e.g., the need for exten- sive flow-distortion corrections (Metzger et al. 2012), the constantly changing flux foot- print along the flight track (Schuepp et al. 1990; Mauder et al. 2008a) and the high costs and security requirements of airborne operations. 3.2.3 Scintillometers Scintillometers also provide spatially-integrated flux measurements, which prompted researchers to use these instruments in order to capture the otherwise neglected flux by non-propagating large eddies. However, these instruments do not measure the heat flux directly and are only sensitive to a part of the turbulence spectrum, i.e. the inertial sub- range, and they rely on the validity of Monin–Obukhov similarity theory in order to deter- mine fluxes from the measured structure parameters with all its implicit assumptions (Ward 2017). The underlying similarity functions have been determined from tower-based EC measurements and therefore are not disconnected from the energy balance closure prob- lem. Nevertheless, scintillometers often yield improved SEB closure, in particular large- aperture scintillometers with a measurement path on the order of kilometres and measure- ment heights on the order of 10 m.i For the LITFASS-2003 field campaign, a combination of an infrared and a microwave scintillometer was deployed together with several SEB stations underneath the measure- ment path. Results show that the scintillometer-based fluxes are considerably larger than the composite of tower fluxes for the same footprint and that the energy balance closure is improved when the scintillometer-based flux are used (Meijninger et al. 2006). According 1 3 3 M. Mauder et al. to these measurements, the underestimation of the EC-based latent heat flux was twice as large as the underestimation of the EC-based sensible heat flux. Moreover, Foken et al. (2010) showed that scintillometer-based fluxes are in close agreement the LES-based spa- tial EC data for that area. to these measurements, the underestimation of the EC-based latent heat flux was twice as large as the underestimation of the EC-based sensible heat flux. Moreover, Foken et al. (2010) showed that scintillometer-based fluxes are in close agreement the LES-based spa- tial EC data for that area. Similar comparisons between scintillometer and EC measurements were conducted for a large-scale field campaigns in China (Liu et al. 2011; Xu et al. 2017b). Here, the SEB closure improved considerably when using the scintillometer-based fluxes, and the flux underestimation was mostly attributed to the sensible heat flux, while the EC-based latent heat flux agreed well with the scintillometer-based latent heat flux. 3.2.4 Lidar Measurements Lidar technology has developed rapidly in recent years, and these remote sensing instru- ments represent another means of obtaining spatially-resolved measurements of atmos- pheric variables. This approach is particularly promising for the future, since a large amount of spatial data can be obtained simultaneously and continuously along the laser path for quite a large spatial extent without interfering with the atmospheric variables to be measured. However, for 3D turbulent wind measurements, a coordinated scan pattern of at least three Doppler lidars is required (Mann et al. 2009) and at the current resolution of Raman and differential absorption lidars it is still challenging to obtain turbulent fluxes directly (Wulfmeyer et al. 2018; Behrendt et al. 2019).i Single lidar measurements by Drobinski et al. (1998) were, more or less, the first to provide observational evidence of organized large eddies in the surface layer. Eder et al. (2015b) provided a more detailed picture of these organised large eddies by using a triple lidar set-up, and related the associated long-wavelength fluxes to the observed pattern in SEB closure at two EC stations for that area. The advective flux of water vapour that results from heterogeneity-induced secondary circulations was quantified by Higgins et al. (2013) using a high-resolution Raman lidar. 3.3 Integrated Studies (2016) using data from the Boundary Layer Late Afternoon and Sunset Turbulence (BLLAST) campaign in the northern foothills of the Pyrenees (Lothon et al. 2014). There, a number of LST fields from different sources and scales were used to estimate the lateral advection in a volume of depth zm, typi- cally taken as the height of the EC system, using the approach described in Sect. 2.4. Land-surface temperature was taken as an approximation of the surface-layer tempera- ture and the wind speed was taken as 1 m s−1, with the intention of comparing the order of magnitude of advection at different scales, using numerical model results, sat- ellite imagery, unmanned aircraft system measurements, and thermal photography. It was found (see their Table 1) that the variability of LST decreases very little with increasing scale and, in consequence, the importance of the advection term increases with resolution. When compared with the imbalance, it is seen that advection is very small for the kilometre scales, but it is much larger for the decametre and metre scales. The hec- tometre scales, which are related to the elements of the landscape, have values of advection of the same order of magnitude as the imbalance, and are candidates in gen- erating non-random transport, in good correspondence with the numerical results of De Roo and Mauder (2018).l These results were further explored with measurements taken at a flat site and with heterogeneous semi-rural land use on the island of Mallorca in 2016. A display of nine masts with temperature and moisture at 2 and 0.2 m above the surface, wind speed and soil measurements, separated by about 150 m, was used to estimate advection for a centrally located SEB station (Simó et al. 2019). Air temperature instead of LST and the actual wind speed were used to overcome some limitations of the previous study. The results found in BLLAST were essentially confirmed for the hectometre scales, with values of advection between 10 and 20 W m−2 in the day in 3-h averages (even larger in 30-min averages), and between 1 and 10 W m−2 at night. These values were consistent with the satellite-derived estimations of Garcia-Santos et al. (2019) for the same experiment. Another kind of integrated experiment was conducted by Cheng et al. (2017), who used high-resolution spatially-distributed temperature data from fibre-optics measure- ments (distributed temperature sensing) in combination with LES. 3.3 Integrated Studies Let us first define what we mean by integrated studies in the context of SEB closure: these are experiments that combine multi-tower measurements with airborne and/or lidar meas- urements and LES in an integrative way to obtain a better understanding of the transport processes in the atmospheric boundary layer leading to the observed lack of SEB closure in EC tower measurements.i The first realistic LES study on energy balance closure for a multi-tower experiment was conducted by Brötz et al. (2014) for the Convective and Orographically induced Pre- cipitation Study (COPS) campaign in summer 2007 (Wulfmeyer et al. 2011). Here, a sodar was used together with several EC towers. The authors found that the energy balance in the surface layer was closed during low wind periods, but was not closed after the onset of a local valley breeze. The associated LES analysis indicates that the missing flux compo- nents of sensible heat are the main reason for the unclosed energy balance in the consid- ered situations. Another example of an integrative study on the SEB closure problem was conducted by Eder et al. (2015a), who deployed two EC towers, one Doppler lidar and a backscat- ter lidar in and around a solitary forest in the Negev desert in Israel. This pine plan- tation has an extent of several kilometres and is surrounded by semi-arid shrubland, 1 3 Surface‑Energy‑Balance Closure over Land: A Review which constitutes a very strong and distinct heterogeneity with respect to albedo and roughness. Interestingly, the energy balance is closed for the tower inside the forest, while an imbalance of roughly 20% is found at the shrubland site, although both sites are expected to be heavily influenced by advection. This systematic distinction between the two sites was reproduced in the associated LES study, which shows that 3D advec- tion and horizontal flux divergence almost cancel each other out over the forest, which has a high sensible heat flux, but they do not in cooler areas, so that the heat fluxes are underestimated using the EC method. This unexpected finding prompted a systematic LES study by De Roo and Mauder (2018), which showed that this behaviour can be generalized if the scale of heterogeneity is in the kilometre range.i A simplified approach to estimating the impact of surface heterogeneity in the SEB equation was made by Cuxart et al. 4 Partitioning of the Energy Balance Residual The residual Imb term of the surface energy balance can be determined when all four main terms are measured independently (Eq. 1). In order to use this residual for correct- ing the measured turbulent heat fluxes, it is critical to know how the otherwise neglected sub-mesoscale transport ought to be partitioned between the sensible and the latent heat fluxes. The most straightforward method preserves the Bowen ratio, i.e., adjusts both the sensible and the latent heat fluxes by the same percentage, based on the 30-min energy balance residual (Twine et al. 2000). At first, this kind of partitioning was chosen in the absence of a better knowledge of the underlying processes and under the assumption of scalar similarity. It was later substantiated by wavelet analysis of the low-wavenumber flux contributions for airborne EC measurements, indicating that the Bowen ratio for mesoscale fluxes is often similar to the small-scale turbulent Bowen ratio (Mauder et al. 2007a; Eder et al. 2014).l Comparisons of EC-based latent heat flux measurements with lysimeter measure- ments as a reference also support an SEB closure correction that is roughly Bowen-ratio preserving (Gebler et al. 2015; Hirschi et al. 2017). Widmoser and Wohlfahrt (2018) agree with this kind of partitioning in principle, and they stress the importance of con- sidering the uncertainty in the measurement of the available energy when such a correc- tion is practically applied for non-ideal sites. For practical reasons, Mauder et al. (2013) proposed a variation of the Bowen-ratio-preserving partitioning method. Instead of clos- ing the energy balance for every 30-min interval, they use daily energy balance ratios as the basis for the adjustment in order to reduce the scatter in the resulting heat fluxes. In addition, they apply this method only during daytime based on the argument that large- scale organized structures generally develop only in convective boundary layers.f There are, however, also studies that point towards a different partitioning of the resid- ual. For example, Wohlfahrt et al. (2010) found that EC measurements at a mountainous grassland site agree best with lysimeters, if the entire residual is attributed to the latent heat flux. In contrast, Ingwersen et al. (2011) compared the measured EC fluxes over an agricultural site with data from a land-surface model, and found best agreement when the entire residual is attributed to the sensible heat flux. Charuchittipan et al. 3.3 Integrated Studies They found that, due to the violation of Taylor’s hypothesis, energy spectra are underestimated in the inertial subrange by 10–30%, and they proposed a correction to compensate for this underestimation, which can be applied to single-tower EC measurements. It is specu- lated that the resulting fluxes would lead to an improved SEB closure, although this remains to be confirmed at the time of writing. 1 3 M. Mauder et al. 4 Partitioning of the Energy Balance Residual (2014) proposed to attribute the residual mostly, but not entirely, to the sensible heat flux. They relate the correction factor to the ratio between the buoyancy flux and the sensible heat flux, so that the fraction of the residual attributed to the sensible heat flux is calculated as (7) fHB = wT wT v = ( 1 + 0.61 ̄Tcp 𝜆Bo )−1 , (7) where w′T′ v is the buoyancy flux computed from the fluctuation of the virtual temperature Tv, λ is the latent heat of vaporization, and Bo is the Bowen ratio. This approach was tested against simulations using a third-order closure model for tall vegetation (Gatzsche et al. 2018), who found good agreement with the buoyancy- flux- based correction for larger Bowen ratios, while the Bowen-ratio-preserving correc- tion shows a better agreement for Bo < 1.5 (Fig. 8). Pointing towards a similar direction, Mauder et al. (2018) found, for a comparison with lysimeters at two grassland sites, that evapotranspiration is overestimated using a Bowen-ratio-preserving adjustment method and underestimated using a buoyancy-flux-based correction method. 1 3 1 Surface‑Energy‑Balance Closure over Land: A Review Fig. 8 Fraction of the residual attributed to the sensible heat flux for the forest site Waldstein–Weidenbrun- nen (DE-Bay), under the assumption that the ACASA model calculated the true Bowen ratio, and according to the correction methods with the Bowen ratio (Twine et al. 2000) and the buoyancy flux (Charuchittipan et al. 2014), after Gatzsche et al. (2018) Fig. 8 Fraction of the residual attributed to the sensible heat flux for the forest site Waldstein–Weidenbrun- nen (DE-Bay), under the assumption that the ACASA model calculated the true Bowen ratio, and according to the correction methods with the Bowen ratio (Twine et al. 2000) and the buoyancy flux (Charuchittipan et al. 2014), after Gatzsche et al. (2018) 6 Summary, Open Questions and Outlook The enigmatic SEB closure problem has inspired a large number of researchers to revisit questions regarding data quality control, flux corrections, and the theoretical foundations of the EC method. Hence, other related fields of research besides micrometeorology, such as wind-energy research, hydrology and ecology have benefited from the substantial scientific progress that has been triggered by this puzzling finding.l i The underlying reasons for the general underestimation of turbulent fluxes are much clearer today than they were 20 years ago when the EBEX-2000 field campaign was organ- ized. We can now rule out instrumental errors as a major contributor to the missing flux; moreover, uncertainties due to the choice of postprocessing software have been minimized. (Sub-)mesoscale transport has been identified as the main reason for the non-closure, which is manifest as a dispersive flux when taking a non-local perspective and as advection by the mean flow and horizontal flux divergence when taking a local perspective. ll This improved understanding about the underlying process and dedicated comparison experiments with independent estimates of the turbulent heat fluxes has also led to a greater clarity on the partitioning of the residual. If availably energy is measured accurately, and if sites are flat and homogeneous within the flux footprint area, the residual can be distributed between the sensible and latent heat fluxes. Recently, evidence tends towards a partition- ing somewhere between a buoyancy-flux-based and a Bowen-ratio-preserving adjustment method, meaning that often sensible heat fluxes ought to be corrected by a larger percent- age than latent heat fluxes. High-resolution LES support this notion, and the dissimilarity between the sensible and latent heat fluxes with respect to large-scale organized structures can be explained by the dissimilarity of the vertical flux and concentration profiles, which is often found in the convective boundary layer (Huang et al. 2009). If we follow this line of argument, we would expect that other trace gas fluxes are also affected by the same transport phenomena, but probably in a dissimilar manner. Systematic high-resolution LES studies appear to be the method of choice for develop- ing a parametrization of the magnitude of the energy balance residual, because the true surface flux is known and instrumental errors can be excluded. The LES-based energy flux correction recently proposed by De Roo et al. (2018) can now be tested and validated for different real-world sites with independent measurements of energy fluxes (tovi.io/collabo- ration). 5 Attempts to Parametrize the Magnitude of the Residual As soon as researchers recognized that the lack of SEB closure is not only an instrumen- tal problem, attempts have been undertaken to find systematic relationships with poten- tial drivers in order to predict the magnitude of the residual. Panin et al. (1998) analyzed data from several micrometeorological experiments and found indications that point to a relationship between the underestimation of turbulent fluxes and terrain inhomogeneity. In order to systematically correct for this effect a scheme is suggested that uses the fetch for different types of surface for the sites surrounding the environment. Later, the principle idea behind this correction was refined by introducing a metric for landscape-scale hetero- geneity rather than the heterogeneity within the flux footprint (Panin and Bernhofer 2008). However, this correction would only predict the average flux underestimation for a certain site and not its variation with changes in stability. Huang et al. (2008) present a correction based on a LES parameter study with purely homogeneous surface forcing. They identify the non-local stability parameter u∗∕w∗ and the normalized measurement height zm∕zi as suitable predictors of the energy balance residual. Note that both scaling variables depend on the boundary-layer height zi. Despite this pioneering work, this parametrization was hardly applied to real-world data, mostly because their correction function is not valid for measurement heights in much of the sur- face layer where EC tower measurements are usually conducted (Eder et al. 2014). This limitation was due to a relatively course grid resolution related to the computational resources available at the time. Recently, De Roo et al. (2018) conducted a similar parameter study, however this time with almost ten times finer grid resolution due to advances in high-performance comput- ing and a newly developed vertical-grid refinement method. They confirmed the principle functional relationship of the residual with u∗∕w∗ and zm∕zi also for lower measurement heights within the surface layer, and they provide separate correction functions for the sensible and the latent heat fluxes, so that a larger part of the residual is attributed to the 1 3 3 M. Mauder et al. sensible heat flux rather than the latent heat flux. This correction is only valid for zi/L < 0 because unstable stratification is a prerequisite for the development of large-scale organised structures in the boundary layer (L is the Obukhov length). 1 3 6 Summary, Open Questions and Outlook This method relies mostly on driving variables, which are measured by an EC sys- tem anyway, i.e. friction velocity, sensible heat flux, air temperature, and the measurement height. In addition, an estimate for the boundary-layer height is required, which can be obtained either from collocated ground-based remote sensing systems, or by using a slab model (e.g. Tennekes 1973; Batchvarova and Gryning 1990), or from reanalysis data. Nevertheless, there is still a number of open questions with respect to the SEB closure problem. For example, it can be expected from Patton et al. (2016) that consideration of the feedback between large-scale coherent structures and plant canopies will improve the predictability of the energy balance residual near the surface. Furthermore, it remains to be explored how the impact of surface heterogeneity on the residual can be incorporated in this model (De Roo and Mauder 2018; Zhou et al. 2019). Surface‑Energy‑Balance Closure over Land: A Review New projects with relevance to the SEB closure problem are ongoing at the time of writing. The integrated studies of CHEESEHEAD (Chequamegon Heterogeneous Ecosystem Energy-balance Study Enabled by a High-density Extensive Array of Detec- tors) and IPAQS (Idealized Planar Array experiment for Quantifying Surface hetero- geneity) aim at employing a spatial EC method by using data from a large number of fast-response tower measurements, which allow the direct determination of dispersive fluxes (Margairaz et al. 2020). These measurements are combined with LES, ground- based remote sensing and airborne measurements, in order to gather as much informa- tion on the 3D transport mechanisms in the boundary layer as possible. Another promis- ing complementary approach is the use of machine-learning techniques for modelling the energy imbalance and the spatial representativeness of single-tower measurements (Xu et al. 2018). Since the lack of SEB closure is not an instrumental problem the rele- vance to a possible correction for trace gas fluxes such as carbon dioxide is still an open question. The progress in the investigation of SEB closure in the last decade could be an impulse to transfer ideas and concepts to the correction of other trace gas fluxes. Acknowledgements Open Access funding provided by Projekt DEAL. We would like to thank the reviewer for detailed and constructive comments. 6 Summary, Open Questions and Outlook 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 Com- mons 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/. 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https://openalex.org/W4245336103	https://www.researchsquare.com/article/rs-151255/v1.pdf?c=1631886988000	English	null	Comparison between Multiple doses and Single-dose Steroids in Preventing the Incidence of Reintubation after Extubation among Critically ill Patients: A Network Meta-analysis	Research Square (Research Square)	2,021	cc-by	4,630	Comparison between Multiple doses and Single-dose Steroids in Preventing the Incidence of Reintubation after Extubation among Critically ill Patients: A Network Meta- analysis Chiwon Ahn Chung-Ang University Min Kyun Na Hanyang University Tae Ho Lim Hanyang University Bo-Hyoung Jang Kyung Hee University Wonhee Kim Hallym University Youngsuk Cho Hallym University Hyungoo Shin Hanyang University Jae Guk Kim Hallym University Juncheol Lee Armed Forces Capital Hospital Kyu-Sun Choi Hanyang University Research Article Page 1/13 Research Article Keywords: Steroid, Planned extubation, Reintubation, Network meta-analysis Posted Date: January 29th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-151255/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/13 Version of Record: A version of this preprint was published at Journal of Clinical Medicine on June 29th, 2021. See the published version at https://doi.org/10.3390/jcm10132900. Page 1/13 Version of Record: A version of this preprint was published at Journal of Clinical Medicine on June 29th, 2021. See the published version at https://doi.org/10.3390/jcm10132900. Page 2/13 Abstract This study aimed to determine the frequency of prophylactic steroid administration to prevent reintubation after extubation in critically ill patients. We systematically searched MEDLINE, Embase, and Cochrane Library for studies regarding the preventive use of multiple doses or single-dose steroids prior to extubation on July 2020, and conducted a network meta-analysis (NMA) to compare these interventions. To assess the risk of bias of each included study, version 2 of the Cochrane risk-of-bias tool for randomized trials was used. Nine randomized control trials comprising 2,098 patients with comparisons of the three interventions were included. Use of multiple doses and single doses showed a significantly lower rate of reintubation compared with placebo (odds ratio [OR]: 0.43, 95% confidence interval [CI]: 0.25–0.72; OR: 0.31, 95% CI: 0.14–0.69). However, the comparison between multiple doses and single doses showed no significant differences (OR: 1.22, 95% CI: 0.32–4.74). According to the surface under the cumulative ranking curve statistic, the treatments should be ranked as follows: single dose (87.1%), high dose (62.8%), and placebo (0.1%). This NMA showed that the multiple doses were not statistically superior to the single dose in lowering the incidence of reintubation after extubation in critically ill patients. Therefore, use of a single-dose steroid can reduce the incidence of reintubation. Introduction Reintubation after extubation failure causes complications such as cardiovascular failure or ventilator-associated pneumonia, and is associated with an increased mortality rate1–3. Extubation should not be considered as a simple reversed process of intubation; the need for extubation should be planned on the day that the patient was intubated4,5. Even if the patients’ medical condition is stable and no potential complications occurred, such as post- extubation stridor, laryngeal edema, or reintubation, the need for extubation should be carefully planned 4,6. The current guideline suggests that the prophylactic use of steroids prior to extubation is recommended as it is effective in reducing inflammatory airway edema, which can cause direct airway injury4,7. A recent meta-analysis also showed that the use of steroids is effective in reducing post-extubation stridor and reducing the incidence of reintubation after extubation8. The difficult airway society guidelines suggest that 100 mg of hydrocortisone (HC) should be administered every 6 hours4. In addition, Lin et al. (2016) showed that the administration of multiple doses is effective in reducing post- extubation airway obstruction than the use of a single dose9. However, despite the suggested steroid dose and administration method, various steroid regimens have been used in previous studies. The diversity in the steroid dose and administration methods used in previous studies has unsettled practicing clinicians. We performed a network meta-analysis (NMA), which allows a coherent analysis of all randomized controlled trials (RCTs), in order to evaluate the association between the administration of prophylactic steroids and the reintubation rate after extubation in intubated critically ill patients. Additionally, we analyzed the efficacy of various steroid doses (multiple doses, single dose, and placebo), by integrating all available direct and indirect evidence in the NMA. Study characteristics Of the six RCTs that were published in East Asia, two were published in France (Table 1). The steroids used for treatment were methylprednisolone (MP), dexamethasone (DM), and HC. Regarding the methods of steroid administration, four studies used multiple doses, three studies used a single dose, and two studies used both single and multiple doses. With regard to the use of multiple doses or single dose, the amount to be administered once was an HC equivalent dose of 100–250 mg. In Lin et al.’s study (2016), the two different amounts of steroids were used in one comparison because both were administrated in multiple doses9. Table 1 Baseline characteristics of included studies Author Year of publication Region Period Population in meta- analysis Age Steroid Dose frequency Equivalent dose of hydrocortisonea Darmon et al. 1992 France 1986– 1987 664 53.16 DM Single 200 Ho et al. 1996 Taiwan 1990 77 62 HC Single 100 Cheng et al. 2006 Taiwan 2002– 2004 128 66.12 MP Multiple or Single 800 or 200 Francois et al. 2007 France 2001– 2002 698 66 MP Multiple 400 Lee et al. 2007 Taiwan 2004– 2006 80 72.55 DM Multiple 500 Baloch et al. 2010 Pakistan 2006– 2008 92 39.65 DM Multiple 500 Cheng et al. 2011 Taiwan 2005– 2006 71 60.49 MP Single 200 Yu et al. 2014 China 2010– 2013 162 67 DM Multiple or Single 250 or 125 Lin et al. 2016 Taiwan 2007– 2010 126 74.09 DM Multiple 1,000 or 500 MP, methylprednisolone; DM, dexamethasone; HC, hydrocortisone; NR, not reported a1 mg MP = 5 mg HC, 1 mg DM = 25 mg HC Ri k f bi Table 1 Results Page 3/13 After the online database search, 1,914 relevant articles were found in MEDLINE, 1,844 in Embase, and 1,871 in the Cochrane Library; meanwhile, two additional articles were found by manual searching. A total of 3,786 studies were identified after removal of duplicates, and 65 potentially relevant articles were retrieved after a full-text review (Fig. 1). The final nine RCTs selected met the eligibility criteria and included 2,098 patients9–17. S d h i i After the online database search, 1,914 relevant articles were found in MEDLINE, 1,844 in Embase, and 1,871 in the Cochrane Library; meanwhile, two additional articles were found by manual searching. A total of 3,786 studies were identified after removal of duplicates, and 65 potentially relevant articles were retrieved after a full-text review (Fig. 1). The final nine RCTs selected met the eligibility criteria and included 2,098 patients9–17. (Fig. 1). The final nine RCTs selected met the eligibility criteria and incl Main analysis: Pairwise meta-analysis Main analysis: Pairwise meta-analysis Each comparison included multiple doses, single dose, and placebo. There were six direct comparisons for multiple doses and placebo, five for single dose and placebo, and two for multiple doses and a single dose (Table 2, Supplementary Fig. S2). The multiple doses showed a significantly lower rate of reintubation compared with placebo (odds ratio [OR]: 0.43, 95% confidence interval [CI]: 0.25–0.72), and the single dose showed a significantly lower rate of reintubation compared with placebo (OR: 0.31, 95% CI: 0.14–0.69). However, the comparison between multiple doses and single doses showed no significant differences (OR: 1.22, 95% CI: 0.32–4.74) (Table 2, Supplementary Fig. S3). There was no significant heterogeneity among the included studies within each comparison. Risk of bias With regard to the overall risk of bias for each included study, five studies had a low risk of bias, while four studies had some risk of bias. In the detailed assessment of all subcategories, the “risk of bias arising from the randomization process” of four studies were evaluated as some concerns risk of bias because the detailed Page 4/13 descriptions of the randomization process were omitted or the baseline difference between intervention and control was shown in supplementary Fig. S1. Quality of evidence We assessed the quality of evidence from each comparison for reintubation using the GRADEpro Guideline Development Tool. The GRADEpro tool revealed a high level of evidence between multiple doses and placebo, and a high level of evidence between single doses and placebo. However, the tool showed a moderate level between multiple doses and placebo due to imprecision (Table 2, Supplementary Table S1). Table 2 Meta-analysis results for pairwise comparisons of steroid administration frequency Tx1 Tx2 No. of studies I2 OR (95% CI) GRADE Multiple doses Placebo 6 0% 0.43 (0.25–0.72) High Single dose Placebo 5 0% 0.31 (0.14–0.69) High Multiple doses Single dose 2 0% 1.22 (0.32–4.74) Moderatea OR, odds ratio aQuality of evidence for direct estimate rated down by one level for serious imprecision Table 2 aQuality of evidence for direct estimate rated down by one level for serious impre Publication bias To assess for publication bias, we created a funnel plot (Supplementary Fig. S5). Overall, the studies showed 95% CIs. In addition, the plot appeared symmetrical in shape. To assess for publication bias, we created a funnel plot (Supplementary Fig. S5). Overall, the studies showed 95% CIs. In addition, the plot appeared symmetrical in shape. Main analysis: Network meta-analysis Figure 2 shows the forest plot of the overall comparison. The inconsistency test at the global and local levels indicated no significant inconsistency (global level: P = 0.9942; local level: P = 0.995, 0.686, and 0.824; Supplementary Fig. S4); there is no problem with accepting the consistency model. Indirect evidence showed that multiple doses or a single dose likely decreased the reintubation rate compared with placebo (Table 3). According to the surface under the cumulative ranking curve (SUCRA) statistic, which calculated the probability of each treatment, the treatment should be ranked in the following order: single dose (87.1 %), multiple doses (62.8 %), and placebo (0.1 %) (Table 4). Results of the statistical analysis showed that the single dose is superior to the multiple doses in terms of treatment effect. Page 5/13 Table 3 League table for networks according to steroid administration frequency Placebo Multiple doses Single dose Placebo - 0.42 (0.25–0.69) 0.31 (0.14–0.67) Multiple doses 2.41 (1.44–4.03) - 0.75 (0.31–1.79) Single dose 3.21 (1.49–6.92) 1.33 (0.56–3.20) - Table 3 Table 4 Rank probability and surface under the cumulative ranking (SUCRA) curve result Rank 1 2 3 Mean Rank SUCRA Single dose 0.741 0.257 0.002 1.3 0.871 Multiple doses 0.259 0.741 0.001 1.7 0.628 Placebo 0.000 0.003 0.998 3.0 0.001 Publication bias Table 4 Discussion Therefore, clinicians might consider the use of steroids to prevent the occurrence of complications after extubation based on the patient’s condition and environment. The last study included in the analysis was conducted in 20169; no RCT has reported the use of steroids to prevent reintubation after extubation. In recent years, noninvasive ventilation (NIV) and high-flow oxygen therapy (HFOT) after extubation to prevent reintubation are used as alternative respiratory supports20–22. The latest NMA reported that NIV is the most effective respiratory support method for preventing reintubation after extubation23. None of the studies included in this meta-analysis used alternative respiratory support after extubation. Steroids used prior to planned extubation and NIV and HFOT used after extubation are not opposite treatments. Although no previous RCTs have used steroids and alternative respiratory support sequentially, these treatment methods can prevent the incidence of reintubation; however, further research is needed to confirm this finding. This study has several limitations. First, the use of three types of steroids can cause bias. Although a standard steroid dose equivalent to the HC dose has been established to achieve the relative anti-inflammatory effect, the interpretation of results is limited due to the differences in the duration of action, time of onset, and duration of potency for each steroid. Second, a slightly direct comparison was performed between multiple doses and single dose. In the network statistics, statistical analysis was performed by adding the values of direct and indirect comparisons; statistically, the consistency was satisfied in this study. However, due to the small number of studies, the results cannot be considered as clinically significant. Hence, additional NMA should be conducted in future studies. Third, the included studies were conducted in China and Taiwan, except for one study, and the generalizability of the results was limited because the studies were only focused on a certain ethnic group and regional area. In conclusion, this NMA showed that multiple doses of prophylactic steroids were not statistically superior to single-dose steroids in reducing the incidence of reintubation after extubation among critically ill patients, and a single dose of steroid might be sufficient to reduce the incidence of reintubation. In addition, both methods are more effective than placebo. Although multiple doses of steroids can increase the steroid concentration and retention time in blood, its effect in preventing reintubation is not superior to that of a single dose. Methods This NMA was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta- Analyses statement for reporting network meta-analyses24. Discussion Therefore, a single dose can be used to reduce the rate of reintubation. Discussion This NMA indicated the necessity of administering steroids in multiple doses or in a single dose to prevent the risk of reintubation after extubation in critically ill patients, and assessed the outcome-specific certainty of evidence using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system compared with previous meta-analysis. The pairwise meta-analysis showed that there was no significant difference between multiple doses and single-dose steroids in terms of reducing the risk of reintubation; however, the NMA showed that the use of a single dose was statistically superior to the use of multiple doses. Additionally, both multiple doses and single-dose steroids have shown significant superiority in lowering the reintubation rate compared with placebo in several studies. The results of this study were inconsistent with those of previous studies, which reported that multiple doses are more effective than single-dose steroids4,9. The duration of action of steroids is at least 8 hours; MP, an intermediate-acting steroid, 12–36 hours; and DM, a long-acting steroid, 36–72 hours18. In the included studies, multiple doses were administered every 4–6 hours. Considering the decrease in body distribution over time and the increase of drug concentration in the blood, multiple doses are a relatively high dose, and the duration of action is longer than that of a single dose. However, the evidence that multiple doses of steroids can lower reintubation than single doses remained unclear. The dosage of steroids and duration of use are important independent risk factors for the occurrence of side effects19. If multiple doses of steroids do not significantly lower the occurrence of reintubation compared with a single dose, this dosing method should not be used as it is cumbersome, is time consuming, and requires more manpower. The steroids used before extubation are short acting and are administered in smaller doses; to date, no study has reported the direct side effects of steroid use during extubation. Even if side effects occur, they are reversible and Page 6/13 The steroids used before extubation are short acting and are administered in smaller doses; to date, no study has reported the direct side effects of steroid use during extubation. Even if side effects occur, they are reversible and Page 6/13 Page 6/13 more likely to be the effect of prolonged use of steroids in critically ill patients rather than the side effect of the use of prophylactic steroids. Study selection Study selection Search strategy MEDLINE, Embase, and Cochrane Library were systematically searched by two independent reviewers (Ahn C and Na MK) for studies regarding the preventive use of multiple doses or single-dose steroids prior to extubation in critically ill medical patients from inception through July 2020. The detailed search strategy is presented in Supplementary Table S2. The reference lists of previous relevant studies and review articles were manually searched to identify other relevant literature. Page 7/13 After excluding duplicate records, two reviewers (Ahn C and Na MK) independently screened the title and abstract of all selected articles to assess for eligible studies. During the preliminary screening, the full text of eligible articles was reviewed to determine whether these studies met the inclusion or exclusion criteria. No restrictions were imposed on the study period or type of steroid. A third reviewer participated in the discussion to adjudicate disagreements. Data extraction Two reviewers (Ahn C and Na MK) independently reviewed the full text of each included study and extracted the data using a standardized form. The abstracted data included the name of the author, publication year, duration of study, setting of the study, sample size, details of the population enrolled, type of steroid used, interval of steroid administration, and equivalent dose of HC that can achieve a relative anti-inflammatory effect [1 mg MP = 5 mg HC, 1 mg DM = 25 mg HC]. When discrepancies occur between reviewers, a discussion was made to reach a consensus. Quality assessment For the quality assessment of the included studies, version 2 of the Cochrane risk-of-bias tool for randomized trials (RoB 2) was used25. RoB 2.0 was divided into the following categories: “risk of bias arising from the interventions,” “risk of bias due to missing outcome data,” “risk of bias in measurement of the outcome,” and “risk of bias in selection of the reported result.” Each subcategory was rated as follows: “yes,” “probably yes,” “no,” “probably no,” and “no information.” Next, using the evaluation result of each subcategory, the risk of bias was evaluated as low, high, or some concerns according to the evaluation algorithm suggested in RoB 2.0. Finally, the risk of bias was determined as low, high, or some concerns according to the overall quality evaluation criteria presented in RoB 2.0. Disagreements between the two reviewers were resolved by discussion. Groups of comparison: multiple doses vs single dose vs placebo The multiple dose group referred to patients who received two or more steroid injections on a regular interval prior to extubation. The single-dose group referred to patients who received one steroid injection. The placebo group referred to patients who did not receive steroid injections. Inclusion and exclusion criteria (1) Studies conducted in adult critically ill medical patients (age ≥ 18 years) admitted in the intensive care unit who used steroids to prevent complications after extubation, (2) studies that reported the outcomes: reintubation, and (3) prospective RCTs were included in the analysis. Meanwhile, (1) non-RCTs, including reviews, cohort studies, and crossover studies; (2) studies conducted in post- surgical patients; (3) studies that enrolled patients who underwent an unplanned extubation; (4) studies that did not report the outcome of interest; and (5) conference abstracts without full-text manuscripts were excluded. Author contributions C.A. and K-S.C. designed the study and performed the data analysis. C.A. and M.K.N. performed the study selection and data extraction. C.A., M.K.N. and K-S.C. conducted the quality assessment and evidence rating. C.A. performed the database search and drafted the manuscript. All authors have read, revised, and approved the final manuscript. Acknowledgements This research was supported by the Chung-Ang University Research Grants in 2021. This work was supported by the research fund of Hanyang University (HY-201800000000589). Data availability The datasets generated during the current study are available from the corresponding author on reasonable request. Statistical analysis The random effects NMA was performed using a frequentist framework to calculate the ORs for dichotomous outcomes and the corresponding 95% CIs. All statistical analyses were performed using the netmeta package in Stata 13.0 (Stata-Corp, College Station, TX, USA). A two-sided P value of less than 0.05 was considered significant. The homogeneity and consistency assumptions underlie the validity of evidence from NMA 26. The inconsistencies between direct and indirect estimates in the entire network for each outcome were assessed locally with a loop- Page 8/13 Page 8/13 specific approach and globally with a design-by-treatment interaction model27. The treatment effects of various respiratory support methods were ranked according to the probabilities of leading to the best results based on the SUCRA for each outcome28. The SCURA value ranged from 0–100%; a higher SUCRA value indicated the effectiveness of the method28. Each comparison was conducted using GRADE analysis (GRADEpro Guideline Development Tool, McMaster University, USA). Finally, we inspected the funnel plot for presence of publication bias using the netfunnel package in Stata 13.0. Competing interests The authors declare that there is no conflict of interests regarding the publication of this paper. 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Methylprednisolone reduces the rates of postextubation stridor and reintubation associated with attenuated cytokine responses in critically ill patients. Minerva Anestesiol. 77, 503-9. (2011). 22. Yu, Y. et al. Use of dexamethasone for preventing postextubation airway obstruction in adults: a prospective randomized study. J. Intern. Med. Concepts Pract. 9, 134-7. (2014). Page 10/13 Page 10/13 23. Schimmer, B. P., & Funder, J. W. ACTH, adrenal steroids, and pharmacology of the adrenal cortex. Goodman and Gilman’s The pharmacological basis of therapeutics 12th ed (McGraw-Hill, New York, 2011). 24. Yasir, M., Goyal, A., Bansal, P., & Sonthalia, S. Corticosteroid adverse effects. (Treasure Island (FL): StatPearls Publishing, 2020) 25. Vargas, F. et al. Intermittent noninvasive ventilation after extubation in patients with chronic respiratory disorders: a multicenter randomized controlled trial (VHYPER). Intensive Care Med. 43, 1626-36. https://10.1007/s00134-017-4785-1. (2017). 26. Fernandez, R. et al. High-flow nasal cannula to prevent postextubation respiratory failure in high-risk non- hypercapnic patients: a randomized multicenter trial. Ann. Intensive Care 7, 47. https://10.1186/s13613-017- 0270-9. (2017). 26. Fernandez, R. et al. High-flow nasal cannula to prevent postextubation respiratory failure in high-risk non- hypercapnic patients: a randomized multicenter trial. Ann. Intensive Care 7, 47. https://10.1186/s13613-017- 0270-9. (2017). 27. Hernández, G. et al. Effect of postextubation high-flow nasal cannula vs conventional oxygen therapy on reintubation in low-risk patients: a randomized clinical trial. JAMA. 315, 1354-61. https://10.1001/jama.2016.2711. (2016). 27. Hernández, G. et al. Effect of postextubation high-flow nasal cannula vs conventional oxygen therapy on reintubation in low-risk patients: a randomized clinical trial. JAMA. 315, 1354-61. https://10.1001/jama.2016.2711. (2016). 28. Zhou, X. Preventive use of respiratory support after scheduled extubation in critically ill medical patients-a network meta-analysis of randomized controlled trials. Crit. Care 24, 370. https://10.1186/s13054-020-03090- 3. (2020). 28. Zhou, X. Preventive use of respiratory support after scheduled extubation in critically ill medical patients-a network meta-analysis of randomized controlled trials. Crit. Care 24, 370. Figure 2 Forest plot of the network meta-analysis for reintubation rate Forest plot of the network meta-analysis for reintubation rate References https://10.1186/s13054-020-03090- 3. (2020). Figures Page 11/13 Figure 1 PRISMA flowchart of the study selection process Page 12/13 Figure 1 PRISMA flowchart of the study selection process PRISMA flowchart of the study selection process PRISMA flowchart of the study selection process Page 12/13 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. PRISMA2009checklist.pdf supplementaryfile.docx supplementaryfile.docx Page 13/13
https://openalex.org/W2073597965	https://europepmc.org/articles/pmc4100165?pdf=render	English	null	MicroRNAs in Brain Metastases: Potential Role as Diagnostics and Therapeutics	International journal of molecular sciences	2,014	cc-by	9,615	Int. J. Mol. Sci. 2014, 15, 10508-10526; doi:10.3390/ijms150610508 Int. J. Mol. Sci. 2014, 15, 10508-10526; doi:10.3390/ijms150610508 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS Keywords: brain metastases; miRNA replacement therapy; antagomirs Keywords: brain metastases; miRNA replacement therapy; antagomirs Keywords: brain metastases; miRNA replacement therapy; antagomirs Samer Alsidawi 1, Ehsan Malek 1,2 and James J. Driscoll 1,2,3,* Samer Alsidawi 1, Ehsan Malek 1,2 and James J. Driscoll 1,2,3,* 1 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA; E-Mails: alsidasr@ucmail.uc.edu (S.A.); maleken@ucmail.uc.edu (E.M.) 2 Division of Hematology and Oncology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA 3 The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA 3 The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA * Author to whom correspondence should be addressed; E-Mail: driscojs@uc.edu; Tel.: +1-513-558-2186 (ext. 123); Fax: +1-513-558-6703. * Author to whom correspondence should be addressed; E-Mail: driscojs@uc.edu; Tel.: +1-513-558-2186 (ext. 123); Fax: +1-513-558-6703. Received: 16 April 2014; in revised form: 22 May 2014 / Accepted: 6 June 2014 / Published: 11 June 2014 Received: 16 April 2014; in revised form: 22 May 2014 / Accepted: 6 June 2014 / Published: 11 June 2014 Abstract: Brain metastases remain a daunting adversary that negatively impact patient survival. Metastatic brain tumors affect up to 45% of all cancer patients with systemic cancer and account for ~20% of all cancer-related deaths. A complex network of non-coding RNA molecules, microRNAs (miRNAs), regulate tumor metastasis. The brain micro-environment modulates metastatic tumor growth; however, defining the precise genetic events that promote metastasis in the brain niche represents an important, unresolved problem. Understanding these events will reveal disease-based targets and offer effective strategies to treat brain metastases. Effective therapeutic strategies based upon the biology of brain metastases represent an urgent, unmet need with immediate potential for clinical impact. Studies have demonstrated the ability of miRNAs to distinguish normal from cancerous cells, primary from secondary brain tumors, and correctly categorize metastatic brain tumor tissue of origin based solely on miRNA profiles. Interestingly, manipulation of miRNAs has proven effective in cancer treatment. With the promise of reduced toxicity, increased efficacy and individually directed personalized anti-cancer therapy, using miRNA in the treatment of metastatic brain tumors may prove very useful and improve patient outcome. In this review, we focus on the potential of miRNAs as diagnostic and therapeutic targets for the treatment of metastatic brain lesions. Int. J. Mol. Sci. 2014, 15 Int. J. Mol. Sci. 2014, 15 Int. J. Mol. Sci. 2014, 15 10509 1. Introduction The treatment of metastatic brain tumors remains a daunting challenge. Metastatic brain lesions are the most frequently occurring intracranial tumors in adults with >170,000 patients diagnosed annually in the US—ten times the incidence of primary brain tumors [1,2]. Brain metastases continue to increase as a result of an aging population, the advent of targeted therapies that have increased the survival of patients with primary tumors and superior methods that allow earlier cancer detection [3]. The majority of brain metastases originate from primary lesions in the lungs (40%–50%), breasts (15%–25%) and melanomatous skin cancers (5%–20%) [4,5]. Median survival for patients with brain metastases is ~2 months if left untreated, but can be extended to 12–15 months with a multi-disciplinary approach, e.g., neurosurgery, radiosurgery and chemotherapy [6]. Irrespective of treatment, prognosis for patients with brain metastasis remains grim. The negative impact of metastatic brain tumors on patients extends beyond that of poor survival to include devastating effects on cognition, language, mobility and emotional well-being of patients and their families. Lung cancer-derived brain metastases are an exceptionally important cause of morbidity and mortality since even small satellite lesions are incapacitating. Nearly 40% of lung cancer patients develop brain metastases during their disease lifetime [7]. At diagnosis, brain metastases can be detected in approximately 10% of all lung-cancer patients and in multiple retrospective series brain metastases are found in 50% of patients [8,9]. Magnetic resonance imaging (MRI) indicates that brain metastases can be detected as either solitary, oligometastases or as multiple lesions distinct from the originating primary tumor (Figure 1). Despite advances in the development of molecularly targeted therapies to treat primary lung tumors, most deaths from lung cancer result from the progressive growth of metastases that are resistant to current therapies. Figure 1. Magnetic resonance imaging (MRI) detection of brain metastasis. (A) Solitary lesion; (B) Oligometastasis; (C) Multiple brain metastases. (A) (B) (C) Figure 1. Magnetic resonance imaging (MRI) detection of brain metastasis. (A) Solitary lesion; (B) Oligometastasis; (C) Multiple brain metastases. Int. J. Mol. Sci. 2014, 15 10510 Metastases develop when tumor cells successfully evade the homeostatic mechanisms within the host to exploit the cytoprotective features provided by the surrounding microenvironment. The “seed-and-soil” hypothesis of metastasis dictates that the successful outgrowth of deadly metastatic tumors depends on permissible, bidirectional interaction between the metastatic cancer cells and host tissue site-specific microenvironment [10]. However, the specific molecular networks, gene expression alterations and cellular signaling pathways needed to establish brain metastases remain poorly defined. Our understanding of the biology of brain metastases has improved dramatically in the last decade as a result of studies implementing animal models inoculated with high-level green fluorescent protein (GFP) labelled tumor cells and monitoring the formation of metastatic tumors in vivo using novel imaging techniques [11–14]. Current models of brain metastasis, such as transgenic and subcutaneous tumors implanted into immunodeficient mice, do not adequately represent the clinical scenario. Specifically, these models do not reflect the precise molecular steps involved in metastasis nor the response to therapeutic agents. To develop improved models, surgical orthotopic implantation (SOI) was developed to transplant histologically-intact human cancer cells or tissue, taken directly from patients, into the corresponding organ of immunodeficient mice. These unique SOI models have been successfully used for innovative drug discovery and mechanistic studies and serve as a bridge to link pre-clinical studies with clinical research and drug development. These highly valuable model systems should also be useful in validating miRNA therapeutics and complement imaging systems in the study of miRNA diagnostics and therapeutics. Histologic examination of tissue from human patient and animal models of brain metastases has revealed that these tumors are surrounded and infiltrated by reactivated astrocytes [15]. Astrocytes are the most common cell type in the brain and contribute to cerebral homeostasis through diverse methods [16]. Astrocytes support the blood–brain barrier (BBB), regulate blood flow, control inflammatory responses and participate in synaptic transmission. Astrocytes have also been shown to control extracellular homeostasis by regulating ion and glucose concentrations, acid-base balance and the supply of metabolites to neurons. Brain metastases surrounded by activated astrocytes are resistant to chemotherapy [15]. The metastatic tumor cells take advantage of the normal protective role of astrocytes which is to protect neural cells from toxins and exploit them to gain protection from chemotherapeutic agents. Figure 1. Magnetic resonance imaging (MRI) detection of brain metastasis. (A) Solitary lesion; (B) Oligometastasis; (C) Multiple brain metastases. Figure 1. Magnetic resonance imaging (MRI) detection of brain metastasis. (A) Solitary lesion; (B) Oligometastasis; (C) Multiple brain metastases. (C) (A) (A) (B) (A) (C) (B) Int. J. Mol. Sci. 2014, 15 Int. J. Mol. Sci. 2014, 15 Int. J. Mol. Sci. 2014, 15 The brain was considered a sacred place and the resistance of metastatic tumor cells in the brain to chemotherapeutic drugs was falsely attributed to the inability to penetrate through the BBB, which is composed of endothelial cells with tight junctions enwrapped with basement membrane, pericytes and astrocytes. However, tumor cells within the brain parenchyma release vascular endothelial growth factor (VEGF) and other cytokines that increase vessel permeability [17,18]. Newer imaging techniques have proven that the BBB is dysfunctional in brain metastases as evidenced by leakage of contrast material into and around the tumors which basically rules out the BBB as the sole mechanism of drug resistance (Figure 2). The formation of brain metastasis reflects the generalized process of cancer metastasis and consists The formation of brain metastasis reflects the generalized process of cancer metastasis and consists of sequential, interlinked, and selective steps. The outcome of each step is influenced by the interaction of metastatic cells with homeostatic factors. Each step of the metastasis is considered rate limiting in that failure of a tumor cell to complete any step effectively terminates the process. Therefore, the formation of clinically relevant metastases represents the survival of unique subpopulations of cells that preexist in primary tumors. The successful formation of clinically significant metastatic tumor is thought to be the final product of survival specific cells within the primary tumor, i.e., metastases-initiating Int. J. Mol. Sci. 2014, 15 10511 cells. A key event of brain metastasis is the migration of cancer cells through the BBB. Although preventing brain metastasis is immensely important for survival, very little is known about the early stage of transmigration and the molecular mechanisms of tumor cells penetrating the BBB. The brain endothelium plays an important role in brain metastasis. Brain Microvascular Endothelial Cells (BMECs) are the major cellular constituent of the BBB and are joined by intercellular tight junctions responsible for maintaining selective permeability. BBB failure is critical in the development and progression of several diseases that affect the central nervous system (CNS), including brain tumor metastasis development. Crossing the BBB is rate limiting in the development of brain metastases. The presence of brain tumors disrupts the normal BBB, and it is now accepted that when a brain lesion grows beyond 1–2 mm the BBB becomes structurally and functionally compromised [19–21]. Int. J. Mol. Sci. 2014, 15 Over-expression of p-glycoprotein, a membrane protein that expels drugs from a cell’s cytoplasm, has also been implicated in chemoresistance [22,23]. Inhibiting p-glycoprotein, however, has not proved successful in reversing chemoresistance. Collectively, these studies indicate that unidentified mechanisms underlie the pro-survival effect of the brain microenvironment which has led to the search for genetic regulators. Figure 2. MRI of the brain to illustrate loss of blood–brain barrier integrity. Patient with metastatic brain lesion in the left cerebral hemisphere (A) before contrast; (B) after contrast. The leakage of contrast material (gadolinium) into and around the tumor rules out the blood–brain barrier as the sole mechanism for drug resistance. (A) (B) 2. MiRNAs and Brain Metastases Genetic and epigenetic changes allow cancer cells to find the brain microenvironment—“the soil”—a favorable niche for tumorigenic “seeds” to implant, grow and blossom [24]. However, the precise manner in which the brain microenvironment promotes the growth of solid tumor cells remains a critical barrier. Understanding the precise micro-environment-mediated genetic events triggered in metastatic tumor cells to promote growth and drug resistance should substantially improve our knowledge base and identify new “druggable” targets. Non-coding (nc) RNAs are master regulators of the human genome and their aberrant expression contributes to tumorigenesis, metastasis and the acquisition of therapeutic resistance. However, the precise role of ncRNAs in brain metastases and the acquisition of drug resistance remained unknown. MiRNAs are endogenously expressed, small, (A) (B) 2. MiRNAs and Brain Metastases Genetic and epigenetic changes allow cancer cells to find the brain microenvironment—“the soil”—a (B) (A) (A) (B) 2. MiRNAs and Brain Metastases Int. J. Mol. Sci. 2014, 15 Int. J. Mol. Sci. 2014, 15 10512 non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level by base pairing to the 3' untranslated region (UTR) of target messenger RNA (mRNA). MiRNAs play a key role in cell development, proliferation, differentiation and apoptosis and, accordingly, alterations in miRNA expression are seen in tissues from all organ systems and contribute to cancers, autoimmune and genetic disorders and infectious processes. The loss of a tumor suppressive miRNA activates inherently oncogenic pathways to promote the generation of a cancer phenotype, tumor initiation, progression and metastasis [25]. Epigenetic alteration of miRNA have also been shown to play a role in cancer since compelling evidence demonstrates that miRNA deregulation promotes generation of a cancer phenotype, tumor initiation, metastatic growth and development of drug resistance [26,27]. Nearly 50% of human miRNA genes are located in areas of the genome associated with carcinogenesis [28]. Studying the miRNA profile of different tumors has gained popularity in the last decade as represents a breakthrough method for tumor classification that can impact cancer diagnosis, prognosis and treatment decisions. For example, miRNA profiling of 103 lymph node negative, breast cancer tumors lead to the identification of miRNA-106b in triple negative tumors and now known to carry a worse prognosis [29]. Different stages of breast cancer were noted to correlate with distinct miRNA profiles, including members of the miR-200 family and miR-9, to suggest that miRNAs are directly involved in tumor progression and metastasis. In colorectal cancer, the detection of circulating miRNA-141 correlated with metastatic disease and poor prognosis [30] and up-regulation of miR-9 was involved in metastases as well through facilitated cell motility and down-regulated α-catenin [31]. Certain miRNAs, e.g., miRNA-34 and let-7, were also found to be directly involved with the survival of tumor-initiating (or metastases-initiating) cancer stem cells (CSCs) [32,33]. MiRNA signatures have been identified within individual tumor types and may improve useful as diagnostics or prognostics of therapeutic response. MiRNA profiling of tumor tissue may facilitate the identification of primary tumors based upon the miRNA profile of the metastatic brain lesion. Even with advanced imaging techniques, a small percentage of metastatic brain tumors remain of unknown origin. A recent study successfully identified the tumor of origin in 84% of brain metastases using a quantitative real-time polymerase chain reaction (qRT-PCR) of 48 different miRNAs [34]. 2. MiRNAs and Brain Metastases Genetic and epigenetic changes allow cancer cells to find the brain microenvironment—“the soil”—a favorable niche for tumorigenic “seeds” to implant, grow and blossom [24]. However, the precise manner in which the brain microenvironment promotes the growth of solid tumor cells remains a critical barrier. Understanding the precise micro-environment-mediated genetic events triggered in metastatic tumor cells to promote growth and drug resistance should substantially improve our knowledge base and identify new “druggable” targets. Non-coding (nc) RNAs are master regulators of the human genome and their aberrant expression contributes to tumorigenesis, metastasis and the acquisition of therapeutic resistance. However, the precise role of ncRNAs in brain metastases and the acquisition of drug resistance remained unknown. MiRNAs are endogenously expressed, small, Int. J. Mol. Sci. 2014, 15 Other studies have shown that miR-92b and miRNA-9/9* are over-expressed in primary brain tumors compared to metastatic brain tumors to aid in the diagnosis of brain lesions [35]. The role of miRNAs in the biology of brain metastases has been established in studies investigating a number of primary tumor types (Table 1) [36–40]. In breast cancer, miR-1258 alterations were directly related to heparanase expression, a known prometastatic enzyme found in brain metastatic breast cancer cells that degrades heparan sulfate chains to affect the cytoskeleton and render cells more capable of crossing the BBB [41,42]. The migratory and invasive capacity of breast CSCs was found to be related to the KLF4 gene expression which is inversely related to miRNA-7 expression [43]. Similarly, in lung cancers, miRNA-145 down-regulation was involved in the growth of lung adenocarcinoma and promoted the formation of brain metastases [44]. MiRNA-328 in non-small cell lung cancer (NSCLC) regulated cell migration and the formation of brain metastases through altered expression of the PRKCA genes [45]. MiRNA-378 promoted brain metastases in NSCLC by increasing expression levels of MMP-7, MMP-9 and VEGF and decreasing levels of Sufu, all key genes involved in angiogenesis and extracellular matrix invasion [46]. MiRNA-200 family members were exclusively elevated in the Int. J. Mol. Sci. 2014, 15 10513 CSF of patients with metastatic brain lesions from various primary tumor types when compared with glioblastoma and non-cancer patients [47]. Table 1. MiRNAs deregulated in brain metastases compared to the primary tumor. Deregulated miRNAs identified in metastatic brain tumor cells compared to their matched primary tumors. NSCLC, non-small cell lung cancer; MMP, matrix metalloproteinase; VEGF, Vascular endothelial growth factor; PTB1b, protein tyrosine phosphatase-1B; HIF-1α: Hypoxia-inducible factor 1-α. Int. J. Mol. Sci. 2014, 15 Deregulated MiRNA Direction of Expression in Brain Metastases Primary Tumor Putative Target miR-1258 [41] Down-regulated Breast Heparanase miR-7 [43] Down-regulated Breast KLF4 gene miR-145 [44] Down-regulated Lung adenocarcinoma 3'-UTR of the JAM-A and fascin miR-146-a [48] Down-regulated Breast B-catenin and hnRNPC miR-768-3p [49] Down-regulated Lung and breast K-RAS miR-19a [50] Down-regulated Breast 3'-UTR of tissue factor transcript [36] miR-29c [50] Down-regulated Breast and melanoma Induced myeloid leukemia cell differentiation protein MCL1 [37] miR-31 [51] Down-regulated Colon p53 [38] miR-328 [45] Up-regulated NSCLC PRKCA gene miR-378 [46] Up-regulated NSCLC MMP-7, MMP-9 and VEGF miR-200 [47] Up-regulated Breast and lung E-cadherin transcriptional repressors ZEB1 and ZEB2 [39] miR-210 [50] Up-regulated Breast and melanoma PTP1b and HIF-1α [40] miR-1, miR-145, miR-146a, miR-143, miR-10b, miR-22 [51] Up-regulated Colon Multiple genes related to apoptosis and oncogenesis The brain micro-environment, represented mainly by the astrocytes, is an active player and key regulator in the increased growth and chemoresistance of metastatic brain tumors (Figure 3). Astrocytes up-regulate a number of survival genes within the neighboring tumor cells and render these cells more aggressive, independent of primary tumor histology or p-glycoprotein activity [15]. MiRNAs are directly involved in the changes that the brain microenvironment implies on the metastatic tumor cells as many studies have shown that the brain microenvironment change the miRNA profile of the tumor cells when compared with the primary tumor. Rhabdoid tumor cells showed different miRNA profiles when originated in the brain compared to the kidney [52]. MiRNA-146a was noted to be suppressed in brain metastases compared to the original tumors in animal models, associated with decreased β-catenin protein levels and increased heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) which may increase migratory and invasive capabilities [48]. MiRNA-768-3p was down-regulated in tumor cells when co-cultured with astrocytes and this was validated in human brain metastatic tissues from lung cancer, breast cancer and melanoma when compared to match-paired Int. J. Mol. Sci. 2014, 15 10514 primary tumor from the same patient. MiRNA-768-3p down-regulation led to an increase in K-ras expression and translated into increased tumor growth and drug resistance [49]. Different miRNA profiles were found between primary colorectal tumors and matched metastatic brain tumors [51] where over-expression of miRNA-145, 1, 146a, 576-5p, 126, HS287, 28-5p, 143, 199b-5p, 199a-5p, 10b, 22, 133b, 145, 199a, 133a, 125b and down-regulation of miRNA-31 and HS170 were observed in brain-metastatic carcinomas. Int. J. Mol. Sci. 2014, 15 Moreover, miRNAs isolated from exosomes of parental breast cancer and melanoma cells were different from those isolated from their corresponding metastatic brain variants. MiRNA-210 was over-expressed while miRNAs-19a and 29c were down-regulated in brain metastases [50]. These studies demonstrate that the brain microenvironment induces changes in the miRNA signature of the tumor cells to activate pro-growth signaling pathways and leads to more aggressive, drug resistant metastatic lesions. Studies suggest that the microenvironment influence on tumor cells that “seed” in the brain may be a universal effect [47]. This represents such an appealing concept to target key miRNAs involved in metastasis. 3. MiRNA Diagnostics Understanding of the role of miRNAs in the biology of brain metastases has generated a greater demand to practically apply this knowledge in clinical practice. MiRNAs hold promise as diagnostics, prognostics and therapeutics to improve cancer patient outcome [53]. For example, miRNAs are being developed to improve detection of the plasma cell dyscrasia multiple myeloma (MM) [54]. Similarly, miRNA-based diagnostics may more readily detect metastatic brain lesions and distinguish primary from metastatic lesions [34]. MiRNA signatures may eventually be incorporated in clinical decision making as prognostic indicators to formulate treatment plans. Multiple miRNA signatures in primary tumors were shown to correlate with more aggressive, invasive, “brain-seeking” behavior. MiRNA-378 in NSCLC is associated with a greater likelihood of tumor seeding within the brain [46]. Clinical trials are needed to determine if miRNA signatures are predictive of worse prognosis. Such signatures could trigger more intensive treatment plans, e.g., prophylactic cranial irradiation or targeted therapy, to prevent the development of metastases or cranial recurrence. Early detection of brain micrometastases may be based upon deregulated miRNAs known to be altered within metastatic brain tumors. Changes in miRNA levels, e.g., loss of miRNA-768-3p signal [49] or increase in miRNA-200 [47], may provide an early signal to prompt aggressive treatment. MiRNAs are also readily detectable and stable within human plasma [30,55,56]. These miRNAs are protected from endogenous RNase activity as free-circulating molecules, within circulating tumor cells (CTCs) or in membrane-derived small membrane vesicles, exosomes, that are released by cells [57]. MiRNAs from plasma, CTCs and exosomes have been successfully detected using RT-PCR techniques and may serve as readily-available diagnsotics [58–60]. Deregulated levels of miRNAs have been detected in the plasma of patients with lymphoma (miRNA-155, 210, 21) [61], leukemia (miRNA-92, 150, 342) [62,63], colon cancer (miRNA-29a, 92a) [64], breast cancer (miRNA-195, 21, 92a, let-7a) [65,66], prostate cancer (miRNA-375, 141) [67], ovarian cancer (miRNA-21, 92, 93) [68], pancreatic cancer (miRNA-155, 196a, 642b, 885, 5p, 22, 16) [69–72], gastric cancer (miRNA-17, 1, 106a, 106b, let-7a and 18a) [73–75] and lung cancer (miRNA-486, 30d, 1, 499 and 375) [58,76,77]. MiRNAs were also found to be stable in the cerebrospinal fluid (CSF) of patients with neoplasms as well as neurologic disorders [47,78]. Given the Int. J. Mol. Sci. 3. MiRNA Diagnostics 2014, 15 10515 relative invasive nature of CSF sampling, the challenge in miRNA diagnostics in brain metastases is the BBB and whether miRNAs (either as free molecules, in CTCs or other form of transport system such as exosomes) are able to cross the BBB and be readily detectable in the serum of patients. Studies in glioblastomas have shown that miRNA signals can be detected within exosomes in the serum of these patients [59]. These results support disruption of the BBB during metastasis [19]. Studies also detected relevant miRNAs in the plasma of Alzheimer’s or Huntington’s disease patients even though the BBB is thought to remain intact in these conditions [79]. Membrane-derived extracellular vesicles (EVs) containing miRNAs originate from CNS tumors and may function as intercellular communication with the microenvironment and across the BBB [80]. Now that it has been established that the miRNA profile of brain metastases is distinct from primary tumors, it would be of great importance to be able to routinely and inexpensively detect these miRNAs in the blood, serum, CSF or urine of patients. 4. MiRNA Therapeutics The delivery of miRNA mimics with tumor suppressor effect into tumor cells have shown to be effective in inducing cell death (Figure 4) [49,94–96]. Figure 3. MiRNA diagnostics and therapeutics for brain metastases. A synthesized antisense nucleotide (antagomir, red) or miRNA replacement (green) is loaded onto a delivery system. The delivery system can be a viral vector such as adenovirus or a non-viral liposome or nanoparticle. The preparation is then administered intravenously to the patient with a metastatic brain tumor and remains stable in the blood stream. The compound crosses the blood–brain barrier and reaches the tumor cells and undergoes endocytosis to the intracellular space. The antagomir is then released from the delivery system which gets degraded. The antagomir binds to the miRNA of interest in blue and antagonizes its oncogenic effect which eventually leads to apoptosis and tumor regression. g g g oncogenic effect which eventually leads to apoptosis and tumor regression. A current dilemma in miRNA therapeutics is an efficient system to guarantee stability in the blood and adequate delivery to tissues of interest. Viral and non-viral delivery methods have been used with variable success. Adenovirus-associated vectors (AAV) emerged as an appealing method since they have acceptable toxicity profiles [97] and were successfully injected intravenously in mouse models to restore miRNA-26 expression in hepatocellular carcinoma cells [98]. Different AAV serotypes can successfully target distinct tumor types. Non-viral delivery methods may be superior to AAV methods given their stable formulations. For example, liposomes composed of phospholipid bilayers were used to deliver miRNA-133b to lung cancer cells in mice [99]. Liposome use, however, is limited by their toxicity, related to their strong cationic charge [100]. Liposomes have gone through multiple levels of development to improve their stability and minimize the side effects. Hyaluronic acid was added to A current dilemma in miRNA therapeutics is an efficient system to guarantee stability in the blood and adequate delivery to tissues of interest. Viral and non-viral delivery methods have been used with variable success. Adenovirus-associated vectors (AAV) emerged as an appealing method since they have acceptable toxicity profiles [97] and were successfully injected intravenously in mouse models to restore miRNA-26 expression in hepatocellular carcinoma cells [98]. Different AAV serotypes can successfully target distinct tumor types. Non-viral delivery methods may be superior to AAV methods given their stable formulations. 4. MiRNA Therapeutics Discoveries in miRNA biology, and their close relationship to oncogenesis in many tumor types, has led to attempts to translate this information into miRNA therapeutics (Figure 3) [81,82]. Currently, however, there is a lack of miRNA-based therapeutics to directly target brain metastases. A well-established feature is that a single miRNA is capable of regulating multiple genes, which makes endogenous miRNAs appealing therapeutic targets. Altering miRNA signatures was also found to sensitize tumor cells to other forms of treatment in, otherwise, chemo-resistant tumors [83]. Two strategies exist for miRNA-based therapeutics: a direct approach which involves either miRNA mimics to replace the loss of a tumor suppressor miRNA or miRNA antagomirs which are antisense oligonucleotides that block oncogenic miRNAs; and an indirect strategy that involves identifying existing agents that modulate the expression and/or processing of miRNAs in traditional compound-library screens. The development of antagomirs went through multiple phases to increase their stability since naked RNA has a very short half-life in the bloodstream and the use of phosphodiester oligodeoxynucleotides (ODNs), without further modification was unsuccessful [84]. The in vivo stability of antagomirs has been augmented by multiple chemical modifications such as the development of phosphorothioate containing oligonucleotides [85], 2'-O-methyl-(2'-O-Me) or 2'-O-methoxyethyl-oligonucleotides (2'-O-MOE) which improves ribonuclease resistance and increases the binding affinity to the miRNA [86], locked nucleic acid (LNA) oligonucleotides where the ribose ring is “locked” by a methylene bridge which further increases the affinity towards single stranded RNAs [87,88], peptide nucleic acids (PNA) which are artificially synthesized polymers similar to RNAs but are resistant to enzyme degradation [89] and fluorine-derivative nucleic acids (FANA and 2'-F) [90]. Similar to antagomirs, miRNA sponges inhibit miRNA where plasmids containing multiple tandem-binding sites to the miRNA of interest are transfected into the cells and help “fool” the miRNA into binding to the sponge instead of its target mRNA [91]. MiRNA masks are single-stranded 2-O-methyl antisense oligonucleotides that are complementary to the supposed miRNA binding sites in the 3'-UTR of the mRNA [92]. MiRNA replacement therapy aims at restoring a tumor suppressor miRNA that is down-regulated in tumor cells with oligonucleotide mimics similar to the original miRNA. Using longer strands that mimic the pre-miRNA have also been proposes but these require different delivery systems to ensure intranuclear Int. J. Mol. Sci. 2014, 15 10516 availability [93]. 4. MiRNA Therapeutics For example, liposomes composed of phospholipid bilayers were used to deliver miRNA-133b to lung cancer cells in mice [99]. Liposome use, however, is limited by their toxicity, related to their strong cationic charge [100]. Liposomes have gone through multiple levels of development to improve their stability and minimize the side effects. Hyaluronic acid was added to Int. J. Mol. Sci. 2014, 15 10517 form polycationic liposome-hyaluronic acid (LPH) which successfully delivered siRNA and miRNA-34a into mouse melanoma models [101]. To overcome the toxicity of liposomes, a neutral lipid emulsion was developed which has a natural predilection to accumulate in the lung compared to the liver predilection of cationic liposomes. The neutral lipid emulsion successfully delivered let-7 and miRNA-34a to lung cancer cells in mice [102]. Liposomes have a short half-life and require continuous infusion or frequent administration which limits their use. Multiple attempts were made to overcome these problems which led to the development of sustained-release polymer formulations [103]. Other forms of non-viral delivery systems include dendrimers which are repetitively-branched perfectly-structured particles that have a high surface to volume ratio and were successfully used in delivering anti-miR-21 and 5-flurouracil to glioblastoma cells in vitro [104]. Other nanoparticles, microspheres and hydrogels have been developed [105] such as the polylactide-co-glycolide (PLGA) particles which are stable particles that allow the delivery of miRNA over time and are highly adaptable and can be used to load multiple cargos. PLGA particles delivered anti-miRNA-155 to malignant pre-B lymphoma cells in mouse models with good results [106]. Figure 4. Model to illustrate the effect of microRNA-based therapeutics for the treatment of brain metastases at the cellular and animal levels. Oncology-directed miRNA replacement therapy. Loss of a tumor suppressor miRNA leads to hyperactivation of inherently oncogenic pathways and tumorigenesis. Administration of a miRNA mimic reinstates the function of the missing tumor suppressor miRNA, suppresses oncogenic pathways and cancer cell growth. MicroRNA Replacement Throughout the different stages of development of gene and miRNA delivery systems, a major obstacle has been crossing the BBB. Although surgically-implanted wafers and intra-thecal routes are established methods to administer chemotherapy, oral or intravenous routes remain the most convenient. The BBB only allows lipophilic molecules, less than 400 Da, to penetrate the CNS [107]. A few novel techniques have been used to overcome this obstacle. 4. MiRNA Therapeutics The Trojan Horse Liposome (THL) system MicroRNA Replacement MicroRNA Replacement MicroRNA Replacement Throughout the different stages of development of gene and miRNA delivery systems, a major obstacle has been crossing the BBB. Although surgically-implanted wafers and intra-thecal routes are established methods to administer chemotherapy, oral or intravenous routes remain the most convenient. The BBB only allows lipophilic molecules, less than 400 Da, to penetrate the CNS [107]. A few novel techniques have been used to overcome this obstacle. The Trojan Horse Liposome (THL) system Int. J. Mol. Sci. 2014, 15 10518 encapsulates the genomic material, i.e., miRNA replacements or antagomirs, within the liposome to protect it from nuclease degradation. The compound is constructed using polyethyleneglycol (PEG) to stabilize the liposome [108]. Part of the PEG can be engineered with peptidomimetic monoclonal antibodies (mAbs) that target specific BBB receptors (such as the insulin receptor or the transferrin receptor) and facilitate the transcytosis of the compound. The THL technology has been used to administer compounds that cross the BBB and deliver genetic material to the CNS [109]. Another novel method to bypass the BBB is through polyethylenimine (PEI)-based delivery systems as are widely used in gene therapy [110]. PEI complexes are positively charged that bind negatively charged nucleic acid, i.e., miRNAs. The compound retains an overall positive charge that interacts with negatively charged polysaccharides on the cell surface. This process is followed by endocytosis of the compound to evade the endosome by inducing an influx of protons and water leading to swelling and disruption of the endosome and release of the compound containing the miRNA in the cytoplasm. PEI-based systems have been modified to cross the BBB by adding a short peptide inspired from the rabies virus glycoprotein (RVG) which binds the acetylcholine receptor [111]. Mannitol also is added to increase the permeability of the BBB [112]. The PEI–RVG compound crossed the BBB and delivered the neuron specific miR-124a to brain cells. Even with rapidly emerging understanding of miRNA biology and the development of novel delivery systems, the clinical use of miRNA therapeutics to treat brain metastases remains limited in pre-clinical development and has yet to be exploited. The previous misconception of the brain as a sanctuary organ that systemic or targeted therapies cannot penetrate has contributed to delays in clinical advancement. 4. MiRNA Therapeutics Future studies are needed to better define miRNA signatures within brain metastases and to correlate these signatures with the miRNA profile of the primary tumor. Advances have been made in pre-clinical and translational studies to identify miRNAs that change after growth in the brain microenvironment but require validation from patient tumor samples [49]. Acknowledgments We thank members of the Driscoll lab (http://www.driscolllab.com/index.html#banner) for critical reading of the review. 5. Conclusions Despite advances in developing miRNA diagnostics and therapeutics, significant challenges remain. Since miRNA are upstream regulators of hundreds of genes, the off-target effects of miRNA therapeutics are a potential limitation. Toxicities associated with miRNA therapeutics are not limited to the delivery system since studies have shown that oversaturating small RNA pathways can be lethal [113]. The induction of interferon-α through the toll like receptor (TLR-7) by short interfering RNA (siRNA) leads to systemic immune responses and poor outcomes [111]. The availability of a reliable delivery system that has minimal toxicities, crosses the BBB and successfully unloads the miRNA therapeutic is needed to promote clinical advancement (Figure 4). In summary, the survival of patients with brain metastases remains poor due to the lack of effective treatments. MiRNAs are key regulators of gene expression and their role in multiple cancer types is well-established. Multiple miRNA signatures are altered in brain metastases relative to the primary tumor and are, in fact, induced through interaction with the brain microenvironment. Identifying miRNA signatures within brain metastases represents a promising approach to target these lesions. However, numerous challenges exist in translating this information into clinical practice. MiRNA Int. J. Mol. 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https://openalex.org/W4315864697	https://vbn.aau.dk/ws/files/513831209/healthcare_11_00251.pdf	English	null	Management of Diabetes during School Hours: A Cross-Sectional Questionnaire Study in Denmark	Healthcare	2,023	cc-by	14,198	Management of Diabetes during School Hours Citation for published version (APA): Nannsen, A. Ø., Kristensen, K., Johansen, L. B., Iken, M. K., Madsen, M., Pilgaard, K. A., Grabowski, D., Hangaard, S., Schou, A. J., & Andersen, A. (2023). Management of Diabetes during School Hours: A Cross- Sectional Questionnaire Study in Denmark. Healthcare, 11(2), Article 251. https://doi.org/10.3390/healthcare11020251 Citation for published version (APA): Nannsen, A. Ø., Kristensen, K., Johansen, L. B., Iken, M. K., Madsen, M., Pilgaard, K. A., Grabowski, D., Hangaard, S., Schou, A. J., & Andersen, A. (2023). Management of Diabetes during School Hours: A Cross- Sectional Questionnaire Study in Denmark. Healthcare, 11(2), Article 251. https://doi.org/10.3390/healthcare11020251 Aalborg Universitet Aalborg Universitet Citation for published version (APA): Nannsen, A. Ø., Kristensen, K., Johansen, L. B., Iken, M. K., Madsen, M., Pilgaard, K. A., Grabowski, D., Hangaard, S., Schou, A. J., & Andersen, A. (2023). Management of Diabetes during School Hours: A Cross- Sectional Questionnaire Study in Denmark. Healthcare, 11(2), Article 251. https://doi.org/10.3390/healthcare11020251 Aalborg Universitet Management of Diabetes during School Hours A Cross-Sectional Questionnaire Study in Denmark Nannsen, Anne Østergaard; Kristensen, Kurt; Johansen, Lise Bro; Iken, Mia Kastrup; Madsen, Mette; Pilgaard, Kasper Ascanius; Grabowski, Dan; Hangaard, Stine; Schou, Anders Jørgen; Andersen, Anette Published in: Healthcare DOI (link to publication from Publisher): 10.3390/healthcare11020251 Creative Commons License CC BY 4.0 Publication date: 2023 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Nannsen, A. Ø., Kristensen, K., Johansen, L. B., Iken, M. K., Madsen, M., Pilgaard, K. A., Grabowski, D., Hangaard, S., Schou, A. J., & Andersen, A. (2023). Management of Diabetes during School Hours: A Cross- Sectional Questionnaire Study in Denmark. Healthcare, 11(2), Article 251. https://doi.org/10.3390/healthcare11020251 Article Anne Østergaard Nannsen 1,* , Kurt Kristensen 1,2 , Lise Bro Johansen 3 , Mia Kastrup Iken 4 , Mette Madsen 5,6, Kasper Ascanius Pilgaard 3,7, Dan Grabowski 3 , Stine Hangaard 5, Anders Jørgen Schou 8,9 and Anette Andersen 1 1 Steno Diabetes Center Aarhus (SDCA), Aarhus University Hospital, 8200 Aarhus N, Denmark y p 2 Department of Pediatrics, Aarhus University Hospital, 8200 Aarhus N, Denmark 2 Department of Pediatrics, Aarhus University Hospital, 8200 Aarhus N, Denmark p y p 3 Steno Diabetes Center Copenhagen (SDCC), Department of Research, Copenhagen University Hospital, 2730 Herlev, Denmark 3 Steno Diabetes Center Copenhagen (SDCC), Department of Research, Copenhagen University Hospital, 2730 Herlev, Denmark 4 Danish Diabetes Association, 2600 Glostrup, Denmark Danish Diabetes Association, 2600 Glostrup, Denmark 5 Steno Diabetes Center North Denmark (SDCN), 9000 Aalborg, Denmark 6 Department of Pediatrics and Adolescent Medicine, Aalborg University Hospital, 9000 Aalborg, Denmark 7 Department of Pediatrics, Copenhagen University Hospital, 2730 Herlev, Denmark 8 Steno Diabetes Center Odense (SDCO), 5000 Odense, Denmark 9 Pediatric Research Unit, Odense University Hospital, 5000 Odense, Denmark * Correspondence: annann@rm.dk 5 Steno Diabetes Center North Denmark (SDCN), 9000 Aalborg, Denmark g 6 Department of Pediatrics and Adolescent Medicine, Aalborg University Hospital, 9000 Aalborg, Denmark 7 Department of Pediatrics, Copenhagen University Hospital, 2730 Herlev, Denmark Department of Pediatrics, Copenhagen University Hospital, 2730 Her 8 Steno Diabetes Center Odense (SDCO), 5000 Odense, Denmark 8 Steno Diabetes Center Odense (SDCO), 5000 Odense, Denmark 9 Pediatric Research Unit, Odense University Hospital, 5000 Odense, Denmark Abstract: Managing diabetes is complicated for many children. It often requires support from an adult during the school day. In Denmark, most children spend 30–35 h a week at school. Nevertheless, diabetes management in schools remains largely uninvestigated. This study aimed to examine the characteristics and organization of diabetes management in Danish primary schools from the personnel’s perspective. All primary schools in Denmark were invited to participate in the study (n = 2129), and 525 schools were included. A questionnaire was constructed and sent by email. Questionnaire data are presented in the descriptive statistics and compared with the ISPAD guidelines. According to 77.2% of respondents, school personnel had received training in diabetes management, and 78.5% of the schools had at least one person available for diabetes support every day. Citation: Nannsen, A.Ø.; Kristensen, K.; Johansen, L.B.; Iken, M.K.; Madsen, M.; Pilgaard, K.A.; Grabowski, D.; Hangaard, S.; Schou, A.J.; Andersen, A. Management of Diabetes during School Hours: A Cross-Sectional Questionnaire Study in Denmark. Healthcare 2023, 11, 251. https://doi.org/10.3390/ healthcare11020251 Academic Editor: Pedram Sendi Received: 9 December 2022 Revised: 6 January 2023 Accepted: 11 January 2023 Published: 13 January 2023 Citation: Nannsen, A.Ø.; Kristensen, K.; Johansen, L.B.; Iken, M.K.; Madsen, M.; Pilgaard, K.A.; Grabowski, D.; Hangaard, S.; Schou, A.J.; Andersen, A. Management of Diabetes during School Hours: A Cross-Sectional Questionnaire Study in Denmark. Healthcare 2023, 11, 251. https://doi.org/10.3390/ healthcare11020251 Keywords: diabetes management; school setting; pediatric diabetes; children with diabetes Received: 9 December 2022 Revised: 6 January 2023 Accepted: 11 January 2023 Published: 13 January 2023 healthcare healthcare healthcare Article Respondents felt prepared to help the students with counting carbohydrates (38.9%), dosing insulin (39.1%), and helping the students during high (52.1%) or low (60.3%) blood sugar levels, insulin chock (35.2%), or during activities (36.3%). Yet, diabetes management was a challenging task. Only 61.7% had an action plan for diabetes management, 37.4% had face-to-face information meetings with the parents, and 55.1% of respondents reported having sufﬁcient time to cooperate with the parents. 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Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. p y If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we w the work immediately and investigate your claim. healthcare 1. Introduction Type 1 diabetes is one of the most frequent chronic conditions in children, with an estimated prevalence of 1.2 million cases worldwide [1]. The European region has the highest number of children and adolescents with type 1 diabetes (n = 295,000) and the highest numbers of incident cases in 2021 (n = 31,000) [1]. In Denmark, 3100 children and adolescents (0–19 years) are diagnosed with type 1 diabetes [1]. Having diabetes may complicate everyday life for the child as diabetes management requires support from an adult [2–5]. Copyright: © 2023 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/). Most children spend many hours at school. In Denmark, children spend an average of 30–35 h a week at school [6]. This transfers the responsibility for diabetes management to the school personnel, and it may be a demanding task to help the children manage diabetes during the school day. A Swedish survey investigating the parents’ perspective of school management of diabetes found that many children did not have a contact person or an https://www.mdpi.com/journal/healthcare Healthcare 2023, 11, 251. https://doi.org/10.3390/healthcare11020251 Healthcare 2023, 11, 251 2 of 24 action plan in case of hypoglycemia, and the parents regularly gave less insulin in the morning due to fear of hypoglycemia during school hours [7,8]. action plan in case of hypoglycemia, and the parents regularly gave less insulin in the morning due to fear of hypoglycemia during school hours [7,8]. The Convention on the Rights of the Child from 1989 is the backbone of many national laws on children’s rights around the world [9]. It states that governments should remove all obstacles for children with disabilities to make them independent and able to enjoy the best possible life in their community [9]. Therefore, Danish national law requires all schools to provide a school day on equal terms with peers for all children [10,11]. Nevertheless, Denmark has no national guidelines on what deﬁnes good diabetes care in a school setting. Diabetes management during school hours relies on the pediatric diabetes departments, schools, and parents to accommodate the individual needs of the child. The International Society for Pediatric and Adolescent Diabetes (ISPAD) issued the latest Clinical Practice Consensus Guidelines (ISPAD guidelines) in 2018 [12]. Whereas the guidelines of the American Diabetes Association include recommendations that are context speciﬁc to the US [13], the ISPAD guidelines are applicable worldwide [12]. It remains uninvestigated how Danish schools accommodate children with diabetes during school hours. Therefore, the aim of the study was to examine the characteristics and the organization of diabetes management in Danish primary schools as seen from the school personnel’s perspective. We used the ISPAD guidelines for diabetes management in school settings to compare the Danish level with the international recommendations. 2.1. Design and Study Population The Kids with Diabetes in School (KIDS) study is a multicenter project, which is conducted in collaboration between the Danish Diabetes Association and the ﬁve Danish Steno diabetes centers. A questionnaire (KIDS questionnaire) was sent to all primary schools in Denmark, including all elementary schools (0th grade, 1st–9th grade and the optional 10th grade), private primary schools (0th–9th grade), boarding schools (9th and 10th grade), and day schools (9th and 10th grade). Our survey required one respondent per school to ﬁll in the questionnaire, and the school was instructed to hand over the questionnaire to the employee with the best knowledge on diabetes management at the given school. 2.2. Data Collection The schools were identiﬁed from the list of schools under the Danish Ministry of Chil- dren and Education. This list comprised 2129 eligible schools at the time of inclusion [14]. The questionnaire was sent to the publicly listed email address of the school. After a reminder by e-mail and then by telephone, 921 schools responded to the initial questions, 841 schools responded to the questions on diabetes status (of which 524 had children with diabetes enrolled) and were ultimately included in the study. See details in Figure 1. 3 of 24 3 of 22 Healthcare 2023, 11, 251 Healthcare 2023, 11, x Figure 1. Flowchart of the inclusion into the study. Figure 1. Flowchart of the inclusion into the study. Figure 1. Flowchart of the inclusion into the study Figure 1. Flowchart of the inclusion into the study. 2.3. Development of the Questionnaire 2.3. Development of the Questionnaire We conducted a literature search to gain insight into questionnaires on diabetes man- agement in school settings from the personnel’s perspective. The search yielded limited results [15]. Therefore, a questionnaire was developed specifically for this study. A thor- ough development process preceded the final questionnaire. The purpose of the question- naire was to explore the organization of diabetes management in Danish primary schools. We used a bottom-up approach to construct a questionnaire specifically for the Danish context based on the literature [15] and the professional expertise from multiple profes- sions, such as medical doctors, anthropologists, and public health researchers with expe- rience in survey studies. We conducted a literature search to gain insight into questionnaires on diabetes management in school settings from the personnel’s perspective. The search yielded limited results [15]. Therefore, a questionnaire was developed speciﬁcally for this study. A thorough development process preceded the ﬁnal questionnaire. The purpose of the questionnaire was to explore the organization of diabetes management in Danish primary schools. We used a bottom-up approach to construct a questionnaire speciﬁcally for the Danish context based on the literature [15] and the professional expertise from multiple professions, such as medical doctors, anthropologists, and public health researchers with experience in survey studies. y The questionnaire was divided into three themes: (1) general demographic infor- mation about the school (6 items), (2) specific questions about diabetes management at the school (29 items), and (3) perceived competences and perceptions of the respondent re- garding diabetes management (7 items) (Appendix A, Table A1). The questionnaire was developed and distributed in Danish and translated to English only for the purpose of this p y The questionnaire was divided into three themes: (1) general demographic information about the school (6 items), (2) speciﬁc questions about diabetes management at the school (29 items), and (3) perceived competences and perceptions of the respondent regarding diabetes management (7 items) (Appendix A, Table A1). The questionnaire was developed and distributed in Danish and translated to English only for the purpose of this publication. publication. 2.4. Analysis 2.4. Analysis Characteristics on participating schools are presented in percent and absolute num- bers (%, n) in Table 1. Values for less than 5 respondents are concealed and displayed as n ≤ 5. Data from the questionnaires were based on self-reports, except for e-mail, school name, municipality, and regional placement. First, we constructed 10 domains based on the content of the ISPAD guidelines. Second, we grouped the specific recommendations from the ISPAD guidelines under these domains. Third, we compared the questions in our questionnaire to the guidelines. The specific guideline and related questionnaire ques- tions were then placed under a domain. This was done to ensure that the questionnaire covered the ISPAD guidelines sufficiently before we analyzed the responses from the Characteristics on participating schools are presented in percent and absolute numbers (%, n) in Table 1. Values for less than 5 respondents are concealed and displayed as n ≤5. Data from the questionnaires were based on self-reports, except for e-mail, school name, municipality, and regional placement. First, we constructed 10 domains based on the content of the ISPAD guidelines. Second, we grouped the speciﬁc recommendations from the ISPAD guidelines under these domains. Third, we compared the questions in our questionnaire to the guidelines. The speciﬁc guideline and related questionnaire questions were then placed under a domain. This was done to ensure that the questionnaire covered the ISPAD guidelines sufﬁciently before we analyzed the responses from the schools. Each domain must be covered with a satisfactory response from the schools to indicate whether Healthcare 2023, 11, 251 4 of 24 or not the schools meet the international standards for diabetes management in schools. Criteria for satisfactory responses from participating schools were determined a priori. For example, the school had to respond with a “yes” to give a satisfactory response to questions regarding individualized diabetes plans. We used the ISPAD guidelines to compare diabetes management in Danish primary schools with international recommendations. An overview of the ISPAD guidelines, the corresponding questions from the KIDS questionnaire, and consensus on satisfactory responses can be seen in detail in Table A1. Table 1. Characteristics of participating schools stratiﬁed on regional location, n = 524. publication. 2.4. Analysis Capital Region of Denmark Central Denmark Region North Denmark Region Region Zealand Region of Southern Denmark Participants, % (n) 19% (101) 29% (150) 13% (66) 12% (65) 27% (142) Institution, % (n) Primary school (public) 63% (64) 67% (100) 65% (43) 58% (38) 49% (69) Primary school (private) 34% (34) 19% (29) 24% (16) 35% (23) 35% (49) Boarding school n ≤5 13% (19) 11% (7) n ≤5 15% (21) Day school n ≤5 n ≤5 0% (0) 0% (0) n ≤5 Number of students per school, % (n) <199 students 15% (15) 31% (46) 45% (28) 31% (20) 34% (48) 200–399 students 19% (19) 23% (35) 26% (17) 31% (20) 31% (44) 400–599 students 21% (21) 21% (21) 18% (27) 25% (16) 19% (27) 600–799 students 24% (24) 19% (29) 11% (7) 12% (8) 11% (16) 800–999 students 13% (13) 7% (10) n ≤5 n ≤5 n ≤5 >1000 students 9% (9) n ≤5 0% (0) 0% (0) n ≤5 Respondents’ occupation, % (n) Administrative personnel 14% (14) 15% (23) n ≤5 15% (10) 12% (17) Teacher 14% (14) 19% (29) 17% (11) 14% (9) 27% (38) Social worker 7% (7) 6% (9) 9% (6) 8% (5) 6% (8) Principal 53% (53) 49% (74) 67% (44) 45% (29) 46% (65) Other manager than principal 11% (11) 5% (7) n ≤5 8% (5) 7% (10) Other, non-manager n ≤5 5% (8) 0% (0) 11% (7) n ≤5 Area (wealth), % (n) Not so wealthy 13% (13) 16% (23) 35% (23) 24% (15) 19% (26) Very wealthy 8% (8) 3% (5) 0% (0) 0% (0) n ≤5 Some wealth 33% (33) 17% (25) 11% (7) 19% (12) 18% (25) Not at all wealthy n ≤5 4% (6) 9% (6) n ≤5 5% (7) Average 44% (44) 60% (89) 45% (30) 51% (32) 57% (80) Table 1. Characteristics of participating schools stratiﬁed on regional location, n = 524. The analysis of non-respondents showed that a higher proportion of non-respondents (compared to respondents) were located in the Capital Region of Denmark and Region Zealand, whereas a higher proportion of respondents (compared to non-respondents) were located in the Central Denmark Region (Table 2). Stratiﬁcation on municipality [16] showed no signiﬁcant differences between respondents and non-respondents (Table 2). The proportion of primary schools was higher among non-respondents (compared to respondents), and the proportion of private schools were higher among respondents (compared to non-respondents). publication. 2.4. Analysis Healthcare 2023, 11, 251 5 of 24 Table 2. Analysis of non-respondents stratiﬁed on region, municipality, and institution, n = 2129. Non-Respondents Respondents n (%) 1052 (66.8%) 524 (33.2%) Region, n (%) Capital Region of Denmark 275 (26.1%) 101 (19.3%) Central Denmark Region 184 (17.5%) 150 (28.6%) North Denmark Region 103 (9.8%) 66 (12.6%) Region Zealand 213 (20.2%) 65 (12.4%) Region of Southern Denmark 277 (26.3%) 142 (27.1%) Municipalities, n (%) Capital municipalities 216 (20.5%) 99 (18.9%) Metropolitan Municipalities 51 (4.8%) 46 (8.8%) Provincial municipalities 238 (22.6%) 95 (18.1%) Commuter Municipalities 227 (21.6%) 112 (21.4%) Rural Municipalities 320 (30.4%) 172 (32.8%) Institution, n (%) Boarding school, live in 95 (9.0%) 53 (10.1%) Primary schools 673 (64.0%) 314 (59.9%) Private primary school 225 (21.4%) 151 (28.8%) Boarding schools 59 (5.6%) 6 (1.1%) 3. Results Most participating schools were located in the Central Denmark Region (n = 150). Across the regional location of the schools, the majority of respondents were school prin- cipals from primary schools in areas of average wealth. Across regions, the schools were fairly evenly distributed, with <199 to 800 enrolled students (Table 1). 3.1. Law and Equal Opportunity In total, 21.7% of schools had guidelines on chronic illness in general, and 25.9% had speciﬁc guidelines on diabetes. A proportion of the schools allocated extra hours for support of students with diabetes in 0th grade (19.5%), 1st–3rd grade (25.0%), 4th–6th grade (23.7%), 7th–9th grade (24.0%), and 10th grade (21.0%). The majority of the respondents experienced the school as being able to include students with diabetes on equal terms with their peers in terms of academic subjects (86.9%), physical education (80.1%), daytime excursions (84.5%), and overnight excursions (74.6%) (Table 3, Figures 2 and 3 and Table A1). Table 3. Overview of single-item questions and subsequent proportion of satisfactory responses. Proportion of Satisfactory Response Does the school have a guideline/policy for handling chronic illness among students? 21.7% Does the school have a guideline/policy for handling diabetes among students? 25.9% Does the school have an action plan, in case a students with diabetes experiences low blood sugar/insulin chock (hypoglycemic reaction)? 61.7% Does the school have one or more persons able to help students with diabetes administer insulin/adjust insulin pump if necessary? 78.5% ble 3. Overview of single-item questions and subsequent proportion of satisfactory responses. Healthcare 2023, 11, 251 6 of 24 Table 3. Cont. Proportion of Satisfactory Response Did teachers and other personnel receive teaching regarding supporting students with diabetes? 77.2% What do you think is the biggest challenge for the school, in regards to management and support to students with diabetes? 54.3% Are there any rules for where and when students with diabetes are allowed to eat/drink? 72.9% Are there activities at school where students with diabetes are limited/restricted? 95.3% It is possible for the school to have extra personnel on hand in some situations, for example excursions? 69.7% Are the students able to measure blood sugar levels at the school? 97.4% Are there any rules for where and when students with diabetes are allowed to use their phones? 53.0% Are there any rules for when and where students with diabetes are allowed to use the restroom? 96.2% Are students able to manage their diabetes, for example eat/drink, measure blood sugar without losing considerable time during class? 85.7% Are students able to manage their diabetes, for example eat/drink, measure blood sugar without losing considerable time during recess? 84.9% How is information from the parents delivered? 37.4% Figure 2. Proportion of schools allocating extra hours to students with diabetes, stratifi grade. day. 3.2. Individualized Diabetes Plan 3.2. Individualized Diabetes Plan The respondents indicated that 61.7% of schools had an action plan in c The respondents indicated that 61.7% of schools had an action plan in case of hypo- glycemia, and 78.5% of schools had one or more personnel present at the school to support the students in diabetes management (Tables 3 and A1). glycemia, and 78.5% of schools had one or more personnel present at the scho the students in diabetes management (Table 3) (Table A1) 3.3. Education and Knowledge 3.1. Law and Equal Opportunity Proportion of participants feeling able to include students with diabetes during a school day. 3.1. Law and Equal Opportunity 19.5% 25.0% 23.7% 24.0% 21.0% 0% 20% 40% 60% 80% 100% Students in 0. grade Students in 1st–3rd grade Students in 4th–6th grade Students in 7th–9th grade Students if 10th grade 100% Figure 2. Proportion of schools allocating extra hours to students with diabetes, stratiﬁed by grade. Proportion of Satisfactory Response Did teachers and other personnel receive teaching regarding supporting students with diabetes? 77.2% What do you think is the biggest challenge for the school, in regards to management and support to students with diabetes? 54.3% Are there any rules for where and when students with diabetes are allowed to eat/drink? 72.9% Are there activities at school where students with diabetes are limited/restricted? 95.3% It is possible for the school to have extra personnel on hand in some situations, for example excursions? 69.7% Are the students able to measure blood sugar levels at the school? 97.4% Are there any rules for where and when students with diabetes are allowed to use their phones? 53.0% Are there any rules for when and where students with diabetes are allowed to use the restroom? 96.2% Are students able to manage their diabetes, for example eat/drink, measure blood sugar without losing considerable time during class? 85.7% Are students able to manage their diabetes, for example eat/drink, measure blood sugar without losing considerable time during recess? 84.9% How is information from the parents delivered? 37.4% Figure 2. Proportion of schools allocating extra hours to students with diabetes, stratif d Figure 2. Proportion of schools allocating extra hours to students with diabetes, stratiﬁed by grade. Figure 2. Proportion of schools allocating extra hours to students with diabetes, stratif grade Figure 2. Proportion of schools allocating extra hours to students with diabetes, stratiﬁed by grade. 7 of 24 es, strati Healthcare 2023, 11, 251 Figure 3. Proportion of participants feeling able to include students with diabetes du day 86.9% 80.1% 84.5% 74.6% 0% 20% 40% 60% 80% 100% Academic subjects Physical education classes Day time excursions Excursions with sleep over Figure 3. Proportion of participants feeling able to include students with diabetes during a school day. Figure 3. Proportion of participants feeling able to include students with diabetes du Figure 3. Proportion of participants feeling able to include students with diabetes during a school day. Figure 3. Proportion of participants feeling able to include students with diabetes du Figure 3. glycemia, and 78.5% of sc the students in diabetes m 3.3. Education and Knowledge 38.4% 44.2% 45.5% 37.9% 37.6% 43.7% 0% 20% 40% 60% 80% 00% Familiarize yourself with diabetes in general Familiarizing yourself with supporting a specific student with diabetes Supporting students with diabetes during school hours Support during physical activity Support during recess Support during excursions Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 5. Proportion of participants with adequate time to support. 38.4% 44.2% 45.5% 37.9% 37.6% 43.7% 0% 20% 40% 60% 80% 100% Familiarize yourself with diabetes in general Familiarizing yourself with supporting a specific student with diabetes Supporting students with diabetes during school hours Support during physical activity Support during recess Support during excursions Figure 5. Proportion of participants with adequate time to support. re 4. Proportion of participants with adequate knowledge on multiple subjects. re 5. Proportion of participants with adequate time to support. 38.4% 44.2% 45.5% 37.9% 37.6% 43.7% 0% 0% 0% 0% 0% 0% Familiarize yourself with diabetes in general Familiarizing yourself with supporting a specific student with diabetes Supporting students with diabetes during school hours Support during physical activity Support during recess Support during excursions Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 5. Proportion of participants with adequate time to support. 38.4% 44.2% 45.5% 37.9% 37.6% 43.7% 0% 20% 40% 60% 80% 100% Familiarize yourself with diabetes in general Familiarizing yourself with supporting a specific student with diabetes Supporting students with diabetes during school hours Support during physical activity Support during recess Support during excursions Figure 5. Proportion of participants with adequate time to support. of participants with adequate time to support. rtion of participants with adequate time to support. Figure 5. Proportion of participants with adequate time to support. of participants with adequate time to support. ortion of participants with adequate time to support. Figure 5. Proportion of participants with adequate time to support. ly communication at school, 59.5% felt well prepared to transfer infor- tes during the day to colleagues, and 44.4% felt well prepared to trans- ubstitute teachers. glycemia, and 78.5% of sc the students in diabetes m 3.3. Education and Knowledge Proportion of participants with adequate knowledge on multiple subjects. sions (43.7%) (Table 3) (Figures 4 and 5) (Table A1). Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 5. Proportion of participants with adequate time to support. 49.0% 57.1% 56.0% 22.4% 61.5% 43.8% 34.1% 39.3% 0% 20% 40% 60% 80% 100% Acute situations Blood sugar levels Physical activity Administering glucagon When to call 112 Diet and carbohydrate counting Dosing insulin Diabetes equipment 38.4% 44.2% 45.5% 37.9% 37.6% 43.7% 0% 20% 40% 60% 80% 100% Familiarize yourself with diabetes in general Familiarizing yourself with supporting a specific student with diabetes Supporting students with diabetes during school hours Support during physical activity Support during recess Support during excursions Figure 5. Proportion of participants with adequate time to support. If the person with the primar responsibilit of the child’s diabetes care was abse Figure 4. Proportion of participants with adequate knowledge on multiple subjects. 49.0% 57.1% 56.0% 22.4% 61.5% 43.8% 34.1% 39.3% 0% 20% 40% 60% 80% 100% Acute situations Blood sugar levels Physical activity Administering glucagon When to call 112 Diet and carbohydrate counting Dosing insulin Diabetes equipment Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 4. Proportion of participants with adequate knowledge on multiple subjects. 49.0% 57.1% 56.0% 22.4% 61.5% 43.8% 34.1% 39.3% 0% 20% 40% 60% 80% 10 Acute situations Blood sugar levels Physical activity Administering glucagon When to call 112 Diet and carbohydrate counting Dosing insulin Diabetes equipment Figure 4. Proportion of participants with adequate knowledge on multiple subjects. 49.0% 57.1% 56.0% 22.4% 61.5% 43.8% 34.1% 39.3% 0% 20% 40% 60% 80% 100% Acute situations Blood sugar levels Physical activity Administering glucagon When to call 112 Diet and carbohydrate counting Dosing insulin Diabetes equipment Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 4. Proportion of participants with adequate knowledge on multiple subjects. 49.0% 57.1% 56.0% 22.4% 61.5% 43.8% 34.1% 39.3% 0% 20% 40% 60% 80% 100 Acute situations Blood sugar levels Physical activity Administering glucagon When to call 112 Diet and carbohydrate counting Dosing insulin Diabetes equipment of participants with adequate knowledge on multiple subjects. Figure 4. Proportion of participants with adequate knowledge on multiple subjects. ortion of participants with adequate knowledge on multiple subjects. ure 4. Proportion of participants with adequate knowledge on multiple subjects. ure 5. Proportion of participants with adequate time to support. glycemia, and 78.5% of sc the students in diabetes m 3.3. Education and Knowledge the students in diabetes management (Table 3) (Table A1). 3.3. Education and Knowledge The respondents indicated that the school personnel had received diab training at 77.2% of the schools. However, the respondents reported to hav knowledge on acute situations (49.0%), blood sugar levels (57.7%), impact o tivity (56.0%), administration of glucagon (22.4%), when to call the emerge The respondents indicated that the school personnel had received diabetes speciﬁc training at 77.2% of the schools. However, the respondents reported to have inadequate knowledge on acute situations (49.0%), blood sugar levels (57.7%), impact of physical activity (56.0%), administration of glucagon (22.4%), when to call the emergency services (61.5%), and the inﬂuence of diet and carbohydrate on blood sugar levels (39.3%). In total, 54.3% of the respondents did not consider it a major challenge to have students with diabetes (Table A1). The respondents reported having adequate time to study or familiarize with diabetes in general (38.4%); to help a speciﬁc student with challenges of diabetes (44.2%); to support students with diabetes during school hours (45.5%); and to support students during physical activity (37.9%), during recess (37.6%), or during school excursions (43.7%) (Table 3, Figures 4 and 5 and Table A1). g Regarding daily communication at school, 59.5% felt well prepared to transfer in- formation about diabetes during the day to colleagues, and 44.4% felt well prepared to transfer information to substitute teachers. Less than half of the respondents reported to have adequate time during the day to transfer information to colleagues (42.5%) or substitute teachers (34.5%). Additionally, respondents reported on the ability to confer with colleagues about the student’s diabetes (Figure 6) (42.6%). Healthcare 2023, 11, 251 ( ); students du ( studen 8 of 24 ur- sions (43.7%) (Table 3) (Figures 4 and 5) (Table A1). Figure 4. Proportion of participants with adequate knowledge on multiple subjects. Figure 5. Proportion of participants with adequate time to support. 49.0% 57.1% 56.0% 22.4% 61.5% 43.8% 34.1% 39.3% 0% 20% 40% 60% 80% 100% Acute situations Blood sugar levels Physical activity Administering glucagon When to call 112 Diet and carbohydrate counting Dosing insulin Diabetes equipment 38.4% 44.2% 45.5% 37.9% 37.6% 43.7% 0% 20% 40% 60% 80% 100% Familiarize yourself with diabetes in general Familiarizing yourself with supporting a specific student with diabetes Supporting students with diabetes during school hours Support during physical activity Support during recess Support during excursions Figure 4. glycemia, and 78.5% of sc the students in diabetes m 3.3. Education and Knowledge Food (Access) I t t l 72 9% f h l did t h l di ti d i ki d i 38.9% 39.1% 52.1% 60.3% 35.2% 36.3% 0% 20% 40% 60% 80% 100% Count carbohydrates Dosing insulin During high blood sugar During low blood sugar During insulin chock/hypoglycemic reaction Dosing insulin during activities Figure 7. Proportion of participants prepared to help the student. insulin (39.1%), helping during high (52.1%) or low (60.3%) blood sugar levels, ins chock (35.2%), or activities (36.3%) (Figure 7 and Table A1). teachers (34.5%). Additionally, respondents reported on the ability to confer with col- leagues about the student’s diabetes (Figure 6) (42.6%). 59.5% 44.4% 42.6% 34.5% 42.6% 51.0% 48.4% 0% 20% 40% 60% 80% 100% Prepared to deliver information to colleagues Prepared to deliver information with substitutes teachers Adequate time to deliver information to colleagues Adequate time to deliver information to substitute teachers Able to discuss/confer with colleagues about the students' diabetes There are other colleagues to take over, if you intentionally are not at school There are other colleagues to take over, if you unplanned are not at school re 2023, 11, x 9 o nts' reports on experiences with colleagues. n with the primary responsibility of the child’s diabetes care was absent 51.0% stated having colleagues who could take on the responsibilities for lanned in advance, and 48.4% stated having colleagues on standby who responsibilities if the absence was more acute (Table A1). The respond- ng prepared to help the students with counting carbohydrates (38.9%), 9.1%), helping during high (52.1%) or low (60.3%) blood sugar levels, .2%), or activities (36.3%) (Figure 7) (Table A1). Figure 6. Participants’ reports on experiences with colleagues. Figure 7. Proportion of participants prepared to help the student. 38.9% 39.1% 52.1% 60.3% 35.2% 36.3% 0% 20% 40% 60% 80% 100% Count carbohydrates Dosing insulin During high blood sugar During low blood sugar During insulin chock/hypoglycemic reaction Dosing insulin during activities Figure 7. Proportion of participants prepared to help the student. ts' reports on experiences with colleagues. with the primary responsibility of the child’s diabetes care was absent 1.0% stated having colleagues who could take on the responsibilities for anned in advance, and 48.4% stated having colleagues on standby who responsibilities if the absence was more acute (Table A1). glycemia, and 78.5% of sc the students in diabetes m 3.3. Education and Knowledge Less than half of the respondents reported to have ng the day to transfer information to colleagues (42.5%) or substitute ng daily communication at school, 59.5% felt well prepared to transfer infor- diabetes during the day to colleagues, and 44.4% felt well prepared to trans- on to substitute teachers. Less than half of the respondents reported to have e during the day to transfer information to colleagues (42.5%) or substitute If the person with the primary responsibility of the child’s diabetes care was absent from the school, 51.0% stated having colleagues who could take on the responsibilities for diabetes care if planned in advance, and 48.4% stated having colleagues on standby who could take on the responsibilities if the absence was more acute (Table A1). The respondents stated feeling prepared to help the students with counting carbohydrates (38.9%), dosing Healthcare 2023, 11, 251 9 of 24 9 of 24 insulin (39.1%), helping during high (52.1%) or low (60.3%) blood sugar levels, ins chock (35.2%), or activities (36.3%) (Figure 7 and Table A1). teachers (34.5%). Additionally, respondents reported on the ability to confer with col- leagues about the student’s diabetes (Figure 6) (42.6%). Figure 6. Participants' reports on experiences with colleagues. If the person with the primary responsibility of the child’s diabetes care was absent from the school, 51.0% stated having colleagues who could take on the responsibilities for diabetes care if planned in advance, and 48.4% stated having colleagues on standby who could take on the responsibilities if the absence was more acute (Table A1). The respond- ents stated feeling prepared to help the students with counting carbohydrates (38.9%), dosing insulin (39.1%), helping during high (52.1%) or low (60.3%) blood sugar levels, insulin chock (35.2%), or activities (36.3%) (Figure 7) (Table A1). 59.5% 44.4% 42.6% 34.5% 42.6% 51.0% 48.4% 0% 20% 40% 60% 80% 100% Prepared to deliver information to colleagues Prepared to deliver information with substitutes teachers Adequate time to deliver information to colleagues Adequate time to deliver information to substitute teachers Able to discuss/confer with colleagues about the students' diabetes There are other colleagues to take over, if you intentionally are not at school There are other colleagues to take over, if you unplanned are not at school Figure 6. Participants’ reports on experiences with colleagues. care 2023, 11, x 9 o Figure 7. Proportion of participants prepared to help the student. 3.4. glycemia, and 78.5% of sc the students in diabetes m 3.3. Education and Knowledge The respond- g prepared to help the students with counting carbohydrates (38.9%), 9.1%), helping during high (52.1%) or low (60.3%) blood sugar levels, 2%), or activities (36.3%) (Figure 7) (Table A1). Figure 6. Participants’ reports on experiences with colleagues. Figure 7. Proportion of participants prepared to help the student. 38.9% 39.1% 52.1% 60.3% 35.2% 36.3% 0% 20% 40% 60% 80% 100% Count carbohydrates Dosing insulin During high blood sugar During low blood sugar During insulin chock/hypoglycemic reaction Dosing insulin during activities Figure 7. Proportion of participants prepared to help the student. s' reports on experiences with colleagues. Figure 6. Participants’ reports on experiences with colleagues. s' reports on experiences with colleagues. Figure 6. Participants’ reports on experiences with colleagues. Figure 7. Proportion of participants prepared to help the student. Figure 7. Proportion of participants prepared to help the student. Healthcare 2023, 11, 251 10 of 24 10 of 24 3.4. Food (Access) p Are there any rules 3.4. Food (Access) p Are there any rules In total, 72.9% of schools did not have rules regarding eating or drinking during the day (Tables 3 and A1). Those who did have rules allowed the students with diabetes to eat or drink as needed if they consulted an adult ﬁrst (data not shown). y to use the restroom? 96.2% Are students able to manage their diabetes, for example eat/drink, measure blood sugar without losing considerable time during class? 85.7% 3.5. Physical Activity Are students able to m blood sugar without lo The majority of schools reported having no diabetes-related limitations in school activities, including physical activity (95.0%). In some situations, for example excursions, 69.7% of schools were able to bring in extra personnel (Tables 3 and A1). blood sugar without losing considerable time during recess? How is information from the parents delivered? 37.4% 3 6 Self-Management 3.6. Self-Management At 85.7% of the School/Home Cooperation In total, 37.4% of the schools had information delivered face-to-face to the parents The respondents reported that 81.4% of parents gave relevant information on diabetes management and that 84.6% gave the necessary supplies for diabetes management. The parents were perceived to be neither too much involved (15.3%) nor too little involved (13.7%). In addition, 55.1% of the respondents reported having adequate time to cooperate with the parents, and 77.4% found it easy to contact parents during the school day (Table 3 Figure 9 and Table A1). Figure 8. Proportion of participants not feeling unsafe. 63.0% 58.7% 59.9% 61.4% 0% 20% 40% 60% 80% 100% Supporting a student with diabetes A student has low blood sugar Advising a student on diet Going on an excursion with a class outside the school Figure 8. Proportion of participants not feeling unsafe. Figure 8 Proportion of participants not feeling unsafe 63.0% 58.7% 59.9% 61.4% 0% 20% 40% 60% 80% 100% Supporting a student with diabetes A student has low blood sugar Advising a student on diet Going on an excursion with a class outside the school Fi 8 P ti f ti i t t f li f Figure 8. Proportion of participants not feeling unsa Figure 8. Proportion of participants not feeling unsafe. 3.6. Self-Management At 85.7% of the At 85.7% of the schools, the respondents estimated that students with diabetes did not lose considerable time during class due to diabetes management, and 84.9% of respondents found that these children did not lose time during recess. The majority of schools made it possible for students to have blood sugar levels measured during the day (97.4%). Most schools had no rules regarding use of restroom (96.2%) or use of mobile phones at all times (53.0%). The respondents reported on the extent to which they felt unsafe in performing tasks related to diabetes management, (1 or 2 on a ﬁve-point scale) for supporting a student with diabetes (63.0%), supporting a student with low blood sugar level (58.7%), supervising a student on diet (59.9%), or going on school excursions with a student with diabetes (61.4%) (Table 3, Figure 8 and Table A1). p not lose considerable time during class due to diabetes management, and 84.9% spondents found that these children did not lose time during recess. The majo schools made it possible for students to have blood sugar levels measured during th (97.4%). Most schools had no rules regarding use of restroom (96.2%) or use of m phones at all times (53.0%). The respondents reported on the extent to which th unsafe in performing tasks related to diabetes management, (1 or 2 on a five-point for supporting a student with diabetes (63.0%), supporting a student with low blood level (58.7%), supervising a student on diet (59.9%), or going on school excursions student with diabetes (61.4%) (Table 3) (Figure 8) (Table A1). Figure 8. Proportion of participants not feeling unsafe. 3.7. School/Home Cooperation In total, 37.4% of the schools had information delivered face-to-face to the p The respondents reported that 81.4% of parents gave relevant information on d management and that 84.6% gave the necessary supplies for diabetes manageme parents were perceived to be neither too much involved (15.3%) nor too little in (13.7%). In addition, 55.1% of the respondents reported having adequate time to co with the parents, and 77.4% found it easy to contact parents during the school day 3) (Figure 9) (Table A1). 63.0% 58.7% 59.9% 61.4% 0% 20% 40% 60% 80% 100% Supporting a student with diabetes A student has low blood sugar Advising a student on diet Going on an excursion with a class outside the school Figure 8. Proportion of participants not feeling unsafe. 3.7. 4 Discussion 4. Discussion 4. Discussion A large proportion of the Danish primary schools were found to have organize diabetes management in accordance with the ISPAD guidelines. The results indica the majority of the schools are capable of including children with diabetes on equal with their peers as stipulated in the Danish national law. The majority of schools least one person available to support diabetes management during the day, but the A large proportion of the Danish primary schools were found to have organized their diabetes management in accordance with the ISPAD guidelines. The results indicate that the majority of the schools are capable of including children with diabetes on equal terms with their peers as stipulated in the Danish national law. The majority of schools had at least one person available to support diabetes management during the day, but the results also suggest several areas for improvement of diabetes management in Danish schools. least one person available to support diabetes management during the day, but the r also suggest several areas for improvement of diabetes management in Danish scho Only a quarter of the schools have adopted specific guidelines for diabetes ma ment, and only around 60% had an action plan in case of hypoglycemia, which can b threating if not treated promptly. At many of the schools, the personnel had receiv abetes specific training. Nonetheless, they still felt inadequate and unprepared in s key aspects of diabetes management, such as managing blood sugar levels, diet, and situations. These results are supported by a German study from 2020 investigating ers’ perceptions of diabetes management, which found low confidence among te when doing daily diabetes management and low institutional support, such as w instructions and diabetes specific policies [17] A Swedish study from 2017 investi gg p g Only a quarter of the schools have adopted speciﬁc guidelines for diabetes manage- ment, and only around 60% had an action plan in case of hypoglycemia, which can be life- threating if not treated promptly. At many of the schools, the personnel had received diabetes speciﬁc training. Nonetheless, they still felt inadequate and unprepared in several key aspects of diabetes management, such as managing blood sugar levels, diet, and acute situations. 4 Discussion 4. Discussion These results are supported by a German study from 2020 investigating teachers’ perceptions of diabetes management, which found low conﬁdence among teachers when doing daily diabetes management and low institutional support, such as written instruc- tions and diabetes speciﬁc policies [17]. A Swedish study from 2017 investigating school management in diabetes from a parental perspective found similar proportions of schools with a written action plan [18]. instructions and diabetes specific policies [17]. A Swedish study from 2017 investi school management in diabetes from a parental perspective found similar proporti schools with a written action plan [18]. Most respondents saw no limitations for children with diabetes in school acti including physical activity. Still, in half of schools, the respondents reported feelin derprepared in supporting the children during physical activity. Less than 25% schools allocate extra hours to support students with diabetes, which indicates that schools do not allocate extra hours for support. This might place a huge burden teachers who support the child as they must do so along with their regular teaching It may also place greater demands on the parents to support the child during th Kingod et al. reported that some parents receive several phone calls during the day th h l t t h l i di b t t d th d i i th d d p Most respondents saw no limitations for children with diabetes in school activities, including physical activity. Still, in half of schools, the respondents reported feeling underprepared in supporting the children during physical activity. Less than 25% of the schools allocate extra hours to support students with diabetes, which indicates that many schools do not allocate extra hours for support. This might place a huge burden on the teachers who support the child as they must do so along with their regular teaching hours. It may also place greater demands on the parents to support the child during the day. Kingod et al. reported that some parents receive several phone calls during the day from the school to get help in diabetes management, and the advice is thus very dependent on the parents receiving diabetes management training [4]. Haslund Thomsen et al. found that lower levels of conﬁdence and knowledge of diabetes management among school personnel greatly affect how much parents have to be a part of the school day [19]. 3 7 School/Home Cooperation 3.7. School/Home Cooperation 3.7. School/Home Cooperation In total, 37.4% of the schools had information delivered face-to-face to the pa The respondents reported that 81.4% of parents gave relevant information on dia management and that 84.6% gave the necessary supplies for diabetes managemen parents were perceived to be neither too much involved (15.3%) nor too little inv (13.7%). In addition, 55.1% of the respondents reported having adequate time to coop with the parents, and 77.4% found it easy to contact parents during the school day In total, 37.4% of the schools had information delivered face-to-face to the parents. The respondents reported that 81.4% of parents gave relevant information on diabetes management and that 84.6% gave the necessary supplies for diabetes management. The parents were perceived to be neither too much involved (15.3%) nor too little involved (13.7%). In addition, 55.1% of the respondents reported having adequate time to cooperate with the parents, and 77.4% found it easy to contact parents during the school day (Table 3, Figure 9 and Table A1). Healthcare 2023, 11, 251 althcare 2023, 11, x 11 of 24 1 Figure 9. Proportion of schools with parent involvement . 55.1% 15.3% 13.7% 77.4% 0% 20% 40% 60% 80% 100% Having adequate time to cooperate with the parents Parents being too involved Parent being too little involved It easy to get in contact with the parents during a school day Figure 9. Proportion of schools with parent involvement . Figure 9. Proportion of schools with parent involveme Figure 9. Proportion of schools with parent involvement . 4.1. Strengths and Limitations To the best of our knowledge, this study is the ﬁrst to explore school personnel’s perspectives on and perceptions of diabetes management in a nationwide Danish school setting. The study is also the ﬁrst to provide insight into the organization and daily management of diabetes in schools in Denmark. g Knowledge on the subject is scarce. We were inspired to use the ISPAD guidelines as it was important to use a questionnaire encompassing a wide range of many aspects of diabetes management in a school setting. Using the ISPAD guidelines ensured that all key themes were considered. The questionnaire in this study included questions related to all aspects of the ISPAD guidelines, except for psychosocial environment. We wanted to explore treatment aspects of diabetes management, knowledge and feelings of safety in diabetes management for school personnel, but we were not able to fairly evaluate the efforts of the school to accommodate the psychosocial aspects of school life for children with diabetes. The student’s psychosocial environment is important and should be explored in future studies. Currently, there are no ofﬁcial guidelines on this topic in Denmark or in the Nordic countries. Therefore, we deemed the internationally applicable ISPAD guidelines suitable for comparison for two reasons. First, they present a set of broad recommendations to schools worldwide. Second, they provide caregivers (at home and in school) with standards to guide diabetes management [12,13]. The guidelines encompass many key aspects of diabetes management, but they may not address the Danish school system in all aspects. For example, primary schools in Denmark are not obligated to provide lunch for the children. Therefore, when we evaluate food access in primary schools according to the ISPAD guidelines, we are evaluating whether the food is accessible in general (as provided by the parents), but not whether the school provides food. Schools are only obligated to provide lunch in boarding schools, where the children live at the premises. Further, our survey included multiple questions on inclusion of children with diabetes during a school day, rather than just asking about inclusion in physical education classes as in the ISPAD guidelines. This provided us with a broader insight into all aspects of a school day. g g y The present study was further strengthened by the pilot-testing of the questionnaire among several groups of potential respondents prior to the actual survey. 4 Discussion 4. Discussion the school to get help in diabetes management, and the advice is thus very depend the parents receiving diabetes management training [4]. Haslund Thomsen et al. that lower levels of confidence and knowledge of diabetes management among personnel greatly affect how much parents have to be a part of the school day [19]. Approximately 80% of schools have one or more personnel present every day means that 20% of the schools may lack experienced support for children with dia Approximately 80% of schools have one or more personnel present every day. This means that 20% of the schools may lack experienced support for children with diabetes. Additionally, only about half of the schools explicitly stated that diabetes management is not considered a challenge, but a considerable proportion of personnel reported to feel unprepared in performing speciﬁc diabetes management tasks. This may place heavy demands on the personnel, both professionally and personally. Our data showed that the Healthcare 2023, 11, 251 12 of 24 12 of 24 respondents in 40% of schools felt less safe when supporting students with diabetes. A Swedish study from 2017 also found limited personnel to support the child with diabetes management during the school day [18]. Lack of training and little knowledge of diabetes management was also reported in a Spanish study, which found that 43.2% of survey respondents did not have enough knowledge about type 1 diabetes although they had previously had or currently had children or adolescents with type 1 diabetes [20]. Our results are similar to a German study, which also found limited communication between teachers and parents about medical needs and a lack of written policies and plans on diabetes management [17]. These results indicate that a discrepancy exists between what the schools convey to provide for the children and the more personal experiences of navigating diabetes management among the school personnel. The results also suggest a lack of structural support in diabetes management, such as appropriate guidelines, action plans, face-to-face meeting with parents, and insufﬁcient time to cooperate with the parents. 4.1. Strengths and Limitations A large pro- portion of the ISPAD guidelines are contained in the Danish national law, such as the legal obligation to provide a school day on equal terms (see Table A1, section “Law and equal opportunity”) [12]. This requires schools to support the child with diabetes man- agement on a daily basis, e.g., measurement of blood sugar level. The ISPAD guidelines state that personnel should be legally authorized to support the child. The Danish law states that schools are legally obligated to support the child. This discrepancy between the ISPAD guidelines and Danish national law makes it difﬁcult to fulﬁl this part of the ISPAD guidelines. Therefore, tailoring the ISPAD guidelines to a Danish context would be appropriate. Healthcare 2023, 11, 251 13 of 24 13 of 24 Our study also has some limitations. As it was not possible to identify where children with diabetes go to school, it was not possible to target the survey at only schools with children with diabetes enrolled. Therefore, we were able to examine only non-respondents for demographic data, but we could not calculate response rates for schools with children with diabetes. There was a risk of misclassifying the respondents based on their reports on students with diabetes enrolled, as it is currently not possible to verify these reports. The self-reported data in this study hold a risk of underreporting or overreporting among respondents. The respondents were school personnel with knowledge on children with diabetes. In many cases (n = 265), the respondent was the school principal and not the child’s closest teacher. Thus, we cannot rule out that this could have inﬂuenced the answers and thereby have underestimated or overestimated certain aspects of diabetes management. y p g We invited all primary schools in Denmark to participate in the study. In total, 524 schools were eligible for the study based on the enrolment of children med diabetes. Several factors could have inﬂuenced the selection into the study. The data collection was conducted during the COVID-19 pandemic, where restrictions burdened the daily management in schools, and new teaching situations could have affected the school’s willingness to participate. Still, primary schools tend to be busy in general, and similar participation rates have been found in other Danish studies [21,22]. Our analysis of non- respondents showed there were more non-respondents in the regions close to the capitol. The reasons for this are unclear. 4.1. Strengths and Limitations One explanation could be that the eastern part of the country was battling higher numbers of COVID-19 cases for an extended period during the data collection for this study and may not have prioritized participation, or it could be a management decision not to participate. More private primary schools participated than public primary schools. We hypothesize that this might be related to variations in available resources during school days between public and private primary schools. Several schools might only have one child with diabetes and may not consider participation as relevant. Lastly, the person receiving the questionnaire initially may not have known whether or not the school had children with diabetes, but this person still had to decide whether or not to participate. Therefore, the study population might underrepresent the true population, which holds a risk of underestimating or overestimating the results. g g Future research using this data should include hypotheses testing, for example con- cerning how the characteristics of diabetes management and the perceptions of the school personnel are associated. 4.2. Perspectives to Future Work in the Field Only a third of the respondents reported having enough time to prepare for and get acquainted with diabetes management. Some schools had appointed a contact person (other than the teacher) for the child. Schools with experience in diabetes management could also serve as advisors for schools without such experience. The situation could be less vulnerable if the school had a small team of teachers and other personnel who could help the child if the assigned contact person was absent (as there would be someone else to cover the diabetes management for a short period of time). Several respondents felt inadequate and unprepared when supporting a student with diabetes. It is essential that school personnel feel capable of caring for the students. One option to strengthen this capability could be setting up different types of learning options to help the personnel, such as e-learning modules, revisiting the training program for diabetes management, apps for managing diabetes, and tele-support from diabetes clinics and other schools with similar experience. Another option could be to organize visits from diabetes clinics at the schools as part of the training of teachers (instead of making the teachers go to the clinics) as this is likely to enhance accessibility and participation, including repeat sessions and brush-up courses. Working with the personnel’s qualiﬁcations and feelings of safety in managing daily diabetes tasks could alleviate the burden on both school personnel and parents. A third option could be to organize network meetings with various stakeholders that are relevant for children with diabetes to explore new ideas for diabetes management and to Healthcare 2023, 11, 251 14 of 24 14 of 24 keep up with new developments in diabetes management. National guidelines need to be developed in Denmark to accommodate the difﬁculties experienced by school personnel and to ensure that the necessary resources are available for the personnel to feel capable of providing diabetes management support. National guidelines are important because they consider the context and the circumstances that are speciﬁc for Denmark. Future studies should also focus on validating the KIDS questionnaire, which has not yet been validated. This is likely because no similar questionnaires seem to exist in Danish to validate against. Validating the questionnaire would make us able to verify the results and conduct follow-up studies with repeated measures. Appendix A Table A1. Overview of the ISPAD guidelines, KIDS questionnaire and satisfactory responses, (n = 524). Table A1. Overview of the ISPAD guidelines, KIDS questionnaire and satisfactory responses, (n = 524). Domain: Law and Equal Opportunity SPAD Guidelines KIDS Questionnaire Items Questionnaire Response Categories Criteria for Satisfactory Response Proportion of Schools with Satisfactory Response • Irrespective of age and ability, all students with diabetes at school must receive the support, encouragement, and supervision of school personnel • Optimal management of diabetes at school is a prerequisite for optimal school performance, including learning, and for the avoidance of diabetes-related complications • Maintaining normo-glycemia during school hours is important and day-to-day glycemic targets should not differ from any other setting • Diabetes is classiﬁed by “common law” as a disability and legal frameworks exist in many nations to ensure the child has equal opportunity to participate in all aspects of school life • Many children with diabetes worldwide do not have ready access to insulin, diabetes supplies, or education. They should be given the same opportunity as other children to obtain an education • School exams or other assessment situations are associated with stress and increased risk of acute transient episodes of hypoglycemia or hyperglycemia that can affect performance • Administration, or careful supervision, of insulin administration requires school personnel to be legally authorized with informed parental consent • Schools should make “reasonable adjustments” to facilitate prescribed medical care to allow for children with type 1 diabetes (T1D) to participate in education on the same basis as their peers. • Schools have a non-delegable duty of care to their students, and school personnel should take reasonable care to protect them from harm that is reasonably foreseeable. Does the school have a guideline/policy for handling chronic illness among students? □ Yes □ No Yes 21.7% Does the school have a guideline/policy for handling diabetes among students? □ Yes □ No Yes 25.9% Does the school allocate extra hours to students with diabetes? State approximate amount of time for each age group: 1. Students in 0th grade 2. Students in 1st–3rd grade 3. Students in 4th–6th grade 4. Students in 7th–9th grade 5. Students in 10th grade Text box >1 h 1. 19.5% 2. 25% 3. 23.7% 4. 24% 5. Appendix A 21% On a scale from 1 to 5 (5 being the highest), to what extent do you experience being able to include students with diabetes in a school day on equal terms with students without diabetes during . . . . 1. Academic subjects 2. Physical education classes 3. Day time excursions 4. Overnight excursions Likert scale: 1–5 For all: 4–5 1. 86.9% 2. 80.1% 3. 84.5% 4. 74.6% 5. Conclusions In this study, diabetes management was found to be a challenging task for the staff in primary schools, as some personnel felt inadequate and unprepared in several key aspects of diabetes management. Our results indicate a lack of structural support in diabetes management. The respondents pointed to the absence of guidelines and action plans, few face-to-face meetings with parents, and little time to cooperate with the parents. Author Contributions: Conceptualization, A.Ø.N. and A.A.; methodology, A.Ø.N. and A.A.; formal analysis, A.Ø.N.; investigation, A.Ø.N.; writing (original draft), A.Ø.N.; writing (revisions), A.Ø.N, A.A., K.K., L.B.J., M.K.I., M.M., K.A.P., D.G., S.H. and A.J.S.; supervision, A.A.; project administration, A.A. and D.G. All authors have read and agreed to the published version of the manuscript. Funding: This study was indirectly funded by the ﬁve Steno diabetes centers in Denmark. These are partly funded by an unrestricted grant from the Novo Nordisk Foundation and the ﬁve Danish regions. The Danish regions and the Novo Nordisk Foundation did not have any part in designing or conducting the study or writing the article. Institutional Review Board Statement: The study was reported to the Danish Data Protection Agency, Central Denmark Region. Informed Consent Statement: There is no formal agency to approve questionnaire-based surveys in Denmark. The participants received written information stating that participation was voluntary and that data were treated conﬁdentially. The study complies with national standards for data protection. Data Availability Statement: Data can be made available upon request. Acknowledgments: We would like to thank all participating schools for taking the time to contribute to the study. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 15 of 24 Healthcare 2023, 11, 251 Table A1. Cont. Table A1. Cont. Table A1. Cont. Domain: Individualized diabetes plan SPAD guidelines KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response • All young people with diabetes at school should have an individualized diabetes management plan (DMP) in place which must be developed and agreed with parents in advance. • The DMP should be reviewed and amended as and when necessary, according to the needs of the young person with diabetes, and/or at least annually. • The type of insulin regimen used at school should be tailored to the needs, ability, and wishes of the child/family and should not be dictated by the school resources. • Whether children can self-manage certain aspects of their diabetes and/or self-administer insulin is not necessarily age dependent and can only be determined by the parent and health care team. • Speciﬁc arrangements may need to be put in place (including access to BG testing equipment; hypoglycemia ﬁrst-aid pack) for exams Does the school have an action plan, in case a students with diabetes experiences low blood sugar/insulin chock (hypoglycemic reaction)? □ Yes □ No □ Don’t know Yes 61.7% Does the school have one or more persons able to help students with diabetes administer insulin/adjust insulin pump if necessary? □ Yes, there is one person at the school □ Yes, there are more than one person at the school □ No □ Don’t know Yes 78.5% Domain: Individualized diabetes plan • All young people with diabetes at school should have an individualized diabetes management plan (DMP) in place which must be developed and agreed with parents in advance. • All young people with diabetes at school should have an individualized diabetes management plan (DMP) in place which must be developed and agreed with parents in advance. • The DMP should be reviewed and amended as and when necessary, according to the needs of the young person with diabetes, and/or at least annually. • The type of insulin regimen used at school should be tailored to the needs, ability, and wishes of the child/family and should not be dictated by the school resources. • Whether children can self-manage certain aspects of their diabetes and/or self-administer insulin is not necessarily age dependent and can only be determined by the parent and health care team. ISPAD Guidelines • Schools should make “reasonable adjustments” to facilitate prescribed medical care to allow for children with type 1 diabetes (T1D) to participate in education on the same basis as their peers. • Schools have a non-delegable duty of care to their students, and school personnel should take reasonable care to protect them from harm that is reasonably foreseeable. Healthcare 2023, 11, 251 16 of 24 Table A1. Cont. Table A1. Cont. • Speciﬁc arrangements may need to be put in place (including access to BG testing equipment; hypoglycemia ﬁrst-aid pack) for exams Healthcare 2023, 11, 251 17 of 24 Table A1. Cont. Domain: Education and knowledge idelines KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response ools are responsible for adequately training their personnel ut diabetes, but the content of the training is the responsibility of health care team and parent. ool personnel should be aware of the signs/symptoms of oglycemia, and a “ﬁrst-aid hypoglycemia” management pack uld be available at all times. Clear instructions for managing oglycemia should be provided. od glucose (BG) monitoring is central to achieving optimal emic control at school and must be familiar to school personnel. asonable adjustments” include school personnel support with ulin administration, as well as understanding and knowledge of betes technologies (including continuous glucose monitoring M] devices and insulin pump settings). ool personnel should be able to manage appropriately the effects ow and high BG levels according to parent and health care team ructions. Did teachers and other personnel receive teaching regarding supporting students with diabetes? □ Yes, all personnel have received teaching □ Yes, personnel, involved with students with diabetes have received teaching □ No □ Don’t know □ Feel free to elaborate:_____ Yes 77.2% What do you think is the biggest challenge for the school, in regards to management and support to students with diabetes? □ It is not considered a challenge to have students with diabetes at the school □ Lack of teaching/instruction in management of diabetes among students □ Teachers cannot/will not take on the responsibility for students with diabetes □ There is not a teacher or pedagogue with knowledge of diabetes present at school every day □ The school nurse is not present every day □ The school has a lot of students with chronical illnesses/health challenges □ The school needs guidelines in this area □ Other:________ It is not considered a challenge to have students with diabetes at the school 54.3% Domain: Education and knowledge Domain: Education and knowledge KIDS questionnaire items • Schools are responsible for adequately training their personnel about diabetes, but the content of the training is the responsibility of the health care team and parent. Table A1. Cont. • Schools are responsible for adequately training their personnel about diabetes, but the content of the training is the responsibility of the health care team and parent. • School personnel should be aware of the signs/symptoms of hypoglycemia, and a “ﬁrst-aid hypoglycemia” management pack should be available at all times. Clear instructions for managing hypoglycemia should be provided. • School personnel should be able to manage appropriately the effects of low and high BG levels according to parent and health care team instructions. Healthcare 2023, 11, 251 18 of 24 Table A1. Cont. Domain: Education and knowledge KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response On a scale from 1–5 (5 being the highest), to what extent do you feel like you have adequate knowledge about . . . 1. Acute situations 2. Blood sugar levels 3. Physical activity 4. Administering glucagon 5. When to call 112 6. Diet and carbohydrate counting 7. Dosing insulin 8. The students diabetes equipment Likert scale: 1–5 For all: 4–5 1. 49.0% 2. 57.1% 3. 56.0% 4. 22.4% 5. 61.5% 6. 43.8% 7. 34.1% 8. 39.3% On a scale from 1–5 (5 being the highest), to what extent do you feel you have adequate time to . . . 1. Familiarize yourself with diabetes in general 2. Familiarizing yourself with supporting a speciﬁc student with diabetes 3. Supporting students with diabetes during school hours 4. Support during physical activity 5. Support during recess 6. Support during excursions Likert scale: 1–5 For all: 4–5 1. 38.4% 2. 44.2% 3. 45.5% 4. 37.9% 5. 37.6% 6. 43.7% Domain: Education and knowledge KIDS questionnaire items • Schools are responsible for adequately training their personnel about diabetes, but the content of the training is the responsibility of the health care team and parent. • School personnel should be aware of the signs/symptoms of hypoglycemia, and a “ﬁrst-aid hypoglycemia” management pack should be available at all times. Clear instructions for managing hypoglycemia should be provided. • Blood glucose (BG) monitoring is central to achieving optimal glycemic control at school and must be familiar to school personnel. • “Reasonable adjustments” include school personnel support with insulin administration, as well as understanding and knowledge of diabetes technologies (including continuous glucose monitoring [CGM] devices and insulin pump settings). Table A1. Cont. • School personnel should be able to manage appropriately the effects of low and high BG levels according to parent and health care team instructions. • School personnel should be able to manage appropriately the effects of low and high BG levels according to parent and health care team instructions. 19 of 24 Healthcare 2023, 11, 251 Table A1. Cont. Domain: Education and knowledge KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response y of k nel. th e of g ects am On a scale from 1–5 (5 being the highest), to what extent do you experience . . . 1. Being well prepared to deliver information to colleagues 2. Being well prepared to deliver information to substitutes teachers 3. You have adequate time to deliver information to colleagues 4. You have adequate time to deliver information to substitute teachers 5. You are able to discuss/confer with colleagues about the students’ diabetes 6. There are other colleagues to take over, if you intentionally are not at school 7. There are other colleagues to take over, if you unplanned are not at school Likert scale: 1–5 For all: 4–5 1. 59.5% 2. 44.4% 3. 42.6% 4. 34.5% 5. 42.6% 6. 51.0% 7. 48.4% On a scale from 1–5 (5 being the highest), to what extent do you feel you are prepared to . . . 1. Help the student with counting carbohydrates 2. Help the student with dosing insulin 3. Help the student during high blood sugar 4. Help the student during low blood sugar 5. Help the student during insulin chock/hypoglycemic reaction 6. Help the student dosing insulin during activities Likert scale: 1–5 For all 4–5 1. 38.9% 2. 39.1% 3. 52.1% 4. 60.3% 5. 35.2% 6. 36.3% Domain: Education and knowledge KIDS questionnaire items • School personnel should be able to manage appropriately the effects of low and high BG levels according to parent and health care team instructions. Healthcare 2023, 11, 251 20 of 24 Table A1. Cont. Table A1. Cont. Domain: Food (Access) uidelines KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response ess to food in schools is an integral part of enabling children to w normally and balance their insulin and food intake of food pictures may help school personnel assess food servings d their estimated carbohydrate content Are there any rules for where and when students with diabetes are allowed to eat/drink? □ Yes, they are only allowed to eat/drink in consultation with personnel □ No, we do not have any rules for □ Don’t know No, we do not have any rules 72.9% Domain: Psychical activity uidelines KIDS questionnaire items Responsecategories in the questionnaire Criteria for satisfactory response Proportion of schools with satisfactory response young people with T1D should be given the same opportunities heir peers to participate safely in all sports and physical activity Are there activities at school where students with diabetes are limited/restricted? □ No, there are no limitation/restrictions □ Yes, daytime excursions with the school □ Yes, overnight excursions □ Yes, physical education (PE) class □ Yes, swimming lessons □ Yes, bigger sports events □ Yes, at after school programs for older students □ Yes, at after school programs for younger students □ Other activities:_____ No, there are no limitations/restrictions 95.3% It is possible for the school to have extra personnel on hand in some situations, for example excursions? □ Yes □ No Feel free to elaborate:_______ Yes 69.7% Domain: Food (Access) Domain: Food (Access) • Access to food in schools is an integral part of enabling children to grow normally and balance their insulin and food intake • Use of food pictures may help school personnel assess food servings and their estimated carbohydrate content • All young people with T1D should be given the same opportunities as their peers to participate safely in all sports and physical activity Healthcare 2023, 11, 251 21 of 24 Table A1. Cont. KIDS questionnaire items Domain: Self-management Domain: Self-management delines KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response ng people with diabetes must be allowed to monitor their BG s, administer insulin, and to treat low/high BG values at any during the school day, with adult supervision if needed Are the students able to measure blood sugar levels at the school? □ Yes, at all places at school □ No, the student is not able to measure blood sugar at school □ Yes, in the classroom □ Yes, in the ofﬁce area □ Yes, at the school nurse ofﬁce □ Other places:_______ Yes 97.4% Are there any rules for where and when students with diabetes are allowed to use their phones? □ No, they are allowed to use their phones in class and recess □ Yes, some rules □ Feel free to elaborate on any rules:____ No, they are allowed to use their phones in class and recess 53.0% Are there any rules for when and where students with diabetes are allowed to use the restroom? □ No □ Don’t know □ Yes, these rules apply:____ No 96.2% Are students able to manage their diabetes, for example eat/drink, measure blood sugar without losing considerable time; 1.During class? 2. During recess? □ Yes □ No □ Don’t know Yes 1. 85.7% 2. 84.9% On a scale from 1–5 (5 being the highest), to what extent do you feel unsafe if... 1. You have to support a student with diabetes 2. A student has low blood sugar 3. You have to advise a student on diet 4. You have to go on an excursion with a class outside the school, where there is a student with diabetes Likert scale: 1–5 For all: 1–2 1.63.0% 2. 58.7% 3. 59.9% 4. 61.4% ISPAD guidelines • Young people with diabetes must be allowed to monitor their BG levels, administer insulin, and to treat low/high BG values at any time during the school day, with adult supervision if needed Healthcare 2023, 11, 251 22 of 24 Table A1. Cont. • Peer relations, local social stigma, racial and religious perspectives can be a burden to patients and families with T1D. KIDS questionnaire items Domain: School/home cooperation Domain: School/home cooperation p SPAD guidelines KIDS questionnaire items Questionnaire response categories Criteria for satisfactory response Proportion of schools with satisfactory response • With a mutually supportive, collaborative approach between parents and the child’s health care team and schools, and with advancements in communication technology, for example, providing sensor glucose data in real time to parents, there is a real opportunity for a truly cooperative approach. • Parents cannot be expected to “ﬁll the gap” of school resources and attend to their child’s medical management during the school day. • Successful diabetes management at school heavily depends on effective communication and problem-solving with the family and schools should clarify expectations and coordinate communication How is information from the parents delivered? □ In a meeting between parents and school □ As written materials □ In AULA or other online platforms □ Don’t know □ Anything else:_____ In a meeting between parents and school 37.4% Do parents of students with diabetes hand out; 1. Relevant information about management of diabetes? 2. The necessary supplies (glucagon, strips for blood glucose measurements, snacks, juice/dextrose? □ Yes □ No □ Some parents do not □ Don’t know Yes 1. 81.4% 2. 84.6% On a scale from 1–5 (5 being the highest), to what extent do you experience . . . 1. Having adequate time to cooperate with the parents 2. Parents being too involved 3. Parent being too little involved 4. It easy to get in contact with the parents during a school day Likert scale: 1–5 For all: 4–5 1. 55.1% 2. 15.3% 3. 13.7% 4. 77.4% KIDS questionnaire items Healthcare 2023, 11, 251 23 of 24 Table A1. Cont. Domain: Psychosocial environment Domain: Psychosocial environment Domain: Psychosocial environment Questionnaire response categories ISPAD guidelines KIDS questionnaire items • Peer relations, local social stigma, racial and religious perspectives can be a burden to patients and families with T1D. • Young people with diabetes have a signiﬁcantly increased risk of being exposed to issues of discrimination, which may impact on self-esteem and cause feelings of stigmatization. • Schools provide a unique opportunity to identify and treat psychological problems in young people with diabetes and close liaison between school personnel and health care professionals is recommended. • Some studies report higher rates of psychological problems such as depression and eating disorders in young people with diabetes. 24 of 24 Healthcare 2023, 11, 251 References 1. The International Diabetes Federation. IDF Diabetes Atlas, 10th ed.; The International Diabetes Federation: Brussels, Belgium, 2021. 1. 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Nurs. 2020, 53, e149–e155. [CrossRef] [PubMed] yp 20. Gutiérrez-Manzanedo, J.V.; Laureano, F.C.-S.; Moreno-Vides, P.; de Castro-Maqueda, G.; Fernández-Santosa, J.R.; Ponce-González, J.G. Teachers’ knowledge about type 1 diabetes in south of Spain public schools. Diabetes Res. Clin. Pract. 2018, 143, 140–145. [CrossRef] [PubMed] hers’ knowledge about type 1 diabetes in south of Spain public schools. Diabetes Res. Clin. Pract. 2018, 14 f] [PubMed] 21. Bast, L.S.; Andersen, H.B.; Andersen, A.; Lauemøller, S.G.; Bonnesen, C.T.; Krølner, R.F. School Coordinators’ Perceptions of Organizational Readiness Is Associated with Implementation Fidelity in a Smoking Prevention Program: Findings from the X:IT II Study. Prev. Sci. 2021, 22, 312–323. y 22. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References Bonnesen, C.T.; Toftager, M.; Madsen, K.R.; Wehner, S.K.; Jensen, M.P.; Rosing, J.A.; Laursen, B.; Rod, N.H.; Due, P.; Krølner, R.F. Study protocol of the Healthy High School study: A school-based intervention to improve well-being among high school students in Denmark. BMC Public Health 2020, 20, 95. [CrossRef] [PubMed] y 22. Bonnesen, C.T.; Toftager, M.; Madsen, K.R.; Wehner, S.K.; Jensen, M.P.; Rosing, J.A.; Laursen, B.; Rod, N.H.; Due, P.; Krølner, R.F. Study protocol of the Healthy High School study: A school-based intervention to improve well-being among high school students in Denmark. BMC Public Health 2020, 20, 95. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4210254132	https://www.frontiersin.org/articles/10.3389/fbioe.2022.769830/pdf	English	null	Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters	Frontiers in bioengineering and biotechnology	2,022	cc-by	9,093	†Present address: †Present address: Karolina Haernvall, AstraZeneca AB, Södertälje, Sweden; Patrik Fladischer, Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; Heidemarie Schoeffmann, Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; Sabine Zitzenbacher, Richard Bittner AG, Feldkirchen in Kärnten, Austria §These authors have contributed equally to this work †Present address: Karolina Haernvall, AstraZeneca AB, Södertälje, Sweden; Patrik Fladischer, Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; Heidemarie Schoeffmann, Boehringer Ingelheim RCV GmbH & Co KG, Wien, Austria; Sabine Zitzenbacher, Richard Bittner AG, Feldkirchen in Kärnten, Austria § Specialty section: This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology Specialty section: This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology Received: 02 September 2021 Accepted: 07 January 2022 Published: 26 January 2022 ORIGINAL RESEARCH published: 26 January 2022 doi: 10.3389/fbioe.2022.769830 Keywords: polyester modiﬁcation, enzyme hydrolysis, genetic code engineering, lipase, Thermoanaerobacter thermohydrosulfuricus, TTL, norleucine Edited by: Edited by: Evangelos Topakas, National Technical University of Athens, Greece ‡ORCID Tea Pavkov-Keller, orcid.org/0000-0001-7871-6680 Karl Gruber, orcid.org/0000-0002-3485-9740 Doris Ribitsch, orcid.org/0000-0002-5822-0204 Georg M. Guebitz, orcid.org/0000-0003-2262-487X Birgit Wiltschi, orcid.org/0000-0001-5230-0951 1Acib–Austrian Centre of Industrial Biotechnology, Graz, Austria, 2Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria, 3Institute of Molecular Biosciences, University of Graz, Graz, Austria, 4BioTechMed-Graz, Graz, Austria, 5Field of Excellence BioHealth—University of Graz, Graz, Austria, 6BASF SE, Ludwigshafen am Rhein, Germany, 7Institute for Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-speciﬁc incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met- 142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in signiﬁcantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These ﬁndings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering. Reviewed by: Reviewed by: Vytas Svedas, Lomonosov Moscow State University, Russia Efstratios Nikolaivits, Chalmers University of Technology, Sweden Correspondence: Doris Ribitsch doris.ribitsch@boku.ac.at ‡ORCID Tea Pavkov-Keller, orcid.org/0000-0001-7871-6680 Karl Gruber, orcid.org/0000-0002-3485-9740 Doris Ribitsch, orcid.org/0000-0002-5822-0204 Georg M. Guebitz, orcid.org/0000-0003-2262-487X Birgit Wiltschi, orcid.org/0000-0001-5230-0951 Reviewed by: Vytas Svedas, Lomonosov Moscow State University, Russia Efstratios Nikolaivits, Chalmers University of Technology, Sweden Correspondence: Doris Ribitsch doris.ribitsch@boku.ac.at Karolina Haernvall 1†§, Patrik Fladischer 1,2†§, Heidemarie Schoeffmann 1†, Sabine Zitzenbacher 1†, Tea Pavkov-Keller 1,3,4,5‡, Karl Gruber 3,4,5‡, Michael Schick 6, Motonori Yamamoto 6, Andreas Kuenkel 6, Doris Ribitsch 1,7‡, Georg M. Guebitz 1,7‡ and Birgit Wiltschi 1,2,4‡ Karolina Haernvall 1†§, Patrik Fladischer 1,2†§, Heidemarie Schoeffmann 1†, Sabine Zitzenbacher 1†, Tea Pavkov-Keller 1,3,4,5‡, Karl Gruber 3,4,5‡, Michael Schick 6, Motonori Yamamoto 6, Andreas Kuenkel 6, Doris Ribitsch 1,7‡, Georg M. Guebitz 1,7‡ and Birgit Wiltschi 1,2,4‡ Citation: highly important issue and target for protein engineering (Shirke et al., 2016; Son et al., 2019). The conventional protein engineering approaches use the 20 canonical amino acids (cAAs) prescribed by the genetic code (Steiner and Schwab, 2012). Still providing a very potential tool, conventional protein engineering is limited by the site-chain chemistry offered by the 20 cAAs. An alternative approach for generating more robust enzymes or altering physico-chemical properties is incorporation of non-canonical amino acids (ncAAs) (Hoesl and Budisa, 2012; Wiltschi et al., 2020). The diverse and unusual side chain chemistries (Dumas et al., 2015) makes them attractive building blocks for protein engineering (Link and Tirrell, 2003; Voloshchuk and Montclare, 2009; Zheng and Kwon, 2012; Wiltschi et al., 2020). Under tightly controlled conditions, many ncAAs can be incorporated into a target protein by exploiting the translational machinery of the host (Cowie and Cohen, 1957). This residue-speciﬁc incorporation (SPI) exploits the natural substrate tolerance of aminoacyl-tRNA synthetases (aaRS) for which various methods have been developed (Budisa, 2004; Hoesl et al., 2011; Ngo and Tirrell, 2011). The non-canonical amino acid norleucine (Nle) is an isosteric carba-analog of methionine (Met) that can be incorporated by the SPI method using a Met auxothrophic strain (Hoesl et al., 2011). The carbon atom of the methylene group of Nle is less electronegative than the corresponding sulfur atom of Met (2.48 vs 2.56) (Xie et al., 1995), resulting in a slightly increased hydrophobicity of the methylene group in comparison to the sulfur atom (Thomson et al., 1994). This subtle difference can exert a cumulative impact when several Met residues are globally exchanged for Nle. For instance, Budisa and co-workers successfully used Nle and other Met analogs to probe protein folding (Budisa et al., 1998) and control the aggregation behavior of prion protein (Wolschner et al., 2009). Nle is a comparably inexpensive ncAA. It can be produced in very good quantities by biosynthesis from glucose and the scalability of this bioprocess was previously demonstrated (Anderhuber et al., 2016). h l d d h h Over the last years, several protein engineering strategies have been reported to tailor the enzymes for the non-natural polymeric substrates, thus improving the enzymatic hydrolysis of synthetic polymers. Different approaches have been applied to adapt the active site to the bulky polymers (Thumarat et al., 2012) and to reduce inhibition effects caused by release products (Roth et al., 2014). Citation: Haernvall K, Fladischer P, Schoeffmann H, Zitzenbacher S, Pavkov-Keller T, Gruber K, Schick M, Yamamoto M, Kuenkel A, Ribitsch D, Guebitz GM and Wiltschi B (2022) Residue-Speciﬁc Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters. Front. Bioeng. Biotechnol. 10:769830. doi: 10.3389/fbioe.2022.769830 With the increasing utilization of synthetic polymers in today’s society, polymer surface functionalization and recycling processes have become enormously important. Surface modiﬁcation is generally performed with harsh chemical methods, photo grafting, or energy- intensive plasma processes (Mozetič, 2019). These conventional methods are often toxic, expensive and adversely affect the mechanical properties of the polymer (Hetemi and Pinson, 2017). Enzymatic surface functionalization of polyesters has received increased attention as an environmentally friendlier and highly speciﬁc process (Pellis et al., 2015; Weinberger et al., 2017; Biundo et al., 2018). Limited enzymatic hydrolysis allows targeted surface functionalization while leaving the January 2022 \| Volume 10 \| Article 769830 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Synthetic Biology for Polymer Hydrolysis Haernvall et al. GRAPHICAL ABSTRACT \| GRAPHICAL ABSTRACT \| GRAPHICAL ABSTRACT \| polymer bulk properties unaffected. Complete enzymatic hydrolysis of polyesters, on the other hand, allows speciﬁc recovery of the valuable building blocks for further synthesis especially from blends and composite materials which is otherwise quite challenging (Gamerith et al., 2017; Wei and Zimmermann, 2017). Enzymatic hydrolysis has so far been reported for various synthetic polymers such as poly-L-lactic acid (PLLA) (Pellis et al., 2015; Hajighasemi et al., 2016; Bonifer et al., 2019), polyethylene terephthalate (PET) (Herrero Acero et al., 2011; Sulaiman et al., 2012; Ribitsch et al., 2013; Kawai et al., 2014; Dimarogona et al., 2015; Barth et al., 2016; Yoshida et al., 2016; Kawabata et al., 2017), polybutylene adipate terephthalate (PBAT) (Biundo et al., 2016; Wallace et al., 2017) and polyurethanes (PU) (Acero et al., 2012; do Canto et al., 2019). To date, most enzymes used for polyester hydrolysis are esterases, lipases or cutinases from typical mesophilic or thermophilic compost microorganisms (Kleeberg et al., 2005; Herrero Acero et al., 2011; Ribitsch et al., 2011; Ribitsch et al., 2012a; Ribitsch et al., 2012b; Pellis et al., 2015). Only few enzymes were isolated from anaerobic (Biundo et al., 2016; Perz et al., 2016a; Perz et al., 2016b) and aquatic bacteria (Haernvall et al., 2017a; Haernvall et al., 2017b; Wallace et al., 2017; Haernvall et al., 2018). Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Enzyme Expression and Puriﬁcation After centrifugation for 45 min at 40,000 x g and 4°C the lysates were ﬁltered through 0.2 µm syringe ﬁlters and puriﬁed on Ni- NTA sepharose according to the manufacturer’s protocol (IBA GmbH, Goettingen, Germany). Finally, the buffer was exchanged for 100 mM Tris-HCl pH 7.0 with PD-10 columns (GE Healthcare, Chicago, IL). Enzyme Expression and Puriﬁcation Enzyme Expression and Puriﬁcation The SPI experiment was performed as described previously (Anderhuber et al., 2016). Brieﬂy, M9 minimal medium (M9 MM) containing M9 salt buffer (48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl and 19 mM NH4Cl) was used supplemented with 1 mM MgSO4, 7 µM CaCl2, 1 mg/L thiamine and trace elements (9 µM FeSO4, 3.5 µM MnSO4, 2.5 µM AlCl3, 2 μM CoCl2, 0.4 µM ZnSO4, 0.5 µM Na2MoO4, 0.4 µM CuCl2 and 0.5 µM H3BO4). 100 mg/L ampicillin was added for plasmid maintenance. 20 mM glucose was provided as the carbon source. For Met depletion at OD600~ 3, 3.5 g/L yeast extract (Carl Roth) were supplemented in the medium. Cells were grown in bafﬂed shake ﬂasks at 37°C with vigorous shaking. After depletion of Met, as indicated by growth arrest, the cultures were supplemented with 1 mM of either Met (cAA, parent TTL [Met]) or Nle (Nle, synthetic TTL [Nle]). Gene expression was induced by adding IPTG (VWR International, Vienna, Austria) to a ﬁnal concentration of 0.5 mM and performed with vigorous shaking for 4 h at 30°C. Cells were harvested at 4,000 g for 20 min at 4°C. In this study, we explored the incorporation of a ncAA into TTL to tune the physicochemical properties of the enzyme for enhanced polyester hydrolysis. As a proof of concept, the methionines (Met) of TTL were exchanged globally by its slightly more hydrophobic analog Nle. By the residue-speciﬁc incorporation of Nle into TTL, we aimed at altering the active site and/or the substrate-entrance channel to better ﬁt the polymeric substrate. Synergistically, the enzyme surface was adapted to promote a better interaction/adhesion to the polymer. Two groups of polyester model substrates with various alkyl and ether diols were subjected to hydrolysis to evaluate the effect of Nle incorporation on polyester hydrolysis. g Cell pellets were resuspended in 30 ml Ni-NTA Lysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 7.4) and incubated for 30 min on ice. Cells were disrupted by sonication on ice (Branson Soniﬁer 250, Emerson Electric, St. Louis, MO). The sonication was performed for 6 min with the following settings: duty cycle: 70%, output: 7-8, sonication tip Φ~1 cm. Chemicals and Substrates Alkyl diols (1,5-pentanediol, 1,8-octanediol and 1,12-dodecanediol) and ether diols (diethylene glycol, triethylene glycol and tetraethylene glycol), terephthalic acid (TA) and 5-sulfoisophthalic acid (NaSIP) were purchased from Sigma-Aldrich (St. Louis, MO). Buffer components, bovine serum albumin (BSA) and methanol (HPLC grade) were also purchased from Sigma-Aldrich. All standard chemicals used in this work were purchased from Sigma-Aldrich, Merck KGaA (Darmstadt, Germany) or Carl Roth (Karlsruhe, Germany), if not stated differently. Norleucine was obtained from IRIS Biotech GmbH (Marktredwitz, Germany). Enzymes for cloning and PCR were from Thermo Fisher Scientiﬁc (Waltham, MA). PCRs were performed using TaKaRa Ex Taq®High-Fidelity DNA polymerase (Clontech Laboratories, Inc., Mountain View, CA) or Dream Taq®DNA polymerase (Thermo Fisher Scientiﬁc). PCR primers were ordered from IDT Inc. (Coralville, IA) in standard desalted quality. Citation: Besides, the adsorption/desorption of the enzymes on the polyester has been improved which turned out to be a crucial and rate-limiting step in enzymatic polymer hydrolysis (Herrero Acero et al., 2013; Ribitsch et al., 2013; Ribitsch et al., 2015). Today it is well known that temperatures higher than the glass transition temperature (Tg) increase the hydrolysis rates due to a higher ﬂexibility of the polymer chains and therefore increased accessibility to enzymes (Kawai et al., 2019). For this reason, the thermostability of polymer hydrolysing enzymes has become a In the past, several groups demonstrated that the incorporation of a palette of different ncAAs into target proteins could improve January 2022 \| Volume 10 \| Article 769830 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 2 Synthetic Biology for Polymer Hydrolysis Haernvall et al. enzyme activity (Parsons et al., 1998; Cirino et al., 2003; Hoesl et al., 2011). NcAA incorporation can increase protein stability (Moroder and Budisa, 2010), especially under harsh conditions, such as tolerance to organic solvents (Deepankumar et al., 2014) or extreme pH values in combination with high temperatures (Votchitseva et al., 2006). For an overview of the engineering of enzymes with ncAAs, the interested reader is referred to (Wiltschi et al., 2020). A whole set of different ncAAs was successfully incorporated into the lipase from the anaerobic extreme thermophilic microorganisms Thermoanaerobacter thermohydrosulfuricus (TTL) by Budisa and coworkers (Acevedo-Rocha et al., 2013), demonstrating the positive effect on enzyme activity and stability in harsh conditions. TTL was ﬁrst described by Royter and coworkers (Royter et al., 2009) as an enzyme with activity at a broad temperature (40°C—90°C) and pH range (pH 6.5–10). However, incorporation of ncAAs resulted not only in enhanced hydrolytic residual activity upon treatment with organic solvents by up to 450% and surfactants by up to 1630% (Hoesl et al., 2011), but has also reduced denaturation, alkylating and inhibition processes (Acevedo-Rocha et al., 2013). and E. coli DH5α (E. coli K-12 F– endA1 supE44 thi-1 recA1 relA1 gyrA96 phoA φ80lacZΔM15 Δ(lacZYA-argF)U169 hsdR17 (rK– mK+) λ–; Thermo Fisher Scientiﬁc) was used for cloning experiments and plasmid propagation. Transformation of E. coli was carried out by electroporation as described by Seidman et al. (2001). pTTL was constructed according to Anderhuber et al. (2016). Brieﬂy, the coding sequence of the lipase from T. thermohydrosulfuricus was PCR-ampliﬁed from synthetic DNA (IDT Inc.) using primers BPp244 and BPp245 (Anderhuber et al., 2016). Intact Protein Mass Determination Intact Protein Mass Determination The protein solutions were desalted using Amicon Ultra 0.5 ml centrifugal ﬁlter units (Millipore, Billerica, MA). A ﬁnal protein concentration of 30 pmol/µL was obtained with water containing 5% acetonitrile and 0.1% triﬂuoroacetic acid. The separation of Strains and Plasmids Protein Quantiﬁcation The Bradford based Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, Munich, Germany) with bovine serum albumin as standard was used to determine protein concentrations of puriﬁed enzymes. The protein assay was performed according to the manufacturer’s instruction. Citation: The PCR fragment was inserted into pQE80L (Qiagen, Hilden, Germany) cut with EcoRI and HindIII by Gibson isothermal assembly (Gibson et al., 2009). Enzyme Expression and Puriﬁcation The SPI experiment was performed as described previously (Anderhuber et al., 2016). Brieﬂy, M9 minimal medium (M9 MM) containing M9 salt buffer (48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl and 19 mM NH4Cl) was used supplemented with 1 mM MgSO4, 7 µM CaCl2, 1 mg/L thiamine and trace elements (9 µM FeSO4, 3.5 µM MnSO4, 2.5 µM AlCl3, 2 μM CoCl2, 0.4 µM ZnSO4, 0.5 µM Na2MoO4, 0.4 µM CuCl2 and 0.5 µM H3BO4). 100 mg/L ampicillin was added for plasmid maintenance. 20 mM glucose was provided as the carbon source. For Met depletion at OD600~ 3, 3.5 g/L yeast extract (Carl Roth) were supplemented in the medium. Cells were grown in bafﬂed shake ﬂasks at 37°C with vigorous shaking. After depletion of Met, as indicated by growth arrest, the cultures were supplemented with 1 mM of either Met (cAA, parent TTL [Met]) or Nle (Nle, synthetic TTL [Nle]). Gene expression was induced by adding IPTG (VWR International, Vienna, Austria) to a ﬁnal concentration of 0.5 mM and performed with vigorous shaking for 4 h at 30°C. Cells were harvested at 4,000 g for 20 min at 4°C. Cell pellets were resuspended in 30 ml Ni-NTA Lysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 7.4) and incubated for 30 min on ice. Cells were disrupted by sonication on ice (Branson Soniﬁer 250, Emerson Electric, St. Louis, MO). The sonication was performed for 6 min with the following settings: duty cycle: 70%, output: 7-8, sonication tip Φ~1 cm. After centrifugation for 45 min at 40,000 x g and 4°C the lysates were ﬁltered through 0.2 µm syringe ﬁlters and puriﬁed on Ni- NTA sepharose according to the manufacturer’s protocol (IBA GmbH, Goettingen, Germany). Finally, the buffer was exchanged for 100 mM Tris-HCl pH 7.0 with PD-10 columns (GE Healthcare, Chicago, IL). Strains and Plasmids E. coli B834 (DE3) (E. coli B F– ompT hsdSB(rB– mB–) dcm+ gal met λ(DE3); Merck KGaA) was the host for the SPI experiment January 2022 \| Volume 10 \| Article 769830 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 3 Synthetic Biology for Polymer Hydrolysis Haernvall et al. possible protein variants was carried out on a capillary HPLC system (1200 Agilent, Agilent Technologies) using a PepSwift RP monolithic column (50 × 0.5 mm, Thermo Fisher Scientiﬁc) at a ﬂow rate of 20 µL/min and a column temperature of 60°C. The gradient of solution A (water +0.05% TFA) and B (ACN +0.05% TFA) was performed as follows: 10% B for 5 min, 10%–100% B for 50 min, 100%–10% B for 1 min, 10% B for 15 min. The injection volume was 5 µL. The Thermo LTQ-FT mass spectrometer (Thermo Fisher Scientiﬁc) was operated with an ESI source in positive mode with the following settings: mass range: 300–2000 m/z, resolution 400000, 500 ms injection time, 1 microscan, source voltage 5 kV, capillary voltage 35 V, sheath gas ﬂow 15. The protein mass spectra were deconvoluted by the Thermo Fisher Scientiﬁc software Protein Deconvolution 2.0, using the Xtract algorithm. The following main parameters were applied: charge carrier, H+; m/z range, minimal 800 to maximal 2000; minimal detected charge state, 4; s/n threshold, 5; relative abundance threshold, 20%. Trace amounts of unlabeled species might be present but fall below the detection limit of the mass spectrometry method (2–5%). Determination of Hydrolysis Products After hydrolysis, samples were analyzed by HPLC-UV on a system consisting of a Dionex UltiMate 181 3000 Pump (Dionex Cooperation, Sunnyvale, United States), a Dionex ASI-100 automated sample injector, a Dionex UltiMate 3000 column compartment and a Dionex UVD 340 U photodiode array detector. The hydrolysis products were separated by a reversed-phase column, [XTerra® RP18, (3.5 μm, 3.0 mm × 150 mm)] (Waters Corporation, Milford, United States) using a non-linear gradient where eluent A consisted of water, eluent B of methanol and eluent C of 0.01 N sulfuric acid. The separation was achieved by a non-linear gradient increased from 15% A to 40% A from 13 to 30 min, followed by an increase to 90% A during 5 min which was kept for 10 min to then be re-established to initial conditions within 1 min and equilibrated for 20 min. Enzyme Adsorption on PET Enzyme Adsorption on PET Enzyme adsorption on PET ﬁlm was monitored as previously described (Ribitsch et al., 2013). Brieﬂy, 0.5 × 1 cm2 PET ﬁlms were washed at 50°C in three consecutive steps of 30 min each with 1.5 ml each of 0.5% (v/v) Triton X-100, 100 mM Na2CO3 and deionized water. The washed membranes were incubated in 1 ml of 0.6 mg/ml enzyme solution for 2 h at 30°C. The ﬁlms were washed twice by dipping into 1.5 ml TBST (25 mM Tris, 0.15 M NaCl, pH 7.6, 0.05% (v/v) Tween®-20) at room temperature. Then, the ﬁlms were incubated with HisProbe (1:2500 dilution in TBST; SuperSignal West HisProbe Kit; Thermo Fisher Scientiﬁc, Waltham, MA) for 40 min at room temperature with shaking. The ﬁlms were washed three times by dipping into 1.5 ml TBST as described above and were developed by incubating in 600 µL of SuperSignal West Pico Substrate Working Solution for 5 min. Chemiluminescence signals were detected using G:box Chemi HR16 and GeneSnap image acquisition software Version 7.05.01 (Syngene, Cambridge, United Kingdom) and quantiﬁed with Colorlite sph850 (Colorlite:Inovative color measurements; Germany). g The structure of the TTL was modeled using the CATALOphore platform of Innophore GmbH (www.innophore.com) employing the program Yasara version 20.4.24.L.64 (www.yasara.org). The model is based on the structure of feruloyl esterase (Est1E) from Butyrivibrio proteoclasticus (PDB 2WTM) (Goldstone et al., 2010) as template, which shares 32% sequence identity and 50% sequence similarity with TTL. The active site cavity in the vicinity of the catalytic triad Ser-113, His-233 and Asp-203 was calculated using the program CavMan (Innophore GmbH, www. innophore.com) employing the LIGSITE algorithm (Hendlich et al., 1997). For the analysis of the hydrophobicity of the cavity, the hydrophobicity module of the program VASCo (Steinkellner et al., 2009) as implemented in CavMan was used. Polyester Synthesis and Characterization The oligomeric model substrate bis-(benzoyloxyethyl) terephthalate (3PET) was synthesized as previously reported (Heumann et al., 2009). Other polyesters were synthesized in a two-step process and characterized as described by Haernvall et al. (2017b). Strains and Plasmids The injection volume was 5 μL, and the ﬂow rate was 0.4 ml/min. The column compartment had a constant temperature of 40°C. The expected release products TA and NaSIP were detected via UV/VIS spectroscopy, and the release products were qualiﬁed and quantiﬁed based on calibration curves. Hydrolysis of Oligomeric and Polymeric Materials The increasing interest in enzymatic polymer functionalization and enzymatic polymer recycling processes has resulted in various proposed strategies to improve the enzymatic hydrolysis of synthetic polymers. Here, in order to evaluate a novel approach, the classical protein engineering strategies were expanded to also include ncAAs. To assess the effects on enzymatic polymer hydrolysis, a lipase from an anaerobic extreme thermophilic microorganism was modiﬁed by exchanging the cAA methionine with the slightly more hydrophobic ncAA norleucine. The effects were evaluated towards oligomeric and polymeric polyester model substrates with systematically varied compositions to enable a mechanistic Model Substrates Polyester powders (10 mg/ml) were incubated in 1 ml of 100 mM Tris-HCl pH 7.0 and in the presence of 1 μM enzyme. The reaction mixture was shaken for 3 h at 70°C and 100 rpm (Infors HT Multitron, Infors AG, Bottmingen, Switzerland). In parallel, enzymes and polymers were incubated in pure buffer as blank reactions. All experiments were run in triplicates. Enzymes were precipitated by addition of ice-cold methanol (1:1 v/v), acidiﬁed with 0.1 M HCl to pH 4 and sedimented in a tabletop centrifuge (15 min, 0°C, 14,000 rpm; Hermle Labortechnik GmbH, Wehingen, Germany). Supernatants were used for HPLC analysis. January 2022 \| Volume 10 \| Article 769830 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 4 Synthetic Biology for Polymer Hydrolysis Haernvall et al. FIGURE 1 \| Hydrolysis of polyester model substrates (A) Hydrolysis of the oligomeric model substrate bis-(benzoyloxyethyl) terephthalate (3PET) with 0.6 µM TTL [Nle] and TTL [Met] at pH 7.0. Total released molecules after 24 h of incubation at 70°C. The release products BA, benzoic acid; HEB, hydroxyethylbenzoate; TA, terephthalic acid and MHET, Mono-(2-hydroxyethyl)terephthalic acid were quantiﬁed by HPLC analysis. Each bar represents the average of three independent samples; error bars indicate the standard deviation. Hydrolysis of polyesters consisting of 70 mol% terephthalic acid (Ta) and 30 mol% 5-sulfoisophthalic acid (NaSIP) and the respective alkyl and ether diols with different chain lengths of (B) alkyl diols (C5, C8, and C12) and (C) ether diols (EG2, EG3, and EG4). Results obtained from hydrolysis with 1 μM TTL [Nle] and TTL [Met] at 70°C represented as the release of terephthalic acid after 24 h of incubation. Each bar represents the average of three independent samples; error bars indicate the standard deviation. Two-tailed p-values from unpaired t-tests were 0.047 (C5), <0.001 (C8), 0.40 (C12), 0.001 (EG2), <0.001 (EG3) and <0.001 (EG4). Hydrolysis of Oligomeric and Polymeric Materials Abbreviations: 1,5-pentanediol (C5), 1,8-octanediol (C8), 1,12-dodecanediol (C12) and ether diols with different chain lengths: EG1: ethylene glycol, EG2: diethylene glycol, EG3: triethylene glycol and EG4: tetraethylene glycol. FIGURE 1 \| Hydrolysis of polyester model substrates (A) Hydrolysis of the oligomeric model substrate bis-(benzoyloxyethyl) terephthalate (3PET) with 0.6 µM TTL [Nle] and TTL [Met] at pH 7.0. Total released molecules after 24 h of incubation at 70°C. The release products BA, benzoic acid; HEB, hydroxyethylbenzoate; TA, terephthalic acid and MHET, Mono-(2-hydroxyethyl)terephthalic acid were quantiﬁed by HPLC analysis. Each bar represents the average of three independent samples; error bars indicate the standard deviation. Hydrolysis of polyesters consisting of 70 mol% terephthalic acid (Ta) and 30 mol% 5-sulfoisophthalic acid (NaSIP) and the respective alkyl and ether diols with different chain lengths of (B) alkyl diols (C5, C8, and C12) and (C) ether diols (EG2, EG3, and EG4). Results obtained from hydrolysis with 1 μM TTL [Nle] and TTL [Met] at 70°C represented as the release of terephthalic acid after 24 h of incubation. Each bar represents the average of three independent samples; error bars indicate the standard deviation. Two-tailed p-values from unpaired t-tests were 0.047 (C5), <0.001 (C8), 0.40 (C12), 0.001 (EG2), <0.001 (EG3) and <0.001 (EG4). Abbreviations: 1,5-pentanediol (C5), 1,8-octanediol (C8), 1,12-dodecanediol (C12) and ether diols with different chain lengths: EG1: ethylene glycol, EG2: diethylene glycol, EG3: triethylene glycol and EG4: tetraethylene glycol. study. Therefore, a set of structurally different polyester substrates was used (Figure 1). The water-insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET, Figure 1A) has been widely used in the past for the detection of PET hydrolysing enzymes such as cutinases (Herrero Acero et al., 2011; Ribitsch et al., 2011; Kawai et al., 2019). ionic phthalic polyesters were used to evaluate the effect of chain length and hydrophilicity/hydrophobicity of polyesters inﬂuencing the hydrolysis. The ﬁrst group of polyesters contained alkyl diols (1,5-pentanediol (C5), 1,8-octanediol (C8) and 1,12-dodecanediol (C12) (Figure 1B). The second group of polyesters contained ether diols (diethylene glycol (EG2), triethylene glycol (EG3) and tetraethylene glycol (EG4)) (Figure 1C), with systematically varied chain lengths. The ratio of TA and NaSIP was kept constant (70:30 mol%). Only few enzymes were identiﬁed so far that show an ability to hydrolyze ionic phthalic acid based polyesters (Haernvall et al., 2017a). Ionic phthalic polyesters are found in many products in our daily life, such as household products. Hydrolysis of Oligomeric and Polymeric Materials In this study, we used model polyesters consisting of the aromatic terephthalic acid (TA) and the ionic aromatic 5-sulfoisophthalic acid (NaSIP), which were linked by altering alkyl and ether diols. Two groups of Structure Modelling of TTL It is conceivable that the replacement of those two methionine residues by norleucine modulates the catalytic activity of the enzyme most likely by altering its substrate binding properties. Most of the remaining methionine residues are part of the hydrophobic core of the protein (Figure 2). Amino acid replacements in this region could change the dynamic properties of the enzyme and its stability. Indeed, Hoesl et al. (2011) observed that the quantitative replacement of the Met residues in TTL resulted in a variant enzyme that was highly active without thermal activation while the parent enzyme containing exclusively Met residues was nearly inactive unless heat activated. Hoesl et al. hypothesized that the global replacement of Met by Nle would increase the hydrophobicity around the substrate binding site, which could enhance the accessibility of the substrate into TTL [Nle] in aqueous solution. To analyze the incorporation efﬁciency of Nle, Met and Nle variants were subjected to intact protein mass analysis (Supplementary Figure S2). In the mass spectrum of TTL [Nle] the protein species with all 11 Met residues exchanged for Nle was the most prominent peak. A minor peak representing lipase with 10 Met residues exchanged for Nle was detected as well. This is in good agreement with previously published results (Hoesl et al., 2011). Hydrolysis of Oligomeric and Polymeric Model Substrates The effect of the incorporating Nle into TTL on polymer degradation was assessed in a ﬁrst step using the oligomeric model substrate 3PET. Interestingly, the global Met→Nle substitution in TTL had a positive impact on the hydrolysis as indicated by a ~30 % higher amounts of hydrolysis products than to the parent enzyme (Figure 1A). Nevertheless, the pattern of the released molecules was similar for the two enzymes TTL (Met) and TTL (Nle). Benzoic acid (BA) was released in the highest concentration, followed by mono-(2-hydroxyethyl) terephthalic acid (MHET), terephthalic acid (TA) and hydroxyethylbenzoate (HEB), respectively. The total amount of released molecules for the hydrolysis of the oligomer model substrates was in the same range as previously reported for Structure Modelling of TTL The modelled structure of the lipase from Thermoanaerobacter thermohydrosulfuricus (TTL) exhibits a typical α/β-hydrolase January 2022 \| Volume 10 \| Article 769830 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 5 Synthetic Biology for Polymer Hydrolysis Haernvall et al. FIGURE 2 \| Cartoon representation of the modelled structure of the lipase from Thermoanaerobacter thermohydrosulfuricus in two, 90°-separated orientations. Amino acids forming the catalytic triad (Ser-113, His-233 and Asp-203) are shown in a cyan stick representation, while methionine residues are shown in magenta. The active site cavity is represented as a semi-transparent surface colored according to hydrophobicity, ranging from blue (hydrophilic) to red (hydrophobic). The ﬁgure was generated using the program PyMOL (www.pymol.org). FIGURE 2 \| Cartoon representation of the modelled structure of the lipase from Thermoanaerobacter thermohydrosulfuricus in two, 90°-separated orientations. Amino acids forming the catalytic triad (Ser-113, His-233 and Asp-203) are shown in a cyan stick representation, while methionine residues are shown in magenta. The active site cavity is represented as a semi-transparent surface colored according to hydrophobicity, ranging from blue (hydrophilic) to red (hydrophobic). The ﬁgure was generated using the program PyMOL (www.pymol.org). marker band as calculated for the theoretical molecular weight of TTL (Supplementary Figure S1). In contrast, the lipase expressed in the presence of Nle appeared as a blurred band at slightly lower molecular weight. In agreement with a previous report (Hoesl et al., 2011), the Nle variant exhibited accelerated electrophoretic mobility compared to the Met variant. The altered migration behavior was a ﬁrst indication for the successful incorporation of Nle into the lipase. Typically, 20 mg/ml TTL [Met] and 10 mg/ml TTL [Nle)] were puriﬁed from 3 to 4 g of cell pellet obtained from cultures in shake ﬂasks. fold with a central eight-stranded, mostly parallel β-sheet ﬂanked by six α-helices (Figure 2). A catalytic triad is formed by the amino acid residues Ser-113, His-233 and Asp-203, with the serine being embedded in a GLSMGG sequence motif. The oxyanion hole is built up by the mainchain amide groups of Phe-37 and Met-114. The active site cavity is located at the upper end of the α/β-hydrolase core and delimited by a small cap domain consisting of residues 140 to 180. The TTL-sequence contains a total of 11 methionine residues, of which Met-114 and Met-142 are in close vicinity to the active site residue (Figure 2). TTL Expression, Puriﬁcation and Characterization TTL was expressed in E. coli and puriﬁed from cleared cell lysates over a C-terminal 6xHisTag by afﬁnity chromatography. Expression in the presence of the canonical Met yielded a deﬁned band migrating at the 29.2 kDa molecular weight January 2022 \| Volume 10 \| Article 769830 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 6 Synthetic Biology for Polymer Hydrolysis Haernvall et al. FIGURE 3 \| Adhesion of TTL [Met] and TTL [Nle] on PET ﬁlm. Transillumination (A); luminescence after detection of the hexahistidine-tag using HisProbe (B). To further evaluate the impact of norleucine incorporation on polyester hydrolysis and to obtain a more detailed mechanistic insights into the hydrolysis behavior of TTL [Nle], a set of structurally different ionic phthalic acid polyesters were investigated. In a ﬁrst step, TTL [Nle] was incubated with polymeric model substrates containing alkyl diols of different chain lengths, C5, C8, and C12 (Figure 1B). TTL [Nle] and the parent enzyme TTL [Met] were both active towards C5 while only a very low activity was detected towards C8 and C12 as indicated by the release of terephthalic acid. TTL [Nle] showed around 5% more activity towards C5 compared to TTL [Met]. However, enzymatic hydrolysis of polymeric substances is inﬂuenced by several parameters, such as water solubility of the substrates, crystallinity, molecular weight, glass transition temperature (Tg) and therefore difﬁcult to predict (Chamas et al., 2020). As described recently (Haernvall et al., 2017b), polyesters C8 and C12 have lower water solubility, higher hydrophobicity and a higher crystallinity of 4 and 12%, respectively, than C5 with crystallinity below 1%, which may have inﬂuenced the hydrolysis. Low water solubility, high hydrophobicity (Okada et al., 1997) and crystallinity have previously been reported to have a negative impact on the enzymatic hydrolysis of polymers (Donelli et al., 2010). However, the glass transition temperature of the polymeric model substrates is decreasing with increasing chain length which would have been expected to have a positive impact on the enzymatic hydrolysis due to the higher ﬂexibility of the polymer chains (Marten et al., 2003). FIGURE 3 \| Adhesion of TTL [Met] and TTL [Nle] on PET ﬁlm. Transillumination (A); luminescence after detection of the hexahistidine-tag using HisProbe (B). FIGURE 3 \| Adhesion of TTL [Met] and TTL [Nle] on PET ﬁlm. Transillumination (A); luminescence after detection of the hexahistidine-tag using HisProbe (B). REFERENCES Terephthalate)-Hydrolyzing Lipase from Pelosinus Fermentans. Appl. Microbiol. Biotechnol. 100 (4), 1753–1764. doi:10.1007/s00253-015-7031-1 Biundo, A., Ribitsch, D., and Guebitz, G. M. (2018). 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Non-canonical Amino Acids as a Useful Synthetic Biological Tool for Lipase-Catalysed Reactions in Hostile Environments. Catal. Sci. Technol. 3 (5), 1198–1201. doi:10.1039/c3cy20712a Sci. Technol. 3 (5), 1198–1201. doi:10.1039/c3cy20712a Budisa, N., Huber, R., Golbik, R., Minks, C., Weyher, E., and Moroder, L. (1998). Atomic Mutations in Annexin V. Thermodynamic Studies of Isomorphous Protein Variants. Eur. J. Biochem. 253 (1), 1–9. doi:10.1046/J.1432-1327.1998.2530001.X Budisa, N., Huber, R., Golbik, R., Minks, C., Weyher, E., and Moroder, L. (1998). Atomic Mutations in Annexin V. Thermodynamic Studies of Isomorphous Protein Variants. Eur. J. Biochem. 253 (1), 1–9. doi:10.1046/J.1432-1327.1998.2530001.X Budisa, N. (2004). Prolegomena to Future Experimental Efforts on Genetic Code Engineering by Expanding its Amino Acid Repertoire. Angew. Chem. Int. Ed. 43 (47), 6426–6463. Angewandte Chemie - International Edition. doi:10.1002/ anie.200300646 Anderhuber, N., Fladischer, P., Gruber-Khadjawi, M., Mairhofer, J., Striedner, G., and Wiltschi, B. (2016). High-level Biosynthesis of Norleucine in E. coli for the Economic Labeling of Proteins. J. Biotechnol. 235, 100–111. doi:10.1016/J. JBIOTEC.2016.04.033 Budisa, N. (2004). Prolegomena to Future Experimental Efforts on Genetic Code Engineering by Expanding its Amino Acid Repertoire. Angew. Chem. Int. Ed. 43 (47), 6426–6463. Angewandte Chemie - International Edition. doi:10.1002/ anie.200300646 Barth, M., Honak, A., Oeser, T., Wei, R., Belisário-Ferrari, M. R., Then, J., et al. (2016). A Dual Enzyme System Composed of a Polyester Hydrolase and a Carboxylesterase Enhances the Biocatalytic Degradation of Polyethylene Terephthalate Films. Biotechnol. J. 11 (8), 1082–1087. doi:10.1002/BIOT. 201600008 Chamas, A., Moon, H., Zheng, J., Qiu, Y., Tabassum, T., Jang, J. H., et al. (2020). Degradation Rates of Plastics in the Environment. ACS Sustain. FUNDING The COMET center: acib: Next Generation Bioproduction is funded by BMVIT, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria und Vienna Business Agency in the framework of COMET–Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG. Scientiﬁc research in a research centre co-funded by BASF. TTL Expression, Puriﬁcation and Characterization Both, TTL [Met] and TTL [Nle] survived well the incubation with PET because their esterase activities after the incubation, e.g., with PET, was not lower than before (Supplementary Information, Supplementary Figure S3). Our ﬁnding corroborates the extraordinary stability of TTL and its Nle reported previously (Hoesl et al., 2011). their substrate entry tunnels and/or active sites. If this is the case, Nle–and eventually also other ncAAs–could complement the existing tools for tuning and improving the activity of enzymes on polymers. CONCLUSION Recently, ncAAs have become a valuable asset for protein engineering, e.g., to introduce non-natural chemical functionalities into enzymes. In this study we have shown for the ﬁrst time that incorporation of the ncAA Nle into a lipase from Thermoanaerobacter thermohydrosulfuricus has a positive impact on the enzymatic hydrolysis of synthetic polyesters. Nle, the carba-analog of Met, is less polar and more hydrophobic than Met while the structures are virtually identical. The global replacement of Met by Nle can tune the hydropathy of a protein in a subtle way that is difﬁcult to attain by exchanging Met with a hydrophobic canonical amino acid. Nle lacks the sulfur atom which is replaced by a methylene group. Due to the lack of the sulfur atom, Nle cannot form sulfoxides protecting the enzymes from oxidative stress and is also claimed to prevent protein aggregation and chemical degradation. Our structural model of TTL predicts Met-114 and Met- 142 to be ocated in close vicinity of the catalytic triad and the active site cavity and two other methionines, Met-147 and Met-158, to be part of the lid. Previously, Budisa et al. (Acevedo-Rocha et al., 2013) hypothesized that replacing methionine residues in the lid could have retained it in an open conformation. This together with the replacement of Met-114 and Met-142 near the catalytic triad might have modiﬁed the environment in the substrate entry tunnel and the catalytic site such that the hydrolysis of the hydrophobic synthetic polymers was enhanced. Further studies reveal whether a similar enhancing effect can be elicited in other polyester hydrolyzing enzymes that accommodate Met residues in AUTHOR CONTRIBUTIONS KH, PF, HS, and SZ performed the experiments. DR, KG, BW, and GG planned the experiments. The manuscript was written through contributions of all authors. All authors have given approval to the ﬁnal version of the manuscript. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. TTL Expression, Puriﬁcation and Characterization In a next step, TTL [Nle] and the parent enzyme TTL [Met] were incubated with the ether diol containing model substrates, EG2, EG3, EG4 (Figure 1C). Surprisingly, an up to 40% increased activity was found for TTL [Nle] towards all three polyesters (EG2, EG3, and EG4) when compared to the parent enzyme TTL [Met]. The highest activity was again detected on the shortest diol chain length, namely EG2, decreasing with increasing chain length. It can also be noted that an overall higher activity was detected for both enzymes towards all three ether diol containing substrates when compared to the alkyl diol analogs. This can be a consequence of the increased water solubility of the ether diol containing polymers and the increased hydrophilicity. It has previously been shown that increased water solubility and increased hydrophilicity is enhancing enzymatic hydrolysis. It has even been suggested as a parameter to tune polymeric biodegradation (Gigli et al., 2013). In none of the cases, NaSIP was detected after hydrolysis, indicating that TTL [Met] and TTL [Nle] have a limited capacity to cleave ester bonds in close vicinity to the ionic monomer NaSIP. These results are in accordance with previously reported data for cutinase A and an arylesterase from Pseudomonas pseudoalcaligenes and a lipase from Pseudomonas pelagia (Haernvall et al., 2017a; Haernvall et al., 2017b). FIGURE 3 \| Adhesion of TTL [Met] and TTL [Nle] on PET ﬁlm. Transillumination (A); luminescence after detection of the hexahistidine-tag using HisProbe (B). We analyzed the adsorption of TTL [Met] and TTL [Nle] on PET ﬁlms as described in the methods section. Adsorbed enzymes were detected by binding of horseradish peroxidase labeled HisProbe to the hexahistidine-fusion tag and chemiluminescence detection. TTL [Nle] adsorbed reproducibly better to the PET ﬁlms than the parent protein (Figure 3). 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https://openalex.org/W4321489384	https://translational-medicine.biomedcentral.com/counter/pdf/10.1186/s12967-023-04000-1	English	null	The relationship of reproductive factors with adiposity and body shape indices changes overtime: findings from a community-based study	Journal of translational medicine	2,023	cc-by	9,119	Amiri et al. Journal of Translational Medicine (2023) 21:137 https://doi.org/10.1186/s12967-023-04000-1 Amiri et al. Journal of Translational Medicine (2023) 21:137 https://doi.org/10.1186/s12967-023-04000-1 Journal of Translational Medicine Open Access The relationship of reproductive factors with adiposity and body shape indices changes overtime: findings from a community‑based study Mina Amiri1, Maryam Mousavi1,2, Fereidoun Azizi3 and Fahimeh Ramezani Tehrani1* Abstract Background Studies focusing on the relationships of adiposity and body shape indices with reproductive factors have reported conflicting results. This study aimed to investigate the influence of reproductive factors on adiposity and body shape indices changes overtime. Materials and methods In this community-based prospective study, 1636 postmenopausal women were selected from Tehran Lipid and Glucose Study (TLGS). The unadjusted and adjusted Generalized Estimating Equation models (GEE) were applied to investigate secular longitudinal trends of adiposity and body shape indices. Results According to the adjusted GEE models, mean changes in body mass index (BMI) in women with early menarche was 1.18 kg/m2 higher than those with normal menarche age (P = 0.030). Moreover, the mean changes in BMI overtime were 0.11 kg/m2 higher in women with premature/early menopausal age than those with normal men- opausal age (P = 0.012). Mean changes of waist circumference (WC) in women with late menopause were 2.27 cm higher than those with normal menopausal age (P = 0.036). We also observed higher mean changes in a body shape index (ABSI) in women with late menopause (P = 0.037), compared to those with normal menopausal age. We found a marginal effect of parity on BMI and WC as well. Conclusions This study demonstrated higher BMI in females with earlier menarche age. We also showed higher values of BMI overtime in women with premature/ early menopause, whereas women with late menopausal age had higher WC and ABSI values. However, more longitudinal studies investigating body composition indices by adjusting all potential confounders are still required to confirm our study findings. Keywords Adiposity, Body mass index (BMI), Waist circumference (WC), A body shape index (ABSI), Age at menarche, Age at menopause, Pregnancy Study design and participants Epidemiologic studies demonstrate that in most popu- lations, the prevalence of adipose tissue disturbances is greater in women than in men [4, 5]. Besides, women have a greater percentage of body fat to prepare for child- bearing and body composition during their reproduc- tive cycle [6] and experience more dissatisfaction with their body shape [7]. These gender differences highlight the role of women’s reproductive characteristics in the formation of the body shape and variations related to adiposity and its indices [6–8]. There is evidence demon- strating that waist circumference (WC) and waist-to-hip ratio (WHR) are associated with sex hormone-binding globulin (SHBG) and sex hormones such as free estra- diol and free testosterone independently of body mass index (BMI). As a result, female sex hormones may regard as one of the main factors affecting fat distribu- tion [9]. Increased concentrations of estrogens or other reproductive hormones during puberty are differentially associated with the activation of the homeobox family (HOX) and other genes to determine regional adipose distribution [10]. Hormonal changes during pregnancy can be also associated with remaining adiposity after pregnancy [11–13]. There is evidence demonstrating that multiparous women have higher BMI than their nul- liparous counterparts [14]. It is also documented that women’s body shape can be affected by their pregnancy status [15]. Previous studies on both populations of pre- and postmenopausal women also highlight the role of the hormonal environment in creating changes related to body fatness, indicating that the rate of reproductive aging is significantly associated with the body fat pattern. It has been shown BMI of women with abnormal levels of sex hormones is more correlated with their hormonal status than their age or menopausal status [16]. For this population-based prospective study, partici- pants were selected from among participants of the Tehran Lipid and Glucose Study (TLGS). TLGS is an ongoing prospective cohort initiated in 1998, in which 15,005 participants aged ≥ 3 years were assessed [31]. In summary, information on various risk factors for non- communicable diseases, demographic variables, and reproductive histories was collected during face-to-face interviews conducted every 3 years in 6 follow-up vis- its. Among TLGS participants, we included all women aged ≥ 20 years who participated in the baseline and at least one follow-up whose reproductive and menopause status was defined. © 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 2 of 13 Amiri et al. Journal of Translational Medicine (2023) 21:137 Study design and participants Out of 5374 postmenopausal women who entered at baseline, 1191 women were excluded due to the missing menarche age and 2547 women due to the missing menopausal age. Finally, a total of 1636 women were eligible and analyzes for the present study. Background therefore, aimed to investigate the influence of repro- ductive history on the adiposity and body shape indices changes overtime after adjusting potential confounders. Adipose tissue disorders, such as obesity and central obe- sity, are increasing worldwide and are associated with an increased risk of adverse health outcomes, in particu- lar cardio-metabolic disturbances [1, 2], leading to an increase in all-cause mortality [3]. Measurements All study participants were interviewed to obtain medi- cal, obstetrics, and family histories using pretested questionnaires [32]. Clinical and anthropometric meas- urements were assessed by trained examiners at each fol- low-up, details of which have been previously published [33]. In summary, weight was measured when they were minimally clothed using a digital scale (Seca 707, Seca GmbH) and rounded to the nearest 100 g. Height was measured without shoes in the standing position with shoulders in normal alignment, using a tape measure. Waist circumference was measured with an unstretched tape measure at the level of the umbilicus without any pressure on the body surface and recorded to the nearest 0.1 cm. Hip circumference was measured at the level of the anterior superior iliac spine without any pressure on the body surface. Although numerous studies have evaluated the rela- tionships of adiposity and body shape indices with repro- ductive factors like age at menarche, menopausal age, and pregnancy history, their results are still conflicting and inconclusive [6, 14–29]. Moreover, these associations have been complicated by reverse causation since most available studies had cross-sectional designs and could not demonstrate causal relationships [6, 14–20]. Further- more, the majority of studies have assessed some adi- posity indices like BMI, waist circumference (WC), and WHR and a limited number of studies have specifically evaluated body shape concerning reproductive factors [15, 19, 30]. This community-based prospective study, Variables Age at menarche was defined as the age at the first men- strual bleeding. Age at menarche < 11 years, 12–15 years, and ≥ 16 were considered as early, normal, and late menarche, respectively. According to the World Health Organization classification, menopause was defined as the absence of spontaneous menstrual bleeding for more than 12 months, for which no other pathologic or physi- ologic cause could be determined [37]. Age at natural menopause was defined as younger than 40 years (pre- mature menopause), 40–44 years (early menopause), 45–54 years (reference category), and 55 years or older (late menopause) [38, 39]. Statistical analysish The baseline characteristics of participants are described by a median, interquartile range (IQR) in non-normal continuous variables, and in cases of non-rejection of the normality assumption, mean (standard deviation) was used. For evaluating the normality hypothesis, we con- ducted the Kolmogorov–Smirnov normality test. The categorical variables were described as frequencies (%). Statistical analysis was performed using Generalized Esti- mating Equation models (GEE) to investigate the secular longitudinal trends of adiposity and body shape indices including BMI, waist, and ABSI and evaluate the effect of reproductive factors on these trends. We used z-scores of ABSI due to the small values of this variable.h The GEE analysis accounts for correlations within sub- jects through a working correlation matrix and enables researchers to accurately estimate the effect size in case of incomplete data (missing variables in some repeated measures), which is common in cohort studies. We assessed the time trends of each adiposity index by fit- ting unadjusted and adjusted GEE models (by adjusting for baseline age, parity, smoking, education, and physical activity status). All statistical analysis was performed in SPSS16 and STATA (version 12; STATA Inc., College Station, TX, USA). The p-values less than 0.05 were considered statis- tically significant. Term definition B d i d Body mass index (BMI) was calculated as weight in kilo- grams (kg) divided by height squared (m2). A body mass index (ABSI) was calculated based on the following for- mula: [WC (cm)/[BMI 2/3 × height (m)1/2] [34]. Smoking status was classified into two categories, including ever smokers (current users and those who used to smoke in the past) and never smokers. For evaluating physical activity, a modified activity questionnaire (MAQ) was used, which is evaluated and validated in the Iranian pop- ulation. According to the questionnaire, physical activity Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 3 of 13 Table 1 Baseline characteristics of the study participants Length of reproduction is the interval duration between age at menarche and Variables Total N = 1636 Baseline agea 57 (52–63) BMI at baseline, Median (IQR) 29.62 (26.70–32.77) Waist circumference at baseline, Median (IQR) 96 (88–103) ABSI in baseline, Median (IQR) 0.08 (0.07–0.08) Educational level (years)b < 6 538 (32.9) 6–12 1058 (64.7) > 12 40 (2.4) Smokingb Never 1539 (94.1) Ever 97 (5.9) Physical activityb,c Low 1135 (69.4) Moderate to high 501 (30.6) Age at menarchea,d 14 (13–15) Early menarcheb 92 (5.6) Normal menarcheb 1345 (82.2) Late menarcheb 199 (12.2) Age at menopausea,e 49 (45–52) Premature/early menopauseb 463 (28.3) Normal menopausal ageb 1029 (62.9) Late at menopausal ageb 144 (8.8) Length of reproductivea 35 (31–39) Graviditya 5 (3–7) Paritya Nulliparousb 5 (3–7) 0 74 (4.5) 1–2 268 (16.4) ≥ 3 1294 (79.1) History of abortionb No 1014 (62) Yes 622 (38) Table 1 Baseline characteristics of the study participants has been specified as low (MET < 600 min/wk), moderate (MET 600–1499 min/wk), and high (MET ≥ 1500 min/ wk) levels [35, 36].ii has been specified as low (MET < 600 min/wk), moderate (MET 600–1499 min/wk), and high (MET ≥ 1500 min/ wk) levels [35, 36].ii Results In this study with a median (IQR) of 16 (15–17) follow- up years, a total of 1636 eligible women were analyzed. Table 1 shows the baseline characteristics of the study participants. The median (IQR) of age and BMI of the study participants were 57.0 (52.0–63.0) years and 29.6 (26.7–32.8) Kg/m2, respectively. Moreover, the median (IQR) of age at menarche and menopause were 14 (13– 15) years and 49 (45–52) years, respectively. The majority of participants had 6–12 years of education and a low level of physical activity. Tables 2–4 show the results of GEE models to esti- mate the effect of reproductive characteristics (age at menarche, age at menopause, parity, and abortion) on the Amiri et al. Results Journal of Translational Medicine (2023) 21:137 Page 4 of 13 Table 2 Effects of menarche age on adiposity indices of participants over time BMI Body mass index; ABSI A body shape index; Ref Reference; CI Confidence interval Model 1: Unadjusted model Model 2: Adjusted for baseline age, smoking, education, and physical activity status Time variable means follow-up visits a Age at menarche < 11 years, 12–15 years, and ≥ 16 were considered as early, normal, and late menarche, respectively b The results of ABSI are presented by z-scores c Significant p-values (p < 0.05) are bolded Reproductive factors Outcomes Model 1 P-value c Model 2 P-value c Coefficient (95% CI) Coefficient (95% CI) Menarche agea (ref: Normal) BMI (kg/m2) Early 1.43 (0.35, 2.50) 0.009 1.18 (0.11, 2.25) 0.030 Late − 1.00 (− 1.75, − 0.25) 0.008 − 1.05 (− 1.79, − 0.31) 0.005 Time − 0.05 (− 0.09, − 0.006) 0.024 − 0.04 (− 0.09, − 0.001) 0.044 Time × early 0.08 (− 0.09, − 0.26) 0.343 0.09 (− 0.09, 0.26) 0.327 Time × late 0.004 (− 0.11,0.12) 0.946 0.006 (− 0.11, 0.12) 0.922 Waist circumference (cm) Early 1.45 (− 1.17, 4.07) 0.278 1.66 (− 0.94, 4.26) 0.211 Late − 1.66 (− 3.46, 0.14) 0.070 − 1.72 (− 3.50, 0.07) 0.059 Time 1.32 (1.19, 1.45) < 0.001 1.33 (1.20, 1.46) < 0.001 Time × early 0.26 (− 0.26, 0.79) 0.316 0.28 (− 0.24, 0.80) 0.295 Time × late − 0.08 (− 0.43, 0.27) 0.647 − 0.08 (− 0.42, 0.27) 0.664 ABSIb Early − 0.19 (− 0.43, 0.04) 0.111 − 0.05 (− 0.28, 0.17) 0.640 Late − 0.02 (− 0.18, 0.15) 0.846 − 0.02 (− 0.17, 0.14) 0.829 Time 0.20 (0.19, 0.21) < 0.001 0.21 (0.20, 0.22) < 0.001 Time × early − 0.003 (− 0.06, 0.05) 0.929 − 0.0004 (− 0.05, 0.05) 0.988 Time × late 0.001 (− 0.04, 0.04) 0.955 0.003 (− 0.03, 0.04) 0.860 Table 2 Effects of menarche age on adiposity indices of participants over time BMI Body mass index; ABSI A body shape index; Ref Reference; CI Confidence interval Model 1: Unadjusted model Model 2: Adjusted for baseline age, smoking, education, and physical activity status Time variable means follow-up visits a Age at menarche < 11 years, 12–15 years, and ≥ 16 were considered as early, normal, and late menarche, respectively b The results of ABSI are presented by z-scores c Significant p-values (p < 0.05) are bolded trend of adiposity indices, i.e. Results BMI, WC, and ABSI with and without adjustment for age, educational level, smok- ing status, and physical activity. Figures 1, 2, 3, 4 illustrate trends of adiposity indices overtime in the different sub- groups of the study participants based on their reproduc- tive factors as well. the mean changes of WC in women with late menarche age were lower than those with normal menarche age (− 1.66 cm; 95% CI − 3.46, 0.14; P = 0.070); this marginal effect remained after adjusting for potential confounders (− 1.72 cm; 95% CI − 3.50, 0.07; P = 0.059). We found no significant association between menarche age and ABSI overtime (Table 2). Main findings of the study i g y By using data from a population-based study with over 15 follow-up years, we attempted to examine the relationships of reproductive factors with adiposity and body shape indi- ces changes overtime. Our study findings showed higher BMI in females with early menarche and higher values of BMI and WC and lower values of ABSI in women with pre- mature/early menopausal age than those with normal age at menopause. A marginal effect of parity on BMI and WC was detected as well, indicating that women with parity ≥ 3 had higher mean changes in these parameters, compared to those with less parity. Discussion changes of z-scores of ABSI in women with late men- opause (0.37; 95% CI 0.18, 0.56; P < 0.001), compared to those with normal menopausal age, finding that remained significant even adjusting confounders (0.19; 95% CI 0.01, 0.38; P < 0.037). After adjusting confound- ers, the interaction effect of time and premature/ early menopause on mean changes of ABSI z-scores was sig- nificant (− 0.03; 95% CI − 0.05, − 0.0009; P = 0.043) (Table 3). Age at menopauseh 1 Trends of adiposity indices overtime based on age at menarche. a BMI trends based on the age at menarche subgr circumference trends based on the age at menarche subgroups; c ABSI trends based on the age at menarche subgroups. B a body shape index Age at menopauseh The mean changes of BMI in women with early menarche were 1.43 kg/m2 higher than those with normal menarche age (95% CI 0.35, 2.50; P = 0.009), finding that remained significant even after adjustment for confound- ers (model 2) (1.18 kg/m2; 95% CI 0.11, 2.25; P = 0.030). On the other hand, the mean changes of BMI in women with late menarche were significantly lower than those with normal age at menarche in both models of unad- justed (− 1.00 kg/m2; 95% CI -1.75, − 0.25; P = 0.008) and adjusted (− 1.05 kg/m2; 95% CI − 1.79, − 0.31; P = 0.005). While BMI had uprising trends in all groups, regardless of their menarche age, the interaction between time and different subgroups of menarche age (early and late) was not significant. We also found a marginally sig- nificant effect of menarche age on WC, indicating that This study revealed that the interaction effect of time and early menopausal age on mean changes in BMI was significant (0.10 kg/m2; 95% CI 0.02, 0.19; P = 0.017), indicating that mean changes in BMI overtime was 0.1 kg/m2 higher in women with premature/ early men- opausal age than those with normal menopausal age, finding that remained significant even after adjusting confounders (0.11 kg/m2; 95% CI 0.02, 0.19; P = 0.012). This study also showed that mean changes of WC in women with late menopause were significantly higher than those with normal menopausal age (2.31 cm; 95% CI 0.19, 4.43; P = 0.033), finding that remained signifi- cant even after adjusting confounders (2.27 cm; 95% CI 0.15, 4.39; P = 0.036). We also observed higher mean Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 5 of 13 Fig. 1 Trends of adiposity indices overtime based on age at menarche. a BMI trends based on the age at menarche subgroups; b Waist circumference trends based on the age at menarche subgroups; c ABSI trends based on the age at menarche subgroups. BMI body mass index; ABSI a body shape index Fig. 1 Trends of adiposity indices overtime based on age at menarche. a BMI trends based on the age at menarche subgroups; b Waist circumference trends based on the age at menarche subgroups; c ABSI trends based on the age at menarche subgroups. BMI body mass index; ABSI a body shape index Fig. Parity and abortion We also found a marginal effect of parity on BMI and WC, indicating that women with parity ≥ 3 had higher mean changes in these parameters, compared to those with less parity. Abortion was not significantly associ- ated with none of the adiposity indices (Table 4). Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 6 of 13 Fig. 2 Trends of adiposity indices overtime based on the age at menopause. a BMI trends based on the age at menopause subgroups; b Waist circumference trends based on the age at menopause subgroups; c ABSI trends based on the age at menopause subgroups. BMI body mass index; ABSI a body shape index Fig. 2 Trends of adiposity indices overtime based on the age at menopause. a BMI trends based on the age at menopause subgroups; b Waist circumference trends based on the age at menopause subgroups; c ABSI trends based on the age at menopause subgroups. BMI body mass index; ABSI a body shape index Possible mechanisms involved in the relationship between reproductive factors, adiposity, and body shape indicesh protective adipose tissue distribution [10]. Indeed, the neuroendocrine system regulates the body fat content of the human body throughout its lifespan [40]. Accord- ingly, women in their different reproductive stages, such as menarche, pregnancy, postpartum, and menopause are exposed to various hormonal changes. These transitions can lead to substantial alterations in metabolic status and body structure [16]. There is evidence demonstrating more prevalence of obesity and other adiposity indices in women than in men worldwide, which highlights the role of women’s reproductive factors in developing these disorders [4, 5]. Although the underlying mechanisms have not been completely elucidated yet, ovarian hormone altera- tions within the reproductive lifespan and menopause transition of women seem to play a major role. In this regard, estrogen has been introduced as a key factor in the causes, consequences, and distribution of fat among women. Estrogens synergize with adipose tissue genes to increase gluteofemoral subcutaneous adipose tissue mass and decrease central adipose tissue mass in reproductive- age women. Deprivation of estrogens after menopause, therefore, independent of aging, is associated with an increase in total adipose tissue mass and a decline in lean body mass. Menopause also partially reverses women’s Age at menarche, adiposity, and body shape indicesi g , p y, y p Earlier studies have demonstrated a significant associa- tion between age at menarche and adiposity and body shape indices, although this relation has been compli- cated by mutual causation [41]. Obesity has been pro- posed as a strong risk factor for age at menarche. Some studies have reported that the typical female pattern of regional adipose tissue distribution emerges after puberty [21, 22], indicating that a normative young adult woman has ~ 18 kg body fat (~ 30% of body weight), compared to a normative young adult man who has ~ 12 kg body fat Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 7 of 13 Fig. 3 Trends of adiposity indices overtime based on the parity status. a BMI trends based on the parity subgroups; b Waist trends based on the parity subgroups; c ABSI trends based on parity subgroups. BMI body mass index; ABSI a body shape index Fig. 3 Trends of adiposity indices overtime based on the parity status. a BMI trends based on the parity subgroups; b Waist trends based on the parity subgroups; c ABSI trends based on parity subgroups. BMI body mass index; ABSI a body shape index Fig. 3 Trends of adiposity indices overtime based on the parity status. a BMI trends based on the parity subgroups; b Waist trends based on the parity subgroups; c ABSI trends based on parity subgroups. BMI body mass index; ABSI a body shape index BMI, which could be explained by weight gain between young adulthood and midlife [25]. The Framingham Heart Study has reported on the association between age at menarche and visceral and subcutaneous adipose tis- sue (SAT), indicating a significant association of earlier age at menarche with greater midlife visceral adipose tissue (VAT) and SAT; these associations were attenu- ated when the authors adjusted for midlife BMI [27]. A population-based prospective study involving 15,807 women aged 40–79 years demonstrated that early age at menarche < 12 years was associated with increased risk of cardiovascular disease events, and its related mor- tality, a relationship which appeared to be only partly mediated by increased adiposity [26]. On the contrary, a prospective study on 1462 Swedish women showed no signification association between age at menarche and subsequent fat distribution [43]. Age at menarche, adiposity, and body shape indicesi Finally, findings from a meta-analysis of 10 cohort studies demonstrated that early menarche (< 12 years of age) was associated with higher BMI; it indicated that the mean BMI in women who experienced early menarche was 0.34 kg m2 higher (~ 15% body weight) [21, 42]. The most likely justifica- tion for this gender variation refers to the influences of ovarian hormones and eating behaviors after menarche. Increased concentrations of estrogens or other reproduc- tive hormones during puberty are differentially associ- ated with the activation of the homeobox family (HOX) and other genes to determine regional adipose distribu- tion [10]. The majority of previous studies have demon- strated an inverse association between age at menarche and adiposity indices, in particular bodyweight [6, 17, 21–27]. In this regard, data from a historical cohort of 3743 Scottish females revealed an inverse relationship between age at menarche and BMI in middle age, which was not explained by early childhood BMI. They con- cluded that age at menarche, as a substantial marker for the sexual maturation process in women, may lead to differences in their adiposity indices [24]. A multicenter, community-based study on 1214 black and white women has demonstrated that earlier menarche is positively associated with visceral and subcutaneous abdominal ectopic fat independent of confounders and young-adult Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 8 of 13 Fig. 4 Trends of adiposity indices overtime based on the abortion status. a BMI trends based on the abortion subgroups; b Waist circumference trends based on the abortion subgroups, c ABSI trends based on the abortion subgroups. BMI body mass index; ABSI a body shape index Fig. 4 Trends of adiposity indices overtime based on the abortion status a BMI trends based on the Fig. 4 Trends of adiposity indices overtime based on the abortion status. a BMI trends based on the abortion subgroups; b Waist circumference trends based on the abortion subgroups, c ABSI trends based on the abortion subgroups. BMI body mass index; ABSI a body shape index between menarche age and ABSI overtime. The ABSI is calculated based on the values of WC, BMI, and height and allows us to estimate visceral obesity and overall adi- posity. It has been shown that this indicator can predict clinical outcomes independently of BMI [44]. Age at menarche, adiposity, and body shape indicesi The lack of significant association between age at menarche and ABSI, therefore, highlights the impact of age at menarche on overall obesity but not visceral adiposity. compared to those who experienced menarche at 12 or more years of age [23]. In agreement with previous studies, the results of the current study indicate that the mean changes in BMI in women with early menarche was 1.43 kg/m2 higher than those with normal menarche age, a finding that remained significant even after adjustment for confounders. On the other hand, the mean changes in BMI in women with late menarche were significantly lower than those with normal age at menarche in both models unadjusted and adjusted. While BMI had uprising trends in all groups, regardless of their menarche age, the interaction between time and different subgroups of menarche age (early and late) was not significant. We also found a margin- ally significant effect of menarche age on WC which remained even after adjusting for potential confound- ers, indicating that the mean changes of WC in women with late menarche age were lower than those with nor- mal menarche age. We found no significant association Menopausal status, adiposity, and body shape indices p p y y p Evidence also indicates that estrogen deprivation during the menopause transition can be independently asso- ciated with an increase in total adipose tissue. On the other hand, hormone therapy with estrogen is related to adiposity loss [17]. Despite reducing energy expendi- ture in the aging period, levels of sex hormones dramati- cally decline during the menopausal transition, and body fat distribution alters with reproductive aging; however, accurate biologic processes involved in changes in body Amiri et al. Menopausal status, adiposity, and body shape indices Journal of Translational Medicine (2023) 21:137 Page 9 of 13 Table 3 Effects of menopausal age on adiposity indices in women of the TLGS over time BMI Body mass index; ABSI A body shape index; Ref Reference; CI Confidence interval Model 1: Unadjusted model Model 2: Adjusted for baseline age, smoking, education, and physical activity status Time variable means follow-up visits a Age at natural menopause was defined as younger than 40 years (premature menopause), 40–44 years (early menopause), 45–54 years (reference category), and 55 years or older (late menopause) b The results of ABSI are presented by z-scores c Significant p-values (p < 0.05) are bolded Reproductive factors Outcomes Model 1 P-value c Model 2 P-value c Coefficient (95% CI) Coefficient (95% CI) Menopause agea (ref: Normal) BMI (kg/m2) Premature/early 0.37 (− 0.18, 0.92) 0.188 − 0.10 (− 0.67, 0.46) 0.722 Late − 0.13 (− 1.01, 0.75) 0.773 0.26 (− 0.62, 1.14) 0.566 Time (year) − 0.07 (− 0.12, − 0.02) 0.005 − 0.07 (− 0.11, − 0.02) 0.006 Time × premature/early 0.10 (0.02, 0.19) 0.017 0.11 (0.02, 0.19) 0.012 Time × late − 0.10 (− 0.24, 0.04) 0.166 − 0.10 (− 0.24, 0.04) 0.163 Waist circumference (cm) Premature/early 0.24 (− 1.10, 1.57) 0.723 0.26 (− 1.01, 1.63) 0.703 Late 2.31 (0.19, 4.43) 0.033 2.27 (0.15, 4.39) 0.036 Time 1.33 (1.18, 1.47) < 0.001 1.34 (1.19, 1.48) < 0.001 Time × premature/early 0.07 (− 0.19, 0.32) 0.611 0.06 (− 0.19, 0.31) 0.643 Time × late − 0.27 (− 0.70, 0.15) 0.209 − 0.28 (− 0.70, 0.15) 0.200 ABSIb Early − 0.10 (− 0.22, 0.02) 0.093 0.11 (− 0.007, 0.23) 0.066 Late 0.37 (0.18, 0.56) < 0.001 0.19 (0.01, 0.38) 0.037 Time 0.21 (0.20, 0.23) < 0.001 0.22 (0.20, 0.24) < 0.001 Time × premature/early − 0.03 (− 0.05, − 0.0004) 0.047 − 0.03 (− 0.05, − 0.0009) 0.043 Time × late − 0.02 (− 0.06, 0.03) 0.390 − 0.02 (− 0.07, 0.02) 0.308 Table 3 Effects of menopausal age on adiposity indices in women of the TLGS over time BMI Body mass index; ABSI A body shape index; Ref Reference; CI Confidence interval Model 1: Unadjusted model Model 2: Adjusted for baseline age, smoking, education, and physical activity status Time variable means follow-up visits a Age at natural menopause was defined as younger than 40 years (premature menopause), 40–44 years (early menopause), 45–54 years (reference category), and 55 years or older (late menopause) b The results of ABSI are presented by z-scores c Significant p-values (p < 0.05) are bolded role of the hormonal environment in creating changes related to body fatness, indicating that the rate of repro- ductive aging is significantly associated with the body fat pattern. Menopausal status, adiposity, and body shape indices In addition, the BMI of women with abnormal levels of sex hormones was more correlated with their hormonal status than their age or menopausal status [16]. A longitudinal study on 213 Czech women showed that the majority of adiposity indices like waist, WHR, hip, and subgluteal thigh circumference increase significantly in the menopausal group [49]. Another study on 300,000 adult Chinese women from 10 diverse areas showed that later age at menopause and longer reproductive years were independently associated with increased adiposity late in life [20]. fat distribution and body shape during the menopausal transition have not been elucidated [16]. Some studies have demonstrated that in both normal and mildly obese women menopause increases body fat by ~ 5% of body weight and decreases fat-free body mass by a slightly smaller amount [45–47]. It has also been suggested that reproductive hormones can significantly modulate these physiological controls of eating which may be influ- enced by cognition alteration after menopause, although further human studies are still required to corroborate these mechanisms [48]. Despite documentation of the proposed mechanisms mentioned above, the associa- tion between age at menopause and body adiposity is still debated and the results of previous studies are con- flicting [16, 17, 20, 27, 28, 49]. The Framingham Heart Study conducted on 522 women revealed no association between menopausal age and several measures of body composition, including BMI and WC [27]. Likewise, a Japanese study conducted on 1022 women found no relationship between BMI and age at natural menopause [28]. In contrast, an observational study on both popula- tions of pre-and postmenopausal women highlights the Our study results indicate that mean changes of BMI overtime were significantly higher in women with prema- ture/ early menopausal age than those with normal men- opausal age, even after adjusting confounders, whereas women with late menopausal age had higher WC and ABSI values. Our study findings, therefore, suggest that having a longer reproductive life span, likely through reproductive events like pregnancy and related factors, Amiri et al. Menopausal status, adiposity, and body shape indices Journal of Translational Medicine (2023) 21:137 Page 10 of 13 Table 4 Effects of parity and abortion on adiposity indices in women of the TLGS over time BMI: Body mass index; ABSI: A body shape index; Ref: Reference; CI: Confidence interval Model 1: Unadjusted model Reproductive factors Outcomes Model 1 P-value b Model 2 P-value b Coefficient (95% CI) Coefficient (95% CI) Parity (ref: 0) BMI (kg/m2) 1–2 0.74 (− 0.63, 2.10) 0.290 0.83 (− 0.51, 2.18) 0.228 ≥ 3 1 (− 0.25, 2.24) 0.117 1.06 (− 0.17, 2.30) 0.092 Time − 0.09 (− 0.29, 0.11) 0.380 − 0.08 (− 0.28, 0.12) 0.413 Time × (1–2) − 0.003 (− 0.22, 0.22) 0.982 − 0.003 (− 0.22, 0.22) 0.978 Time × (≥ 3) 0.06 (− 0.15, 0.26) 0.595 0.05 (− 0.15, 0.26) 0.607 Waist circumference (cm) 1–2 1.67 (− 1.72, 5.06) 0.335 1.75 (− 1.61, 5.11) 0.307 ≥ 3 3.09 (− 0.008, 6.19) 0.051 2.84 (− 0.23, 5.92) 0.070 Time 1.18 (0.58, 1.79) < 0.001 1.20 (0.60, 1.80) < 0.001 Time × (1–2) 0.19 (− 0.48, 0.87) 0.573 1.19 (− 0.48, 0.86) 0.577 Time × (≥ 3) 0.15 (− 0.47, 0.77) 0.644 1.13 (− 0.48, 0.75) 0.673 ABSIa 1–2 − 0.03 (− 0.35, 0.28) 0.849 − 0.05 (− 0.35, 0.25) 0.749 ≥ 3 0.14 (− 0.15, 0.43) 0.346 0.06 (− 0.21, 0.34) 0.660 Time 0.21 (0.15, 0.28) < 0.001 0.22 (0.16, 0.28) < 0.001 Time × (1–2) 0.01 (− 0.06, 0.08) 0.778 0.01 (− 0.06, 0.08) 0.785 Time × (≥ 3) − 0.01 (− 0.08, 0.05) 0.666 − 0.01 (− 0.08, 0.05) 0.674 Abortion (ref: No) BMI (kg/m2) Abortion 0.35 (− 0.15, 0.85) 0.169 0.25 (− 0.25, 0.75) 0.319 Time − 0.04 (− 0.09, 0.01) 0.127 − 0.03 (− 0.08, 0.01) 0.165 Time × abortion − 0.02 (− 0.1, 0.05) 0.575 − 0.02 (− 0.09, 0.06) 0.651 Waist circumference (cm) Abortion 0.41 (− 0.79, 1.61) 0.502 0.19 (− 1.03, 1.37) 0.783 Time 1.30 (1.15, 1.45) < 0.001 1.31 (1.16, 1.46) < 0.001 Time × abortion 0.07 (− 0.17, 0.30) 0.572 0.1 (− 0.17, 0.29 0.618 ABSIa Abortion − 0.02 (− 0.13, 0.09) 0.716 − 0.04 (− 0.14, 0.07) 0.481 Time 0.19 (0.18, 0.21) < 0.001 0.20 (0.19, 0.22) < 0.001 Time × abortion 0.02 (− 0.007, 0.04) 0.160 0.02 (− 0.009, 0.04) 0.208 Table 4 Effects of parity and abortion on adiposity indices in women of the TLGS over time can lead to increasing WC values overtime, the hypoth- esis needs to confirm by further well-design longitudinal studies. Menopausal status, adiposity, and body shape indices also evidence estimating that multiparous women have higher BMI than their nulliparous counterparts [14]. On the other hand, women’s body shape may be influ- enced by their pregnancies due to the mobilization of polyunsaturated fatty acids (PUFAs) from the lower parts of their bodies to meet the needs of the develop- ing fetus [15]. A large number of studies have assessed a link between parity and excess body weight after child- birth, and the majority of these studies reported a pos- itive association [6, 14, 17, 18, 20, 29]. In this regard, a cross-sectional study revealed that adiposity was Parity, abortion, adiposity, and body shape index On the contrary, data from a cross-sectional study on 508 Chilean women revealed that BMI, but not other adiposity parameters like WC, WHR, and waist-to-height ratio (WHtR), was modestly affected by parity after controlling by individual, reproductive, and metabolic confounders, finding suggesting a little or no influence of parity on the adiposity indices, in particu- lar central [14]. While at the initiation of the study, we expected to observe a strong association between parity and adi- and conflicting results might be explained by a lack of consideration of some potential confounders like age at first pregnancy, lactation, birth interval, etc. associated inversely with age at first birth. In addition, the mentioned study reported that there was a non- linear positive association between adiposity and parity [20]. Likewise, data from the Stockholm Pregnancy and Weight Development Study (SPAWN), after 15 years of follow-up, showed an increment of 0.5 kg body weight per pregnancy [50]. The Coronary Artery Risk Devel- opment in Young Adults (CARDIA) study with over 10 years of follow-up demonstrated greater values of WC and WHR in multiparous women compared to nulliparous women [51]. Findings from the Million Women Study indicated that childbearing patterns had a persistent effect on BMI, indicating that women with more births had higher BMI than nulliparous women. On the other hand, at every parity level, a reduction in BMI is associated with just 6 months of breastfeed- ing in women [18]. A prospective study on Swedish women reported similar findings, indicating that par- ity was positively associated with total as well as cen- tral obesity, and lactation time was positively related to abdominal fat cell diameter [43]. Findings from the Third National Health and Nutrition Examination Survey (NHANES III) demonstrated that after adjust- ing age and BMI, an increasing parity in women was significantly associated with a relative decrease in hip circumference and an increase in WC after controlling for age and BMI [30]. A cross-sectional survey of 4130 white British women, using three-dimensional pho- tonic scanning showed that parous women ≤ 40 years, parity, independent of age and BMI, was associated with increased abdominal dimensions and reduced hip and thigh dimensions compared to nulliparous, inde- pendent of age and BMI, although the effects of parity on shape diluted over time [19]. This finding indicates that the effect of pregnancy is to accelerate an under- lying age-associated fat redistribution in younger adult females. Conclusionh This study demonstrated higher BMI in females with earlier menarche age. We also showed higher values of BMI overtime in women with premature/ early meno- pause, whereas women with late menopausal age had higher WC and ABSI values. A marginal effect of par- ity on BMI and WC was detected as well, indicating that women with parity ≥ 3 had higher mean changes in these parameters, compared to those with less par- ity. However, more longitudinal studies investigating body composition indices by adjusting all potential confounders are still required to confirm our study findings. Strengths and limitationsh The greatest strength of our study is its design as a com- munity-based study with a long-term follow-up, which enables us to make causal inferences about trends of adiposity and body shape indices overtime considering reproductive factors in an unselected population. This study has also some limitations. First, while we tried to adjust all potential confounders, other risk factors like lifestyle, socioeconomic status, nutrition patterns breastfeeding patterns, and a history of medical inter- vention for the management of obesity could not be considered due to the unavailability of data. Second, we could not assess body composition indices since their data were not available. Finally, it should be also con- sidered that the TLGS has a representative sample of an urban Iranian population, caution should be used when attempting to generalize findings to rural people. Acknowledgements g The authors would like to thank the National Institute for Medical Research Development (NIMAD) for approval of this project and its support as a research project. Parity, abortion, adiposity, and body shape index On the contrary, data from a cross-sectional study on 508 Chilean women revealed that BMI, but not other adiposity parameters like WC, WHR, and waist-to-height ratio (WHtR), was modestly affected by parity after controlling by individual, reproductive, and metabolic confounders, finding suggesting a little or no influence of parity on the adiposity indices, in particu- lar central [14]. Author contributions MA: Study conception, design, execution, literature search, data collection, analysis and interpretation of data, critical discussion, manuscript drafting, and revising the manuscript. MM: Statistical analyses, interpretation of data, and drafting and revising statistical methods and figures. FA: Critical discussion, editing. FRT: Study design, interpretation of data, and critical discussion. All authors read and approved the final manuscript. While at the initiation of the study, we expected to observe a strong association between parity and adi- posity measurements, our study findings showed just a marginal effect of parity on BMI and WC, indicating that women with parity ≥ 3 had higher mean changes of these parameters, compared to those with less par- ity. Abortion was not significantly associated with none of the adiposity indices. However, it should be kept in mind that the reason for this weak difference Funding h f d g The funding sources had no involvement in the study. Parity, abortion, adiposity, and body shape index It is well-known that pregnancy triggers weight gain and obesity in women [12] as a result of hormonal changes during pregnancy, increased dietary intake, changes in energy balance, heritable characteristics, adverse lifestyle risk factors associated with childrear- ing, and other postpartum behaviors [11–13]. There is Amiri et al. Journal of Translational Medicine (2023) 21:137 Amiri et al. Journal of Translational Medicine (2023) 21:137 Page 11 of 13 associated inversely with age at first birth. In addition, the mentioned study reported that there was a non- linear positive association between adiposity and parity [20]. Likewise, data from the Stockholm Pregnancy and Weight Development Study (SPAWN), after 15 years of follow-up, showed an increment of 0.5 kg body weight per pregnancy [50]. The Coronary Artery Risk Devel- opment in Young Adults (CARDIA) study with over 10 years of follow-up demonstrated greater values of WC and WHR in multiparous women compared to nulliparous women [51]. Findings from the Million Women Study indicated that childbearing patterns had a persistent effect on BMI, indicating that women with more births had higher BMI than nulliparous women. On the other hand, at every parity level, a reduction in BMI is associated with just 6 months of breastfeed- ing in women [18]. A prospective study on Swedish women reported similar findings, indicating that par- ity was positively associated with total as well as cen- tral obesity, and lactation time was positively related to abdominal fat cell diameter [43]. Findings from the Third National Health and Nutrition Examination Survey (NHANES III) demonstrated that after adjust- ing age and BMI, an increasing parity in women was significantly associated with a relative decrease in hip circumference and an increase in WC after controlling for age and BMI [30]. A cross-sectional survey of 4130 white British women, using three-dimensional pho- tonic scanning showed that parous women ≤ 40 years, parity, independent of age and BMI, was associated with increased abdominal dimensions and reduced hip and thigh dimensions compared to nulliparous, inde- pendent of age and BMI, although the effects of parity on shape diluted over time [19]. 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https://openalex.org/W1991644120	https://europepmc.org/articles/pmc1913059?pdf=render	English	null	A patient survey of out-of-hours care provided by Emergency Care Practitioners	BMC emergency medicine	2,007	cc-by	5,678	BioMed Central BioMed Central Received: 25 September 2006 Accepted: 15 June 2007 C Emergency Medicine 2007, 7:4 doi:10.1186/1471-227X-7-4 © 2007 Halter et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Emergency Medicine Open Access A patient survey of out-of-hours care provided by Emergency Care Practitioners Mary Halter†1, Tom Marlow†2, Daryl Mohammed3 and Geo Address: 1Faculty of Health and Social Care Sciences, Kingston University and St George's, University of London, Grosvenor Wing, St George's Hospital, Cranmer Terrace, London SW17 0RE, UK, 2Community Services Development, 95 Beaconsfield Road, Surbiton, KG5 9AW, UK and 3London Ambulance Service NHS Trust, 8-20 Pocock Street, London, SE1 0BW, UK Email: Mary Halter - mhalter@hscs.sgul.ac.uk; Tom Marlow - Tom.Marlow@neuro.gu.se; Daryl Mohammed - daryl.mohammed@lond- amb.nhs.uk; George TH Ellison - gellison@hscs.sgul.ac.uk * Corresponding author †Equal contributors Background lance calls. The diploma involves dedicated modules on physical assessment, clinical decision-making, minor ill- ness, chronic conditions, pharmacology, paediatric care and the health of older people. At the same time, ECPs also undertake supervised clinical placements with a vari- ety of other practitioners as part of their continuing pro- fessional development, including: GPs; emergency department physicians; community nurses; and social care professionals. More recently they have started to use 'Patient Group Directives' to administer a limited number of medicines, although this had not been introduced at the time the present study took place. g The delivery of out-of-hours primary health care has changed substantially in recent years and now includes a range of service models, including: deputising services; telephone triage; primary care centres; walk-in centres; emergency departments; and/or cooperatives [1]. A new model, which has been positively received by policy mak- ers [2,3], involves Emergency Care Practitioners (ECPs) working with GPs, particularly in the delivery of primary care through out-of-hours home visits. For this and their other enhanced roles, ECPs undertake additional training to develop autonomous practice which aims to enable them to assess and treat the patient at the point of access (wherever possible) and thereby avoid the use of hospital- based emergency departments (wherever appropriate), with subsequent gains in capacity in the hospital sector. Additionally, in the GP environment, the aim has been to reduce waiting times in primary care by visiting patients for GPs [2]. Since December 2004 ECPs have also been deployed to conduct out-of-hours home visits between 09h00 and 21h00 at weekends and on public holidays. When the present study took place (February to April 2005) London ECPs were working alongside GPs in just one such out-of- hours service, in a single NHS-defined geographical area – Bromley Primary Care Trust. This service involves patients who contact their out-of-hours GP service by telephone. Each such call is logged by a call handler who passes the call on to a GP based at the out-of-hours primary care cen- tre. This GP telephones the patient back to elicit any clin- ical information required and to make an initial assessment of any clinical needs. At this point the patient may be given advice on the telephone, or asked to attend the out-of-hours primary care centre. Background But if a home visit is deemed necessary, the GP decides if the patient's condi- tion is suitable for an ECP, or whether a GP is required (although the patient will not ordinarily know which type of practitioner will be visiting them). This decision is based on strict criteria, developed by the GP lead for the ECP out-of-hours scheme, which determines which con- ditions are suitable for home visits by ECPs. There has been a paucity of research into alternative mod- els of out-of-hours primary care, leading to a lack of evi- dence about clinical outcomes [4]. What studies there have been have focused on patient satisfaction and have found that patients were less satisfied when they did not receive the care they were expecting [5-7]. Related research on alternative models of pre-hospital emergency care, where Emergency Medical Technicians or Paramedics have adopted an ECP-type role (which allowed them to 'treat and release' patients at the scene – albeit without the additional training that ECPs receive), suggested that there were a number of unresolved concerns about patient safety [8-10]. The aim of the present study was therefore to evaluate the care provided to patients receiving out-of-hours home vis- its from ECPs in London from the patients' perspective and assess their wellbeing following the visit. We antici- pated that the potential existed for ECPs to make inappro- priate care decisions, given that ECPs are comparatively inexperienced providers of out-of-hours care. And although this was not intended to be a survey of patient satisfaction, we expected that patients' experiences of care might be negatively affected by the fact that this was deliv- ered by an ECP, given that patients who call their out-of- hours GP service would not have expected an ECP to carry out their home visit. At the time of the present study any information provided by the patient calling the out-of-hours service, together with the assessment made by the GP based at the out-of- hours centre, was transmitted electronically to a computer in the ECP's response car. And after every home visit, whether by a GP or ECP, the patient's own GP received a faxed copy of the 'call sheet' which contained the attend- ing practitioner's record of the assessment they had made, any treatment they had provided and any further care they recommended. Background This could include a recommendation to the patient's GP that they should be followed up urgently, but this was not always the case. The management infor- mation available for analysis in the present study there- fore included: the age and gender of the patient; their presenting complaint; subsequent disposal (i.e. treatment at home with/without referral, or conveyance to the out- of-hours primary care centre/hospital emergency depart- ment); and the time spent with the patient. http://www.biomedcentral.com/1471-227X/7/4 BMC Emergency Medicine 2007, 7:4 Abstract Background: Emergency Care Practitioners (ECPs) have recently been deployed to provide out- of-hours primary care home visits – a practice development that has been supported by policy makers. The aim of the study was to evaluate the care provided to patients receiving out-of-hours home visits from ECPs in London from the patients' perspective and to assess their wellbeing following the visit. Methods: A bespoke telephone-administered questionnaire was designed to survey all patients who had received out-of-hours care in Bromley Primary Care Trust from ECPs during a ten week period in 2005 (n = 174). Results: Sixty three patients (36.2%) were excluded because: no telephone number was available; they had a diagnosis of dementia; or had not received a study information sheet. The remainder (n = 111) were contacted 3–5 days after the home visit, and 81 of these (73.0%) completed the survey. Of those respondents treated at home who gave unequivocal answers (n = 60), all but one (8.3%) reported that they felt that their treatment had been 'right' and/or had followed any advice given. However, overall only 86.4% reported that they had been clear about their ECP's assessment, and only 58.0% reported that their health was now 'better'. Those who reported that they were not clear about their assessment were less likely to report that their health was 'better' (p = 0.03) and more likely to have subsequently used hospital-based health services (p = 0.03). Conclusion: Most patients treated at home by ECPs appeared satisfied and compliant with the care provided, according to the measures used in this study. However, it appears that a sizeable minority of patients were unclear about ECP assessments and it remains to be seen whether these patients had pre-existing health complaints which made them less likely to recover and more likely to seek hospital care, or whether the lack of clarity about their assessment undermined their subsequent recovery and necessitated hospital care. Further research is required to establish if the assessments provided by ECPs are less clear than those provided by other practitioners, and whether it is possible to ensure that all such assessments are clear to all patients. Patients hold a mainly positive view of out-of-hours home visit care provided by ECPs, although a lack of clarity about their assessment was evident, with a possible impact on their continuing health. Page 1 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-227X/7/4 http://www.biomedcentral.com/1471-227X/7/4 Survey questionnaire hours within 3–5 days of their ECP home visit (i.e. as soon as possible thereafter). hours within 3–5 days of their ECP home visit (i.e. as soon as possible thereafter). y q A bespoke questionnaire was developed by members of the research team (MH and TM), drawing on discussions with the ECP-, GP- and management-leads for out-of- hours care in Bromley Primary Care Trust, and focusing on concerns raised before the scheme became opera- tional. The questionnaire was subsequently examined by these leads who concluded that it had both face and con- tent validity, and had successfully covered all of their prin- cipal concerns. The questionnaire used both closed- and open-ended questions and focussed on the following aspects of out-of-hours care: what had happened to the patient during and after the ECP's visit, with questions tai- lored to two groups -those patients who had received advice (had they followed the advice) and those who had been treated, referred or taken to another facility (had this treatment felt 'right'); clarity about the ECP's assessment (i.e. what the outcome of care would be and when any subsequent care might happen); and whether their health had felt 'better' following the ECP's visit. Data collected during the first week of the study were used to pilot and evaluate the questionnaire, but no issues arose with administering the survey during this period, and since no modifications were made, the data from this initial week were included in the final analyses. Analysis R Responses to the questionnaire were transcribed verbatim during each telephone interview, and were subsequently categorised by two of the authors (MH and TM) prior to analysis. Data were managed in Microsoft Access and were statistically analysed using SPSS version 12 to conduct t tests, χ2 tests and Fisher's exact tests. Methods Study setting The ECPs currently practising in London are all Emer- gency Medical Technicians or Paramedics who have undertaken additional diploma-level training, part-time over a two year period. During this time they continue to practice, primarily by responding to emergency ambu- Page 2 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-227X/7/4 BMC Emergency Medicine 2007, 7:4 http://www.biomedcentral.com/1471-227X/7/4 Results Of the 174 eligible participants, 63 (36.2%) were excluded because: they had a diagnosis of dementia (n = 2); no telephone number was available (n = 6); or there was no record that they had received a study information sheet (n = 55; see Table 1). There were no significant dif- ferences in age (χ2 = 2.76, d.f. = 1, p > 0.05), gender (χ2 = 0.02, d.f. = 1, p > 0.05), disposal (χ2<0.01, d.f. = 1, p > 0.05) or visit duration (t = 1.07, n = 112, p > 0.05) between patients included and excluded from the survey. However, there were significant differences in presenting complaint (χ2 = 29.20, d.f. = 20, p = 0.02), with fewer of those excluded having urological and pain-related com- plaints and fewer of those included having neurological and gastrointestinal complaints. Participant recruitment All patients who had received an ECP out-of-hours home visit between 26th February and 15th May 2005 were eligi- ble for recruitment into the study. ECPs were asked to give these patients an information sheet at the end of their home visit, which explained that they might receive a tel- ephone call from a researcher, at which point the research governance approval allowed the researchers to contact the participants and request informed consent to partici- pate in the study. The records of each of these patients were then collected from the GP out-of-hours centre and potential participants were telephoned during office Only one of the 111 patients included in the study declined to take part, but 29 others failed to answer their phone call (see Table 1). Nonetheless, there were no sig- nificant differences in age (χ2 = 3.85, d.f. = 1, p < 0.05), gender (χ2 = 0.81, d.f. = 1, p < 0.05), type of complaint (χ2 = 25.2, d.f. = 18, p < 0.05), or visit duration (t = 1.07, n = Table 1: Reasons for excluding potential participants and non-respondents Exclusion stage Reason for exclusion n % within stage Prior to contacting patients (of n = 174) No telephone number recorded 6 3.5 Dementia documented 2 1.1 No record that the patient had received a study information sheet1 55 31.6 Subtotal 63 36.2 On patient contact (of n = 111) No reply to telephone call 29 26.1 Patient declined to participate 1 0.9 Subtotal 30 27.0 1Careful examination of patients who had not received a study information sheet found that the majority had received visits on seven specific days within the thirty day study period, suggesting that these exclusions reflect a general failure to deliver study information sheets rather than the selective exclusion of specific types of patients during the course of the study. Research governance Research governance approval for the study was granted by the London Ambulance Service and Bromley Primary Care Trust. Participant recruitment = 1, p < 0.01; see Table 2) and may therefore have been easier to contact by phone within 3– 5 days. 112, p > 0.05) between the 81 participants who completed the questionnaire and the 30 who did not, although the former were significantly more likely to have been treated at home (χ2 = 9.09, d.f. = 1, p < 0.01; see Table 2) and may therefore have been easier to contact by phone within 3– 5 days. From Table 3, which summarises the experiences of patients captured by the survey questionnaire, it is clear that all but one of those who had not been conveyed to hospital and who had given unequivocal answers (n = 59 of n = 60; 98.3%) reported that they had felt the treatment they had received had been 'right' and/or had followed the ECP's advice. Nonetheless, within the 3–5 days since their home visit, only 61.5% of the 26 patients that had been referred on by their ECP had actually been seen by these services, and 38.1% of those who had not been referred on by their ECP had subsequently been seen by a GP, nurse or hospital-based practitioner. Indeed, overall only 86.4% of the 81 participants reported that they had been clear about their ECP's assessment, and only 58.0% reported that their health was now 'better'. Those who reported that they were not clear about their assessment (n = 9) were less likely to report that their health was now 'better' (22.2% vs 62.9%; (χ2 = 5.41, d.f. = 1, p = 0.03), and these respondents were also more likely to have sub- sequently used hospital-based health services (33% vs 3.3%; χ2 = 6.39, d.f. = 2, p = 0.03). From Table 2 it is clear that most of the respondents were female (67.9%) and aged 60 or above (59.3%). More than two thirds had presented with respiratory infections (27.2%), urological complaints (17.3%), back pain (12.3%) or medical conditions (12.3%; including: arthri- tis; hypertension; influenza; and heart failure), while comparatively few had gastro-intestinal complaints (7.4%), pain (other than chest or back pain; 6.2%), minor injuries (5.0%), dizziness (2.5%) or neurological com- plaints (2.5%). Few of these conditions resulted in imme- diate conveyance to the hospital emergency department (12.3%) or out-of-hours primary care centre (3.7%) and most were treated at home, with (32.1%) or without (51.9%) referral to additional services. Participant recruitment Table 1: Reasons for excluding potential participants and non-respondents Exclusion stage Reason for exclusion n % within stage Prior to contacting patients (of n = 174) No telephone number recorded 6 3.5 Dementia documented 2 1.1 No record that the patient had received a study information sheet1 55 31.6 Subtotal 63 36.2 On patient contact (of n = 111) No reply to telephone call 29 26.1 Patient declined to participate 1 0.9 Subtotal 30 27.0 1Careful examination of patients who had not received a study information sheet found that the majority had received visits on seven specific days within the thirty day study period, suggesting that these exclusions reflect a general failure to deliver study information sheets rather than the selective exclusion of specific types of patients during the course of the study. Table 1: Reasons for excluding potential participants and non-respondents 1Careful examination of patients who had not received a study information sheet found that the majority had received visits on seven specific days within the thirty day study period, suggesting that these exclusions reflect a general failure to deliver study information sheets rather than the selective exclusion of specific types of patients during the course of the study. 1Careful examination of patients who had not received a study information sheet found that the majority had received visits on seven specific days within the thirty day study period, suggesting that these exclusions reflect a general failure to deliver study information sheets rather than the selective exclusion of specific types of patients during the course of the study. 1Careful examination of patients who had not received a study information sheet found that the majority had received visits on seven specific days within the thirty day study period, suggesting that these exclusions reflect a general failure to deliver study information sheets rather than the selective exclusion of specific types of patients during the course of the study. http://www.biomedcentral.com/1471-227X/7/4 BMC Emergency Medicine 2007, 7:4 hours (148 minutes), and averaged just under an hour (50 minutes). hours (148 minutes), and averaged just under an hour (50 minutes). 112, p > 0.05) between the 81 participants who completed the questionnaire and the 30 who did not, although the former were significantly more likely to have been treated at home (χ2 = 9.09, d.f. Participant recruitment phic and service-related characteristics of respondents and non-respondents Table 2: Sociodemographic and service-related characteristics of respondents and non-respondents BMC Emergency Medicine 2007, 7:4 http://www.biomedcentral.com/1471-227X/7/4 Table 3: Self-reported outcomes following the ECP home visit Patient disposal Question Reported outcome n % Remained at home (n = 68) Followed advice given or treatment felt right (n = 68) Yes 59 86.8 No 1 1.5 Unclear from responses 8 11.8 Overall services used (n = 68) GP or nurse 29 42.6 Emergency department 1 1.5 Hospital admission 3 4.4 None reported 35 51.5 Follow up to the ECP's referral (n = 26)1 Not seen yet (GP = 9, physiotherapist = 1)1 10 38.5 Seen and remained at home (GP = 11, nurse = 2, GP and nurse = 1, emergency department = 1)1 15 53.8 Seen and admitted to hospital (GP) 2 7.7 No referral reported but services used (n = 16) Seen and remained at home (GP = 14, nurse = 1) 15 93.8 Seen and admitted to hospital (GP) 1 6.2 Conveyed (n = 13) Outcome following conveyance Discharged from out-of-hours centre 0 0 Discharged from the emergency department 3 23.1 Admitted to hospital 10 76.9 All (n = 81) Clear about the outcome at the end of the assessment Yes 70 86.4 No 9 11.1 No response to this question 2 2.5 Clear about when the next steps might happen? Yes 47 58.0 No 2 2.5 Unclear from responses 4 4.9 No response to this question 28 34.6 How are you now? Better 47 58.0 Managing 20 24.7 Needed help 7 8.6 Worse 7 8.6 1One patient was referred to both the hospital emergency department and to a physiotherapist. Table 3: Self-reported outcomes following the ECP home visit One patient was referred to both the hospital emergency department and to a physiotherapist. 1One patient was referred to both the hospital emergency department and to a physiotherapist. in presenting complaint amongst those excluded and included in the study, and it is unclear whether patients selected for ECP visits presented with the same level of complexity or severity of presenting condition, the subse- quent diagnoses of respondents in our study suggests that they were broadly representative of patients receiving sim- ilar care elsewhere. Participant recruitment Finally, there was substantial variation in the duration of ECP home visits, which ranged from just 11 minutes to more than two Table 2: Sociodemographic and service-related characteristics of respondents and non-respondents Characteristic Respondents Non respondents All n % n % n % 81 73.0 30 27.0 111 100 Age Median 82 years 78 years 80 years Aged less than 60 31 39.2 17 60.7 48 44.9 Aged 60 and over 48 60.8 11 39.3 59 55.1 Not known 2 - 2 - 4 - Gender Female 55 67.9 23 76.7 78 70.3 Male 26 32.1 7 23.3 33 29.7 Presenting complaint Respiratory infection 22 27.2 4 13.3 26 23.4 Urological 14 17.3 6 20.0 20 18.0 Medical condition1 10 12.3 10 33.3 20 18.0 Back pain 10 12.3 1 3.3 11 9.9 Gastrointestinal 6 7.4 2 6.7 8 7.2 Pain (other than chest or back) 5 6.2 2 6.7 7 6.3 Minor injuries 4 5.0 0 0 4 3.6 Fainted/dizziness 2 2.5 0 0 2 1.8 Neurological/stroke 2 2.5 0 0 2 1.8 Other 6 7.4 5 16.7 11 9.9 Disposal Treated at home2 42 51.9 12 40.0 54 48.6 Treated at home and referred 26 32.1 5 16.7 31 27.9 Conveyed to the out-of-hours primary care centre 3 3.7 2 6.7 5 4.5 Conveyed to the hospital emergency department 10 12.3 11 36.7 21 18.9 Time spent with the patient (minutes) Median (where known, n = 81) 50 (11–148) 47 (15 – 94) 50 (11–148) 1Medical conditions included arthritis, hypertension, influenza, and heart failure. 2Respondents were significantly more likely to have been treated at home (χ2 = 9.09, d.f. = 1, p < 0.01). Participant recruitment Indeed, the respondents in the present study had a similar range of diagnoses [11] and outcomes (such as subsequent self-reported health [12] and hospital admission [13]) to those observed by previous studies of out-of-hours care. Nonetheless, the findings of this study are only strictly applicable to patients with similar pre- Page 5 of 7 (page number not for citation purposes) Discussion Further research is therefore warranted to establish whether this might reflect ECPs' relative inexperience in out-of-hours primary care, and whether this might improve with appropriate experi- ence, clinical supervision or training. Further research is also required to establish if a lack of clarity is only found when ECPs provide such care, or whether it might be expected from any practitioner dealing with difficult con- ditions or patients with confusion. Either way, additional support and training might be appropriate for ECPs and GPs to improve patient understanding of the out-of-hours assessments they provide. Meanwhile, a second potential limitation is that the study used a bespoke questionnaire which relied on self- reported outcomes rather than more objective measures which might have provided a more reliable assessment of the quality of care provided by ECPs [14]. However, the use of self-reported outcomes is entirely appropriate for addressing the aim of the present study, which was to assess patients' experiences of the care they had received, and this approach was crucial for identifying an important minority of patients who were unclear about the assess- ments provided by ECPs. As such the questionnaire suc- cessfully captured sufficient variation in perceived care and subsequent wellbeing to identify important differ- ences in these outcomes amongst different groups of respondents, even though there was insufficient variation in their response to advice, services used or conveyance to hospital to permit a detailed investigation thereof (see Table 3). Notwithstanding these potential limitations, the present study does provide a degree of reassurance that ECPs were capable of delivering care that was considered acceptable, or advice that was followed by patients, at out-of-hours home visits, according to the measures we used. Moreo- ver, most of our respondents were treated at home which suggests that our interpretation of findings is particularly relevant to this group. Setting aside the eight respondents who provided equivocal answers, all but one of the remaining 60 respondents who had not been conveyed to hospital felt that the care they had received from their ECP had been 'right' or that they had followed the advice the ECP had given. This high level of agreement with the care provided by ECPs, and of self-reported compliance with the advice offered by ECPs, mirrors the high level of satis- faction found in a recent evaluation of ECPs working in emergency care [15]. Discussion Before laying too much store by the results of the present study there are two potential limitations that need to be taken into account. First and foremost, the survey drew on a modest sample of respondents, a disproportionate number of whom were women, treated at home and aged ≥60. However, since older people are the principal recipi- ents of home visits in this out-of-hours service, and since most of those attended at home by GPs remain there after their visit, it seems likely that respondents were broadly similar to patients receiving out-of-hours home visits from GPs. And although there were significant differences Page 5 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-227X/7/4 BMC Emergency Medicine 2007, 7:4 patients were sufficiently satisfied with the care received from ECPs to overcome any disappointment at not being visited by a GP. senting conditions subject to similar selection for subse- quent care, and who were primarily treated at home rather than in hospital. Despite these reassuring findings, the present study did identify a sizeable minority of respondents who were unclear about their ECP's assessment. These patients were less likely to report that their health was 'better' and more likely to have subsequently sought hospital care. While it is not possible to establish the reasons for these associa- tions from the cross-sectional survey used in the present study, it is possible to identify two possible explanations and suggest fruitful avenues for future research. The first explanation is that the ECPs did not always provide a clear assessment of what would happen next for each of the patients they visited, and that a lack of clarity undermined the recovery of these patients and increased their risk of requiring or seeking hospital-based care. The second explanation is that health conditions which are more dif- ficult to assess and explain to patients are less likely to improve and more likely to require additional, hospital- based care. Certainly, patients with lower self-reported health status have been found to be significantly less sat- isfied with their out-of-hours care in the past [16], and it is likely that it is difficult for ECPs and GPs to clearly assess or explain all conditions to all patients. Nonetheless, the present study found that ECPs' assessments can be unclear to out-of hours primary care patients, despite the fact that they spend longer with them than GPs. Conclusion Th The present study found high levels of agreement with the care provided or self-reported compliance with advice provided by ECPs during out-of-hours primary care home visits. However, it also found a sizeable minority of patients who were not clear about their ECP's assessment, and this was associated with subsequent wellbeing and use of services. Discussion This suggests that the ECP model, particularly its focus on treating patients at the point of contact and avoiding conveyance to hospital wherever appropriate, is well-received by most patients in both con- texts. And while the present study was not intended to be a survey of general satisfaction with ECP-delivered out-of- hours primary care, it is worth considering such studies in similar settings to inform what we might have expected to find here. In particular, while patient satisfaction is gener- ally higher with home visits than with other models of out-of-hours primary care [4,5], we did not expect such a positive response to the ECP home visit because the patients were likely to have been expecting a GP, and pre- vious studies have found higher satisfaction with out-of- hours care when this met patients' prior expectations [5- 7]. With this in mind the present study suggests that References 1. Department of Health: Raising standards for patients: new part- nerships in out-of-hours care An independent review of GP out-of-hours services in England commissioned by the Department of Health. London , Department of Health; 2000. p , p ; 2. NHS Modernisation Agency: Right Skill, Right Time, Right Place The ECP Report. London , Department of Health,; 2004. p p 2. NHS Modernisation Agency: Right Skill, Right Time, Right Place The ECP Report. London , Department of Health,; 2004. p , p ,; 3. Department of Health: Taking Healthcare to the Patient. Transforming NHS Ambulance Services. London , Depart- ment of Health; 2005. p p 3. Department of Health: Taking Healthcare to the Patient. Transforming NHS Ambulance Services. London , Depart- ment of Health; 2005. 4. Leibowitz R, Day S, Dunt D: A systematic review of the effect of different models of after-hours primary medical care serv- ices on clinical outcome, medical workload, and patient and GP satisfaction. Family Practice 2003, 20(3):311-317. y ( ) 5. Thompson K, Parahoo K, Farrell B: An evaluation of a GP out-of- hours service: meeting patient expectations of care. Journal of Evaluation in Clinical Practice 2004, 10(3):467-474. Evaluation in Clinical Practice 2004, 10(3):467 474. 6. McKinley RK, Roberts C: Patient satisfaction with out of hours primary medical care. Quality in Health Care 2001, 10(1):23-28. p y Q y , ( ) 7. McKinley RK, Stevenson K, Adams S, Manku-Scott TK: Meeting patient expectations of care: the major determinant of satis- faction with out-of-hours primary medical care? Family Practice 2002, 19(4):333-338. ( ) 8. Snooks HA, Dale J, Hartley-Sharpe C, Halter M: On-scene alterna- tives for emergency ambulance crews attending patients who do not need to travel to the accident and emergency department: a review of the literature. Emergency Medicine Jour- nal 2004, 21(2):212-215. ( ) 9. Pringle RP Jr., Carden DL, Xiao F, Graham DD Jr.: Outcomes of patients not transported after calling 911. Journal of Emergency Medicine 2005, 28(4):449-454. ( ) 10. Hauswald M, Raynovich W, Brainard AH: Expanded emergency medical services: the failure of an experimental community health program. Prehospital Emergency Care 2005, 9(2):250-253. p g p g y ( ) 11. Brogan C, Pickard D, Gray A, Fairman S, Hill A: The use of out of hours health services: a cross sectional survey. BMJ 1998, 316(7130):524-527. ( ) 12. Competing interests p g None. There was no specific funding for the study. MH and TM carried out the study whilst employed at London Ambulance Service NHS Trust. Page 6 of 7 (page number not for citation purposes) Page 6 of 7 (page number not for citation purposes) Page 6 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-227X/7/4 http://www.biomedcentral.com/1471-227X/7/4 http://www.biomedcentral.com/1471-227X/7/4 BMC Emergency Medicine 2007, 7:4 Pre-publication history p y The pre-publication history for this paper can be accessed here: MH, TM and DM designed the study. TM carried out the patient interviews. MH, TM and GTHE carried out the data analysis and interpretation. MH led preparation of the paper with GTHE. TM and DM commented substantially on all drafts. http://www.biomedcentral.com/1471-227X/7/4/prepub http://www.biomedcentral.com/1471-227X/7/4/prepub Acknowledgements We are grateful to the participating patients; to Richard Mintern and the Bromley ECP team at London Ambulance Service for providing data and informing patients of the study; and to Mary Cooke at EMDoc for providing access to data. Caro van Uden, Gerard Bury and Edward Krupat all pro- vided helpful suggestions for improving the manuscript following review. References McKinley RK, Cragg DK, Hastings AM, French DP, Manku-Scott TK, Campbell SM, Van F, Roland MO, Roberts C: Comparison of out of hours care provided by patients' own general practition- ers and commercial deputising services: a randomised con- trolled trial. II: The outcome of care. BMJ 1997, 314(7075):190-193. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 7 of 7 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge ( ) 13. Salisbury C: Observational study of a general practice out of hours cooperative: measures of activity. BMJ 1997, 314(7075):182-186. 14. Rao M, Clarke A, Sanderson C, Hammersley R: Patients' own assessments of quality of primary care compared with objec- tive records based measures of technical quality of care: cross sectional study. BMJ 2006, 333(7557):19. y J ( ) 15. Halter M, Marlow T, Tye C, Ellison GTH: Patients' experiences of care provided by emergency care practitioners and tradi- tional ambulance practitioners: a survey from the London Ambulance Service. Emerg Med J 2006, 23(11):865-866. g J ( ) 16. Glynn LG, Byrne M, Newell J, Murphy AW: The effect of health status on patients' satisfaction with out-of-hours care pro- vided by a family doctor co-operative. Family Practice 2004, 21(6):677-683.
https://openalex.org/W3040652137	http://dspace.bsu.edu.ru/bitstream/123456789/46586/1/Zyryanov_Medication.pdf	English	null	Medication adherence in patients with stable coronary artery disease in primary care	Research results in pharmacology	2,020	cc-by	5,706	Medication adherence in patients with stable coronary artery disease in primary care Sergey K. Zyryanov1, Sergey B. Fitilev1, Alexander V. Vozzhaev1, Irina I. Shkrebniova1, Natalya N. Shindryaeva2, Dmitry A. Klyuev1, Liusine N. Stepanyan1, Nikolay N. Landyshev1, Yana G. Voronko1 1 RUDN University, Medical Institute, Department of Pharmacology and Clinical Pharmacology, 6 Miklukho-Maklaya St., Moscow 117198, Russia 2 City Polyclinic #2 of Moscow Healthcare Department, 12 Fruktovaya St., Moscow 117556, Russia Corresponding author: Alexander V. Vozzhaev (vozzhaev-av@rudn.ru) Academic editor: Tatyana Pokrovskaya ♦ Received 11 April 2020 ♦ Accepted 15 May 2020 ♦ Published 3 Academic editor: Tatyana Pokrovskaya ♦ Received 11 April 2020 ♦ Accepted 15 May 2020 ♦ Published 30 June 2020 Citation: Zyryanov SK, Fitilev SB, Vozzhaev AV, Shkrebniova II, Shindryaeva NN, Klyuev DA, StepanyanLN, Landyshev NN, Voronko YG (2020) Medication adherence in patients with stable coronary artery disease in primary care. Research Results in Pharmacology 6(2): 97–103. https://doi.org/10.3897/rrpharmacology.6.54130 Research Results in Pharmacology 6(2): 97–103 UDC: 615.331 DOI 10.3897/rrpharmacology.6.54130 Research Results in Pharmacology 6(2): 97–103 UDC: 615.331 DOI 10.3897/rrpharmacology.6.54130 Abstract Introduction: Lack of research targeting non-adherence to cardiovascular medications in Russia prevents from devel oping effective interventions to improve adherence. The aim was to study medication adherence in patients with stable coronary artery disease in primary care. Material and methods: The study was conducted in a primary care setting of Moscow. Demography, medical history, pharmacotherapy data were obtained retrospectively from 386 coronary patients’ medical records. Medication adher ence was measured by 8-item Morisky Medication Adherence Scale (MMAS-8). A statistical analysis was performed using SPSS Statistics V16.0. Results and discussion: According to the results from MMAS-8, 188 (48.7%) coronary patients had high medication adherence, 135 (35.0%) – moderate, and 63 (16.3%) – low. By the dichotomous interpretation: 48.7% (n = 188) – were adherent, 51.3% (n = 198) – were non-adherent. These groups were similar in gender distribution, age, and medical history profile (p > 0.1 for all variables). Smokers prevailed in the non-adherent group (13.6 vs. 5.3%; p = 0.009). Both groups were equally prescribed beta-blockers, antiplatelets, and statins (p > 0.1 for all). Use of fixed dose combinations (11.7 vs. 5.6%; p = 0.048) and the number of pills taken (mean 5.64 ± 1.52 vs. 5.99 ± 1.62; p = 0.029) were associated with better adherence. Higher values of total cholesterol (mean 5.2 ± 1.4 vs. 4.7 ± 1.2 mmol/L; p < 0.001) and low-den sity lipoprotein cholesterol (mean 2.9 ± 1.2 vs. 2.4 ± 0.9 mmol/L; p < 0.001) were revealed in non-adherents. Subjects with suboptimal adherence visited general practitioners more frequently (median 5 vs. 3 visits; p = 0,003). Conclusion: Medication non-adherence in coronary outpatients exceeded 50%. High adherence was associated with more frequent use of fixed dose combinations and fewer pills taken by patient. Smoking and poorer control of blood lipids prevailed in non-adherents, who also caused higher load on general practitioners. Keywords Keywords coronary artery disease, medication adherence, Morisky scale, primary care. coronary artery disease, medication adherence, Morisky scale, primary care. nov SK et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permi e, distribution, and reproduction in any medium, provided the original author and source are credited. Zyryanov SK et al.: Medication adherence in coronary outpatients 98 to measure adherence. The patients were interviewed via telephone. Introduction The famous quote of American surgeon Dr. C. Everett Coop – “Drugs don’t work in patients who don’t take them” (1985) – has become especially relevant over the recent years to highlight an important role of adequate medication adherence in effective pharmacotherapy. De veloped countries have already built upscale healthcare systems, so a further increase in pharmacological per formance strongly depends on improvement of patient adherence to evidence-supported medications. The glo bal nature of this problem was first raised by The World Health Organization back in 2003 stating that “adherence to long-term therapy for chronic illnesses in developed countries averages 50%. In developing countries, the rates are even lower” (De Geest and Sabate 2003). According to later studies, medication adherence in pa tients with coronary artery disease (CAD) is on average 30–70% (Chowdhury et al. 2013; Chen et al. 2015). Such a situation was described as «non-adherence pandemic» (Kolandaivelu et al. 2014). Meanwhile, there is enough evidence that poor adherence is associated with unfavora ble clinical outcomes (Du et al. 2015; Lenzi et al. 2017) and higher treatment costs (Bitton et al. 2013) in CAD patients. The data from the medical records and questionnaires were transferred to patients’ case report forms. The stu dy database was constructed in MS Excel. Statistical data processing was performed using IBM SPSS Statistics V16.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as mean (M), standard deviation (SD), first (Q1), second (median, Q2) and third (Q3) quartiles. Categorical variables were expressed as frequencies and percentages. Significance of the differences between the groups was estimated by standard statistical tests (two-si ded). Independent t-tests for two independent samples were used for continuous variables distributed approxi mately according to the normal law; Wilcoxon rank-sum tests were used for continuous variables not distributed approximately according to the normal law. Kolmogor ov-Smirnov normality tests were used to check the nor mality of distribution. Chi-square tests were used for cate gorical variables. The level of statistical significance was set at p < 0.05. In the Russian outpatient care practice, adherence to cardiovascular medications is far from being optimal, and this issue lacks proper scientific attention (Bochkareva et al. 2019). It seems difficult to develop and implement ef fective interventions to improve adherence without such information. So, the aim of this research was to study medication adherence in patients with stable CAD at the primary care level. Abstract Patient self-report tools (questionnaires, scales) are broadly used for exploring adherence to cardiovascu lar medications because they are inexpensive, simple, and quite accurate (Shi et al. 2010; Culig and Leppée 2014; Nguyen et al. 2014). One of such self-report tools is Mo risky scale (4-item or 8-item), which was originally deve loped and validated in patients with arterial hypertension. The 8-item version has better validation parameters com pared with the 4-item scale: internal consistency reliability (described by Cronbach’s alpha) 0.83 vs. 0.61, sensitivity 0.93 vs. 0.81, specificity 0.53 vs. 0.44. It also has strong correlation with validation criteria (Morisky et al. 1986, 2008). Interpretation of MMAS-8 was performed in a stan dard way. The patients were asked to answer questions 1–7 as “yes” (0 points) or “no” (1 point), except question 5, which was scored the opposite. Question 8 had a five-point Likert response scale and was scored 1 point for the ans wer “never”. Summing up the points, adherence was as sessed as low (less than 6 points), moderate (6–7 points) or high (8 points). The simplified dichotomous interpretation of MMAS-8 (adherent – 8 points, non-adherent – less than 8 points) was also used (Tan et al. 2014). Material and methods The study was conducted in a large outpatient healthcare facility of Moscow city as part of the Pharmacoepidemio logic Quality Improvement Program of Pharmacotherapy of Stable CAD in Primary Care. The study was approved by the Ethics Committee of this medical institution. At the first stage, 2000 medical records of cardiologic pa tients treated at the facility were randomly selected. Out of this sample, 805 outpatient records were included into a retrospective analysis in line with the following crite ria: age 30 years and older, verified CAD, non-partici pation in any ongoing clinical trial. The following data were obtained: demographics, medical histories including any documented cardiovascular behavioural risk factors, available results of laboratory tests (lipid profile and gly cemic status), pharmacotherapy prescribed to patients by cardiologists, and additional medication maintenance sta tuses. The information on the number of visits to the car diologist and general practitioner over the twelve-month period was also collected. Results and discussion Data from 386 patients with established stable CAD who gave full and unambiguous replies to all MMAS-8 questions were included into the analysis. According to MMAS-8 scoring, 188 coronary patients had high medi cation adherence, 135 – moderate, and 63 – low (Fig. 1). It is notable that in the outpatient cardiovascular registry “PROFILE” the researchers also measured medication ad herence (n = 130) by MMAS-8 tool. The results revealed that 40.8% of patients had high adherence, 36.9% – mode rate, and 22.3% – low (Lukina et al. 2018). But the popula tion was different in some variables, like age and medical history profile from the one described in this study. Data from 386 patients with established stable CAD who gave full and unambiguous replies to all MMAS-8 questions were included into the analysis. According to MMAS-8 scoring, 188 coronary patients had high medi cation adherence, 135 – moderate, and 63 – low (Fig. 1). It is notable that in the outpatient cardiovascular registry “PROFILE” the researchers also measured medication ad herence (n = 130) by MMAS-8 tool. The results revealed that 40.8% of patients had high adherence, 36.9% – mode rate, and 22.3% – low (Lukina et al. 2018). But the popula tion was different in some variables, like age and medical history profile from the one described in this study. At the second stage, the assessment of medication adhe rence in coronary patients was conducted. The 8-item Mo risky Medication Adherence Scale (MMAS-8) was used Research Results in Pharmacology 6(2): 97–103 99 to this finding because it makes the primary direction of possible interventions to improve medication adherence obvious. Analyzing the type of non-adherence is crucial as it should be addressed in different ways, like educatio nal and physician-focused activities to modify intentional non-adherence or behavioral patient-focused strategies to target unintentional non-adherence. Figure 1. Medication adherence in patients with stable coronary artery disease (n = 386). The dichotomous interpretation of MMAS-8 was ap plied for further analysis. The patients with moderate and low adherence formed the group of non-adherents (51.3%). Such a distribution partly matches available Russian data concerning outpatients with arterial hyper tension and CAD, treated at Moscow primary care faci lities. According to that data, 61.1% of patients had poor medication adherence (≤ 3 points by 4-item Morisky sca le). Results and discussion Yet, the target population was younger, and prevalen ce of CAD was less than 50% (Fofanova et al. 2017). Figure 1. Medication adherence in patients with stable coronary artery disease (n = 386). The detailed analysis of non-adherent patients’ respon ses to specific questions MMAS-8 revealed signs of un intentional non-adherence due to forgetfulness (Fig. 2A), which could be expected with regard to the age profile of the study population (mean age 68.9 ± 9.9 years; share of patients ≥ 65 years – 66.8%). However, quite a large num ber of patients turned out to have intentional non-adheren ce due to feeling worse (34.2%) or better (31.7%) when taking the prescribed medications (Fig. 2B). Almost one- third of the participants felt hassled about sticking to their treatment regimens. The investigators of the “PROFILE” registry mentioned above reported 23.5% and 23.6% of the patients were prone to breaking the recommended treatment plan when feeling worse or better, respectively (Lukina et al. 2018). The category of patient-related factors of non-adheren ce is most well studied by now. But there is still no single opinion on this issue. Among possible predictors of poor adherence to cardiovascular medications, the following were mentioned: younger and older age, male sex, low level of income, smoking, forgetfulness due to cognitive disorders, distrust of a healthcare provider, lack of faith in successful treatment outcome, etc. (Warren et al. 2013; Kolandaivelu et al. 2014; Khatib et al. 2019). First, the groups of adherent and non-adherent patients were compared in respect to the demographic and medical history data (Table 1). The number of male subjects was similar in both groups. No statistically significant diffe rences were identified in prevalence of concomitant con ditions, so the groups were comparable in respect to the medical history profiles.fi Besides, it seemed interesting to analyze the respon ses of moderately adherent patients, who were kind of “one step” away from being highly adherent. What pre vented them from making this “step”? It turned out the intentional non-adherence was the first to “be blamed” for that (Fig. 3). It is important to pay specific attention Unfortunately, it appeared to be difficult to analyze prevalence of cardiovascular risk factors in adherent and A B Figure 2. Reasons of unintentional (A) and intentional (B) non-adherence among non-adherent patients (n = 198). B Figure 2. Figure 3. Reasons of unintentional (A) and intentional (B) non-adherence among moderately adherent patients (n = Table 1. Demographics and Medical History Profile of Adherent and Non-adherent Patients with Stable Coronary Artery Disease. Table 2. Pharmacotherapy Patterns of Adherent and Non-adher ent Patients with Stable Coronary Artery Disease. Variable Adherent (n = 188) Non-adherent (n = 198) p Beta-blockers, n (%) 144 (76.6) 156 (78.8) 0.605 Calcium channel blockers, n (%) 87 (46.3) 91 (46.0) 0.950 Long-term nitrates, n (%) 18 (9.6) 10 (5.1) 0.087 Antiplatelet agents, n (%) 135 (71.8) 133 (67.2) 0.323 Oral anticoagulants, n (%) 58 (30.9) 78 (39.4) 0.079 Statins, n (%) 159 (84.6) 166 (83.8) 0.843 ACE† inhibitors, n (%) 93 (49.5) 82 (41.4) 0.112 ARBs‡, n (%) 82 (43.6) 92 (46.5) 0.574 ACE† inhibitors or ARBs‡, n (%) 175 (93.1) 173 (87.4) 0.060 Fixed dose combinations, n (%) 22 (11.7) 11 (5.6) 0.048 Number of pills taken, 5.64 ± 1.52 5.99 ± 1.62 0.029 M ± SD (Q1, Q2, Q3) (2, 6, 10) (2, 6, 12) Note: †angiotensin-converting enzyme; ‡angiotensin II receptor blockers. Variable Adherent (n = 188) Non-adherent (n = 198) p Gender: Male, n (%) 66 (35.1) 72 (36.4) 0.797 Female, n (%) 122 (64.9) 126 (63.6) Age (years), 69.9 ± 8.9 70.4 ± 9.2 0.909 M ± sd (Q1, Q2, Q3) (64, 70, 77) (63, 70, 78) Myocardial infarction, n (%) 86 (45.7) 87 (43.9) 0.721 Revascularization, n (%) 80 (42.6) 75 (37.9) 0.349 Stable angina, n (%) 128 (68.1) 142 (71.7) 0.437 Arterial hypertension, n (%) 174 (92.6) 185 (93.4) 0.734 Chronic heart failure, n (%) 168 (89.4) 175 (88.4) 0.760 Atrial fibrillation, n (%) 54 (28.7) 66 (33.3) 0.385 Diabetes, n (%) 44 (23.4) 44 (22.2) 0.782 Dyslipidemia, n (%) 31 (16.5) 37 (18.7) 0.571 Chronic kidney disease, n (%) 40 (21.3) 33 (16.7) 0.248 non-adherent patients due to the low registration rate of such information in medical records. Only a smoking sta tus was an exception. So, 3-time higher prevalence of smo king was identified in the group of non-adherent patients (13.6 vs. 5.3%; p = 0.009). The possible explanation here could be the “healthy adherer effect”, which describes the association of better medication adherence with a patient’s healthier lifestyle (Gehi et al. 2007; Ladova et al. 2014). of these medicines (above 70% of patients were on novel OACs) and a risk of bleeding. Results and discussion Reasons of unintentional (A) and intentional (B) non-adherence among non-adherent patients (n = 198). Zyryanov SK et al.: Medication adherence in coronary outpatients 100 A B Statins were prescribed to 84.2% (n = 325) of participants with similar rates in ad herents and non-adherents. Renin-angiotensin-aldosterone system (RAAS) inhibitors were recommended to 90.2% (n = 348) of the patients. No statistical significance was identified in prescription rates of angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor bloc kers (ARBs) between the groups. However, there was an obvious tendency (p = 0.060) to the association of RAAS inhibitors intake with better adherence, possibly, due to a rather favorable safety profile of these medications.i Another key factor of medication adherence is the qua lity of prescribed pharmacological treatment (Lukina et al. 2017). Therefore, the next step was to compare the phar macotherapy patterns of adherent and non-adherent co ronary patients in regard with prescription rates of relevant medications (Table 2). The positive aspect was that almost all the patients were recommended drugs to prevent throm bosis. Prescription rates of oral anticoagulants (OACs) correlated with prevalence of atrial fibrillation. The ten dency (p = 0.079) to higher rates of OACs in non-adherent patients was revealed. It might be explained by high costs The first line medications – beta-blockers – were jus tifiably top recommended (77.7%, n = 300) among anti anginal drug therapies. Long-term nitrates were used only in 7.3% (n = 28) of cases. And calcium channel blockers were prescribed to 46.1% (n = 178) of patients. The pat terns of antianginal pharmacotherapy were similar in ad herent and non-adherent subjects. The only exception was Research Results in Pharmacology 6(2): 97–103 101 Table 3. Modifiable Risk Factors in Adherent and Non-adherent Patients with Stable Coronary Artery Disease. nitrates having the tendency (p = 0.087) to association with better adherence. The proven strategy to improve a patient’s adherence is to prescribe fixed dose combinations (Castellano et al. 2014; Fuller et al. 2017). Unfortunately, such an approach was applied only to 8.5% (n = 33) of participants. This must be admitted as a negative sign considering the re sults of comparison between the groups. The use of fixed dose combinations was associated with higher adherence (p = 0.048). Besides, the number of pills taken by a pa tient was less in the adherent group (p = 0.029), which partly confirmed the role of polypharmacy as an impor tant predictor of non-adherence. Furthermore, the study by Khatib et al. Bitton A, Choudhry NK, Matlin OS, Swanton K, Shrank WH (2013) The impact of medication adherence on coronary artery disease costs and outcomes: a systematic review. The American Journal of Medicine 126(4): 357.e7–357.e27. https://doi.org/10.1016/j.am jmed.2012.09.004 [PubMed] Bochkareva EV, Butina EK, Kim IV, Kontsevaya AV, Drapkina OM, Leon D, McKee M (2019) Adherence to antihypertensive medication in Russia: a scoping review of studies on levels, determinants and in tervention strategies published between 2000 and 2017. Archives of (2019) demonstrated that the number of medicines taken by coronary patients (n = 503) was an independent predictor of intentional non-adherence (odds ratio 1.18; 95% CI 1.07–1.31). Variable Adherent (n = 188) Non-adherent (n = 198) p Body mass index (kg/m2), 29.6 ± 4.8 29.3 ± 4.6 0.788 M ± SD (Q1, Q2, Q3) (26.2, 29.3, 32.6) (26.3, 29.2, 32.1) Systolic blood pressure (mm Hg), 135 ± 18 134 ± 20 0.588 M ± SD (Q1, Q2, Q3) (120, 130, 141) (120, 130, 150) Diastolic blood pressure (mm Hg), 79 ± 9 78 ± 10 0.380 M ± SD (Q1, Q2, Q3) (70, 80, 80) (70, 80, 80) Total cholesterol (mmol/L), 4.7 ± 1,2 5.2 ± 1.4 <0.001 M ± SD (Q1, Q2, Q3) (3.7, 4.5, 5.4) (3.6, 5.0, 5.8) LDL† cholesterol (mmol/L), 2.4 ± 0.9 2.9 ± 1.2 <0.001 M ± SD (Q1, Q2, Q3) (1.8, 2.2, 2.8) (1.9, 2.5, 3.5) Triglycerides (mmol/L), 1.7 ± 0.9 1.9 ± 1.5 0.755 M ± SD (Q1, Q2, Q3) (1.0, 1.4, 2.0) (1.0, 1.4, 2.1) HDL‡ cholesterol (mmol/L), 1.4 ± 0.3 1.3 ± 0.3 0.706 M ± SD (Q1, Q2, Q3) (1.1, 1.4, 1.6) (1.2, 1.4, 1.5) Glycosylated hemoglobin (%), 6.9 ± 1.4 6.9 ± 1.2 0.677 M ± SD (Q1, Q2, Q3) (6.1, 6.6, 7.3) (6.3, 6.6, 7.1) Fasting plasma glucose (mmol/L), 6.7 ± 3.1 6.1 ± 1.5 0.710 M ± SD (Q1, Q2, Q3) (4.7, 5.3, 7.5) (5.1, 5.6, 6.6) Note: †low-density lipoprotein; ‡high-density lipoprotein. ; ) Thus, the groups of adherent and non-adherent pa tients with stable CAD turned out to be similar in demo graphics, medical history profiles and pharmacotherapy patterns, which made it especially interesting to compare the results of cardiovascular risk factors management be tween these groups. In that regard, the available clinical and laboratory data were analyzed (Table 3). The groups were similar in body mass index. Mean figures of blood pressure also were comparable, although this might be explained by the fact that the patients followed physician recommendations more precisely shortly before the visit to the cardiologist (Feinstein 1990). The studied groups were similar in the glycemic status and some parameters of the lipid profile (triglycerides, high-density lipoprotein cholesterol). However, the levels of total cholesterol and low-density lipoprotein (LDL) cholesterol were higher (p < 0.001) in non-adherent patients.i Note: †low-density lipoprotein; ‡high-density lipoprotein. Table 4. Conclusion The prevalence of medication non-adherence in patients with stable CAD at a primary care setting was more than 50%. Patient’s age and sex, medical history profile and pharmacotherapy pattern were not associated with better or worse adherence. High adherence was related to the use of fixed dose combinations and to fewer cardiovascu lar medications taken by the patient. Smoking and poorer control of blood lipids (total cholesterol and LDL choles terol) prevailed in non-adherent patients, who also caused a higher load on general practitioners. An additional me dication maintenance status had no influence on medicati on adherence of coronary patients. Further local research is needed to address this serious problem. The final task of this research was to calculate the num ber of visits to the cardiologist and general practitioner over the twelve-month period and define the prevalence of additional medication maintenance in adherent and non-adherent coronary patients (Table 4). So, the parti cipants from the both groups were visiting cardiologists with the same frequency, probably due to comparable severity of their cardiological medical histories. But non-adherent patients needed to visit general practitio ners more often (p = 0.003), which might be a sign of more frequent complaints and episodes of feeling unwell. The status of additional medication maintenance provided no better adherence. The study by Fofanova et al. (2017) revealed no association either between the additional me dication maintenance status and adherence in outpatients with arterial hypertension and CAD. Conflict of interest The authors have neither funding nor support to report. The authors have no competing interests to declare. Use of Primary Care Facility Resources by Adherent and Non-adherent Patients with Stable Coronary Artery Disease. Table 4. Use of Primary Care Facility Resources by Adherent and Non-adherent Patients with Stable Coronary Artery Disease. 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British Journal of Clinical Pharmacology 77(3): 427–445. https://doi. org/10.1111/bcp.12194 [PubMed] [PMC] Du L, Cheng Z, Zhang Y, Li Y, Mei D (2017) The impact of med ication adherence on clinical outcomes of coronary artery disease: A meta-analysis. European Journal of Preventive Cardiology 24(9): 962–970. https://doi.org/10.1177/2047487317695628 [PubMed] Oganov RG, Simanenkov VI, Bakulin IG, Bakulina NV, Barbarash OL, Boytsov SA, Boldueva SA, Garganeeva NP, Doshchitsin VL, Ka rateev AE, Kotovskaya YV, Lila AM, Lukyanov MM, Morozova TE, Pereverzev AP, Petrova MM, Pozdnyakov YM, Syrov AV, Tarasov AV, Tkacheva ON, Shalnova SA (2019) Comorbidities in clinical practice. Algorithms for diagnostics and treatment. Cardiovascular Therapy and Prevention [Kardiovaskulyarnaya Terapiya i Profilaktika] 18(1): 5–66. https://doi.org/10.15829/1728-8800-2019-1-5-66 [in Russian] Feinstein AR (1990) On white-coat effects and the electronic moni toring of compliance. Archives of Internal Medicine 150(7): 1377– 1378. https://doi.org/10.1001/archinte.150.7.1377 [PubMed] Fofanova TV, Ageev FT, Smirnova MD, Deev AD (2017) Adherence to therapy in the outpatient setting: the ability to identify and assess the effectiveness of therapy. Cardiology [Kardiologiia] 57(7): 35–42. https://doi.org/10.18087/cardio.2017.7.10004 [PubMed] [in Russian] Shi L, Liu J, Fonseca V, Walker P, Kalsekar A, Pawaskar M (2010) Cor relation between adherence rates measured by MEMS and self-reported questionnaires: a meta-analysis. Health and Quality of Life Outcomes 8: 1–99. https://doi.org/10.1186/1477-7525-8-99 [PubMed] [PMC] Fuller RH, Perel P, Navarro-Ruan T, Nieuwlaat R, Haynes RB, Huffman MD (2018) Improving medication adherence in patients with cardiovascular disease: a systematic review. Heart (British Cardiac Society) 104(15): 1238–1243. https://doi.org/10.1136/heart jnl-2017-312571 [PubMed] Tan X, Patel I, Chang J (2014) Review of the four item Morisky Medication Adherence Scale (MMAS-4) and eight item Morisky Medication Adherence Scale (MMAS-8). Innovations in Pharmacy 5(3): Article 165. https://doi.org/10.24926/iip.v5i3.347 Gehi AK, Ali S, Na B, Whooley MA (2007) Self-reported medication adherence and cardiovascular events in patients with stable coronary heart disease: the heart and soul study. Archives of Internal Medicine 167(16): 1798–1803. References Zyryanov SK et al.: Medication adherence in coronary outpatients 102 Public Health 77: 1–43. https://doi.org/10.1186/s13690-019-0366-9 [PubMed] [PMC] Public Health 77: 1–43. https://doi.org/10.1186/s13690-019-0366-9 [PubMed] [PMC] Khatib R, Marshall K, Silcock J, Forrest C, Hall AS (2019) Adher ence to coronary artery disease secondary prevention medicines: ex ploring modifiable barriers. Open Heart 6(2): e000997. https://doi. org/10.1136/openhrt-2018-000997 [PubMed] [PMC] Khatib R, Marshall K, Silcock J, Forrest C, Hall AS (2019) Adher ence to coronary artery disease secondary prevention medicines: ex ploring modifiable barriers. Open Heart 6(2): e000997. https://doi. org/10.1136/openhrt-2018-000997 [PubMed] [PMC] Khatib R, Marshall K, Silcock J, Forrest C, Hall AS (2019) Adher ence to coronary artery disease secondary prevention medicines: ex ploring modifiable barriers. Open Heart 6(2): e000997. https://doi. org/10.1136/openhrt-2018-000997 [PubMed] [PMC] Boitsov SA, Pogosova NV, Bubnova MG, Drapkina OM, Gavrilova NE, Eganyan RA, Kalinina AM, Karamnova NS, Kobalava ZhD, Koncevaja AV, Kuharchuk VV, Luk’janov MM, Maslennikova GJa, Marcevich SJa, Metel’skaja VA, Meshkov AN, Oganov RG, Popo vich MV, Sokolova OJu, Suhareva OJu, Tkacheva ON, Shal’nova SA, Shestakova MV, Jufereva JuM, Javelov IS (2018) Cardiovascu lar prevention 2017. National guidelines. Russian Journal of Cardi ology [Rossiiskii Kardiologicheskii Zhurnal] (6): 7–122. https://doi. org/10.15829/1560-4071-2018-6-7-122 [in Russian] Kolandaivelu K, Leiden BB, O’Gara PT, Bhatt DL (2014) Non-adher ence to cardiovascular medications. European Heart Journal 35(46): 3267–3276. https://doi.org/10.1093/eurheartj/ehu364 [PubMed] Ladova K, Vlcek J, Vytrisalova M, Maly J (2014) Healthy adherer effect – the pitfall in the interpretation of the effect of medication ad herence on health outcomes. Journal of Evaluation in Clinical Prac tice 20(2): 111–116. https://doi.org/10.1111/jep.12095 [PubMed] Ladova K, Vlcek J, Vytrisalova M, Maly J (2014) Healthy adherer effect – the pitfall in the interpretation of the effect of medication ad herence on health outcomes. Journal of Evaluation in Clinical Prac tice 20(2): 111–116. https://doi.org/10.1111/jep.12095 [PubMed] Castellano JM, Sanz G, Peñalvo JL, Bansilal S, Fernández-Ortiz A, Alvarez L, Guzmán L, Linares JC, García F, D’Aniello F, Arnáiz JA, Varea S, Martínez F, Lorenzatti A, Imaz I, Sánchez-Gómez LM, Roncaglioni MC, Baviera M, Smith Jr SC, Taubert K, Pocock S, Brotons C, Farkouh ME, Fuster V (2014) A polypill strategy to im prove adherence: results from the FOCUS project. Journal of the American College of Cardiology 64(20): 2071–2082. https://doi. References https://doi.org/10.1001/archinte.167.16.1798 [PubMed] [PMC] Warren JR, Falster MO, Fox D, Jorm L (2013) Factors influencing ad herence in long-term use of statins. Pharmacoepidemiology and Drug Safety 22(12): 1298–1307. https://doi.org/10.1002/pds.3526 [PubMed] Research Results in Pharmacology 6(2): 97–103 103 Author Contributions Sergey K. Zyryanov, Doctor Habilitated of Medical Sciences, Professor, Head of the Department of Pharma cology and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: sergey.k.zyryanov@gmail.com, ORCID ID https://orcid.org/0000-0002-6348-6867. The author was the research program supervisor and contribu ted to the concept of the study. Sergey K. Zyryanov, Doctor Habilitated of Medical Sciences, Professor, Head of the Department of Pharma cology and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: sergey.k.zyryanov@gmail.com, ORCID ID https://orcid.org/0000-0002-6348-6867. The author was the research program supervisor and contribu ted to the concept of the study. Sergey B. Fitilev, Doctor Habilitated of Medical Sciences, Professor, Professor of the Department of Pharmaco logy and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: fitilevsb@yandex.ru, ORCID ID https://orcid.org/0000-0001-8395-419X. The author contributed to the concept and design of the study, analysis and interpretation of the study results, and final editing of the article. Sergey B. Fitilev, Doctor Habilitated of Medical Sciences, Professor, Professor of the Department of Pharmaco logy and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: fitilevsb@yandex.ru, ORCID ID https://orcid.org/0000-0001-8395-419X. The author contributed to the concept and design of the study, analysis and interpretation of the study results, and final editing of the article. Alexander V. Vozzhaev, Candidate of Biological Sciences, Associate Professor of the Department of Pharmaco logy and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: vozzhaev-av@rudn.ru, ORCID ID https://orcid.org/0000-0002-2687-5986. The author had the primary role in data collection, statistical data proces sing, analysis and interpretation of the study results, prepared the draft version of the article, and translated the final version of the article into English. Alexander V. Vozzhaev, Candidate of Biological Sciences, Associate Professor of the Department of Pharmaco logy and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: vozzhaev-av@rudn.ru, ORCID ID https://orcid.org/0000-0002-2687-5986. The author had the primary role in data collection, statistical data proces sing, analysis and interpretation of the study results, prepared the draft version of the article, and translated the final version of the article into English. Irina I. Shkrebniova, Candidate of Medical Sciences, Associate Professor of the Department of Pharmacology and Clinical Pharmacology, Medical institution, RUDN University, Candidate of Medical Sciences, Associate Pro fessor, e-mail: ishkrebneva@yandex.ru, ORCID ID https://orcid.org/0000-0002-0070-3115. The author contribu ted to optimization of data collection tools, contributed to interpretation of the results of the study and scientific editing of the article. Irina I. Author Contributions Shkrebniova, Candidate of Medical Sciences, Associate Professor of the Department of Pharmacology and Clinical Pharmacology, Medical institution, RUDN University, Candidate of Medical Sciences, Associate Pro fessor, e-mail: ishkrebneva@yandex.ru, ORCID ID https://orcid.org/0000-0002-0070-3115. The author contribu ted to optimization of data collection tools, contributed to interpretation of the results of the study and scientific editing of the article. Natalya N. Shindryaeva, Doctor of Medical Sciences, Professor,Head Physician of City Polyclinic #2 of Moscow Healthcare Department, e-mail: mont76@mail.ru, ORCID ID https://orcid.org/0000-0001-6560-2756. The author was the scientific consultant and provided assistance in organization of the study conduct and clinical data collection. Natalya N. Shindryaeva, Doctor of Medical Sciences, Professor,Head Physician of City Polyclinic #2 of Moscow Healthcare Department, e-mail: mont76@mail.ru, ORCID ID https://orcid.org/0000-0001-6560-2756. The author was the scientific consultant and provided assistance in organization of the study conduct and clinical data collection. Dmitry A. Klyuev, post-graduate student at the Department of Pharmacology and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: dmitrijkluev070496@gmail.com, ORCID ID https://orcid.org/0000-0003- 2400-3938. The author contributed to the data collection and statistical processing. Dmitry A. Klyuev, post-graduate student at the Department of Pharmacology and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: dmitrijkluev070496@gmail.com, ORCID ID https://orcid.org/0000-0003- 2400-3938. The author contributed to the data collection and statistical processing. Liusine N. Stepanyan, post-graduate student at the Department of Pharmacology and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: lyusine.stepanyan@yandex.ru, ORCID ID https://orcid.org/ 0000- 0002-6012-8917. The author contributed to optimization of data collection tools and data collection. Liusine N. Stepanyan, post-graduate student at the Department of Pharmacology and Clinical Pharmacology, Medical Institute, RUDN University, e-mail: lyusine.stepanyan@yandex.ru, ORCID ID https://orcid.org/ 0000- 0002-6012-8917. The author contributed to optimization of data collection tools and data collection. Nikolay N. Landyshev, student of Medical Institute, RUDN University, e-mail: nikolay.landyshev@gmail.com, ORCID ID https://orcid.org/0000-0002-9289-6849. The author contributed to optimization of study database and data collection. Nikolay N. Landyshev, student of Medical Institute, RUDN University, e-mail: nikolay.landyshev@gmail.com, ORCID ID https://orcid.org/0000-0002-9289-6849. The author contributed to optimization of study database and data collection. Yana G. Voronko, student of Medical Institute, RUDN University, e-mail: yana9voronko@yandex.ru, ORCID ID https://orcid.org/0000-0003-0779-5742. The author contributed to the data collection. Yana G. Voronko, student of Medical Institute, RUDN University, e-mail: yana9voronko@yandex.ru, ORCID ID https://orcid.org/0000-0003-0779-5742. The author contributed to the data collection.
https://openalex.org/W2796244011	https://zenodo.org/records/2100803/files/article.pdf	English	null	Romanza and Allegretto for Violoncello and Pianoforte; Or Viola and Pianoforte	Musical times and singing class circular	1,900	public-domain	1,567	Source: The Musical Times and Singing Class Circular, Vol. 41, No. 692 (Oct. 1, 1900), p. 676 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/3368879 Accessed: 14-12-2015 08:51 UTC [Novello and Company, Limited.] [Novello and Company, Limited.] p y MR. WILFRED BENDALL'S 'Song Dances' are a very attractive series of choric measures in three vocal parts, first and second soprano and contralto, with an independen accompaniment for pianoforte solo or duet, or orchestra So independent are the accompaniments and so complete in themselves that, played alone, they would form a number of pleasing instrumental solos or duets. The songs are six in number, and the words of three, 'To the Nightingale,' 'The rain is falling,' and 'Song should breathe,' are by Barry Cornwall. The first of these is set in minuet rhythm, the second in waltz time, and the third in tempo di Mazurka, the characteristics of each dance form being pleasingly accentuated. The names of the other numbers are, 'To the Skylark,' words by James Hogg; 'Twilight,' words anonymous; and ' Dametus, his jigge in praise of his love,' by an unknown poet of an unknown period. The last-named is a 'right merrie measure,' the poet insisting that 'love lasts for aye,' and the composer vigorously supporting this amorous opinion. MR. WILFRED BENDALL'S 'Song Dances' are a very attractive series of choric measures in three vocal parts, first and second soprano and contralto, with an independen accompaniment for pianoforte solo or duet, or orchestra So independent are the accompaniments and so complete in themselves that, played alone, they would form a number of pleasinginstrumental solos or duets. SWalpurgis Night (on April 25). A concert to be looked forward to with pleasure and regret is the 'farewell' of Mr. Edward Lloyd, which will take place at the Royal Albert Hall, on December 12. pleasing The songs are six in number, and the words of three, 'To the Nightingale,' 'The rain is falling,' and 'Song should breathe,' are by Barry Cornwall. The first of these is set in minuet rhythm, the second in waltz time, and the third in tempo di Mazurka, the characteristics of each dance form being pleasingly accentuated. The names of the other numbers are, 'To the Skylark,' words by James Hogg; 'Twilight,' words anonymous; and ' Dametus, his jigge in praise of his love,' by an unknown poet of an unknown period. The last-named is a 'right merrie measure,' the poet insisting that 'love lasts for aye,' and the composer vigorously supporting this amorous opinion. ST. JAMES'S HALL. p SOME few years ago the attention of musicians was drawn by the late Major Day's book,' Music in Southern India,' to Eastern scales, and subsequent investigation revealed to Western composers the possibilities of increas- ing the resources of their art. The subject seems to have appealed with peculiar force to Mr. Granville Bantock, and the present series of songs are a part outcome of his endeavours to combine Eastern melodic idiom with Western form and harmony. Each book, which, by the way, is got up in artistic fashion, comprises a cycle of six songs, and, as will be expected, possesses considerable distinctiveness. That this distinctiveness is always pleasing, or, indeed, acceptable, can scarcely be said; but several of the songs are very charming, a few are really beautiful, and cultured vocalists in search of music out of the beaten track may be recommended to examine these books. It should be added that the text is thoroughly Eastern in spirit, picturesquely suggestive, and emotional, and that English singers who reasonably doubt their ability to articulate their own language distinctly can take refuge in a German translation. s The Saturday Popular Concerts re-commence on Novem- ber 3 and continue weekly till December 15. The Ballad Concerts at this hall will be resumed on November 7, continuing weekly during the month. Other Afternoon Concerts are announced by Miss Clara Butt and Mr. Kennerley Rumford; Madame Marie Brema, Mr. Henry Bird, and Mr. Vert; and Recitals by Herr Reisenauer (3), Mr. Frederick Dawson, Mr. Donald Tovey (3), Herr Rosenthal, Miss Marguerite Elzy, and Madame Marchesi, with Messrs. Wolff and Schonberger. e y Schonberger. In the evening three Richter Concerts will be given, commencing on the 22nd inst.; those of the Curtius Concert Club will be resumed on November 7. Mr. Henry Such will give an Orchestral Concert, and Herr Kupper- schmidt a Recital, while the Saturday Orchestral Concerts (now under the direction of Mr. Edward O'Brien) will re-commence on the 6th inst. [Novello and Company, Limited.] [Novello and Company, Limited.] QUEEN'S HALL. n Mr. Robert Newman announces eight Symphony Concerts, on the afternoons of October 27, November io and 24, December 8, January 26, February g, and March 2 and 16; some Ysaye Concerts, details of which are yet to come, in November; a St. Andrew's Day Concert on November 30; and the usual Christmas Day, Ash Wednesday, and Good Friday Concerts. Mr. Henry J. Wood will conduct throughout. The so-called 'London Musical Festival' will take place from April 29 to May 4. The Sunday Concert Society's Sunday Afternoon Concerts will be held every Sunday afternoon at 3.30. Songs of China, Yapan, India, Arabia, Persia, and Egypt. Words by Helen F. Schweitzer. Music by Granville Bantock. [Breitkopf and Hairtel.] THE COMING SEASON. Romanza and Allegretto for Violoncello and Pianoforte or Viola and Pianoforte. By W. Wolstenholme. [Novello and Company, Limited.] Romanza and Allegretto for Violoncello and Pianoforte or Viola and Pianoforte. By W. Wolstenholme. SINCE the end of June, with some few unimportan exceptions, the voice of the ' Divine Art' has been prac- tically silent until the commencement of the Promenade Concerts on August 25, a record of which will be found in another column. Entrepreneurs are now, however, bestirring themselves for the coming winter season, and the cessation of' wars' alarms' and the return of peace certainly make the outlook brighter and more hopeful than it was at this time last year. The first prospectus to claim our attention is that of the p y Two little pieces very suitable for teaching purposes; the Romanza will prove a general favourite. The Allegretto suffers from a bald accompaniment to the middle section. The composer has arranged the above pieces for the viola, for which they seem very suitable. STEINWAY HALL THIS is a new issue with sundry decided improvement of a convenient arrangement which is familiar to many lovers of Beethoven. The English text, by Lady Macfarren has been revised and partly re-written; moreover, each vocal part has now its own stave, independent of the admirably arranged pianoforte accompaniment made by the late Berthold Tours. The new edition is, therefore, not only useful for home use, but for employment in the ranks of the chorus, or for following a performance of this gigantic work. Now that the 'Choral' is no longer regarded as ' impossible,' but, on the contrary, may be said to have become popular, this issue is certainly opportune. will, as usual, be the locale of Mr. Clifford Harrison's Recitals, which were to be resumed on the 29th ult., while at s , ST. GEORGE'S HALL Mr. Charles Fry's Costume Recitals, with Miss Olive Kennett and company, now in their seventh season, will be given on the Saturday afternoons in November, commencing on the ioth. Mr. Edward O'Brien will direct the music, which is a prominent feature at these performances. This content downloaded from 130.130.37.84 on Mon, 14 Dec 2015 08:51:48 UTC All use subject to JSTOR Terms and Conditions Review Source: The Musical Times and Singing Class Circular, Vol. 41, No. 692 (Oct. 1, 1900), p. 676 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/3368879 Accessed: 14-12-2015 08:51 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Musical Times Publications Ltd. is collaborating with JSTOR to digitize, preserve and extend access to The Musical Times and Singing Class Circular. http://www.jstor.org This content downloaded from 130.130.37.84 on Mon, 14 Dec 2015 08:51:48 UTC All use subject to JSTOR Terms and Conditions 676 THE MUSICAL TIMES.-OCTOBER I, 1900. ROYAL CHORAL SOCIETY. Song Dances. Vocal suite for female voices. Composed by Wilfred Bendall. t Seven Subscription Concerts are announced, with the addition of the customary Good Friday performance of 'The Messiah.' The series opens on November 8 with SElijah,' followed by 'Judas Maccabmus' (December 6), 'Messiah ' (January I), ' Scenes from the Song of Hiawatha' (January 24), Parker's ' Hora Novissima' and Beethoven's Choral Symphony (February 2o),' Israel in Egypt' (March 14), and the 'Hymn of Praise' and SWalpurgisNight' (on April25) THE CRYSTAL PALACE celebrated Saturday Concerts, as such, are now, alas ! only a memory; but the veteran conductor will direct two Concerts on the 27th inst. and November io; Mr. Newman's orchestra, under Mr. Henry J. Wood, will be responsible for Concerts on the 13th and 20th inst. and November 3; and on November 17 Dr. Richter's orchestra, under their distinguished chief, will give a Wagner Concert. , Beethoven's Choral Symphony. Pianoforte arrangemen with vocal parts in open score. By Berthold Tours.
https://openalex.org/W2283728539	https://europepmc.org/articles/pmc4950013?pdf=render	English	null	The genetics of neuroticism and human values	Genes, brain and behavior	2,016	cc-by	6,316	George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ contrasts motives to follow the status quo (conservation) against motives to pursue personal intellectual and emo- tional interests in uncertain directions (openness). One impor- tant characteristic of this circumplex model is that it makes speciﬁc predictions about sinusoidal associations between social values and external variables. As shown in Fig. 1b, this sinusoidal waveform becomes evident if the values are ordered according to their positions along the value circle: an external variable that is most positively related to a par- ticular value should manifest less positive and progressively more negative correlations until reaching the opposing value type. This prediction has received support in many studies ﬁnding that values at opposite ends of the circular model exhibit opposing relations to other judgements and behaviour (see Schwartz 1996) and in one study observing a sinusoidal pattern in relations between values and personality traits (Parks-Leduc et al. 2015). This sinusoidal waveform supports the model’s assumptions about latent motivational conﬂicts between values. †School of Psychology, ‡Neuroscience and Mental Health Research Institute, and §MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Cardiff, UK *Corresponding author: G. Zacharopoulos, School of Psychology, Cardiff University, Tower Building 70 Park Place, Cardiff CF10 3AT, UK. E-mail: zacharopoulosg@cardiff.ac.uk Human values and personality have been shown to share genetic variance in twin studies. However, there is a lack of evidence about the genetic components of this association. This study examined the interplay between genes, values and personality in the case of neuroti- cism, because polygenic scores were available for this personality trait. First, we replicated prior evidence of a positive association between the polygenic neuroticism score (PNS) and neuroticism. Second, we found that the PNS was signiﬁcantly associated with the whole human value space in a sinusoidal waveform that was consistent with Schwartz’s circular model of human values. These results suggest that it is useful to consider human val- ues in the analyses of genetic contributions to personal- ity traits. They also pave the way for an investigation of the biological mechanisms contributing to human value orientations. However, the exact genetic loci driving this association between values from Schwartz’s model and personality have remained obscure. This association can be investigated by utilizing a growing body of knowledge on personality genet- ics. As complex psychological dispositions, human values and personality traits are both likely to be affected by numerous genes simultaneously (in addition to strong environmental inﬂuences). George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ To capture the genetic inﬂuence of complex traits and values, it is therefore useful to focus on genetic indices that reﬂect the contribution of a great number of single nucleotide polymorphisms (SNPs), such as polygenic scores derived from Genome-Wide Association Studies (GWAS). Keywords: Genetics, human values, neuroticism, personality, polygenic score Thus far, a polygenic score has been identiﬁed only for one trait: neuroticism. A polygenic neuroticism score (PNS) is available through a recent meta-analysis of GWAS of personality traits (N = 63 661) (Genetics of Personality Consortium et al. 2015). Neuroticism is a personality factor ranging from emotional stability to high nervousness, ten- sion and moodiness. In the meta-analysis, a neuroticism score (NS) was derived from a number of measures includ- ing the NEO Personality Inventory, the Eysenck Personality Questionnaire, the International Personality Item Pool inven- tory, harm avoidance scores in Cloninger’s Tridimensional Personality Questionnaire and negative emotionality scores in the Multidimensional Personality Questionnaire. The meta-analysis showed that 0.6% of the variance in this NS was explained by the PNS. Although this low percentage suggests only a small genetic component, it was reliable and potentially important, making it a relevant candidate for studying genetic contributions to neuroticism and other individual differences related to neuroticism. Received 28 November 2015, revised 27 January 2016, 19 February 2016, accepted for publication 21 February 2016 Received 28 November 2015, revised 27 January 2016, 19 February 2016, accepted for publication 21 February 2016 The beliefs people have about ideals that are important in life, their ‘values’, are reliably associated with certain personality traits (Parks-Leduc et al. 2015; Rim 1984). Extending this con- nection, studies of twins have found that the shared variance between human values and personality has a signiﬁcant her- itable component (Schermer et al. 2008, 2011). Schermer et al.’s (2008, 2011) analyses of shared genetic variance between traits and values utilized Schwartz’s (1992) circular model of values. This model is supported by data from over 70 nations with a range of cross-sectional, lon- gitudinal and experimental methods (Maio 2010; Schwartz et al. 2012). The model posits the existence of 10 types of social values (Fig. 1a), with each expressing speciﬁc motives. These motives are organized along two dimen- sions. © 2016 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd. 361 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Genes, Brain and Behavior (2016) 15: 361–366 doi: 10.1111/gbb.12286 Genes, Brain and Behavior (2016) 15: 361–366 doi: 10.1111/gbb.12286 Genes, Brain and Behavior (2016) 15: 361–366 doi: 10.1111/gbb.12286 The genetics of neuroticism and human values George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ Subjects A l f j A total of 81 right-handed Caucasian university students aged between 19 and 42 (50 females; mean ± SD age = 23.85 ± 3.71) par- ticipated in the study, which was approved by the Ethics Committee in the School of Psychology, Cardiff University. Participants were informed that the study examined the connection between value–morality judgements and biological indices. They took part individually in the laboratory, wherein they completed the measures of human values and personality, provided a saliva sample, and were then debriefed. The sample used consisted of an existing sample collected for behavioural analysis. In this study we included all the participants from the existing sample for which the human value score, personality score and the genetic score were available. However, a different possibility emerges if we consider relevant research examining links between neuroticism and relevant affective states and attitudes. Neuroticism is asso- ciated with a higher likelihood of anxiety and depression, which are two hallmarks of emotional instability that lead people to withdraw from the world around them (Angst et al. 2003; Thompson et al. 2011). This pattern suggests that emotional instability may cause people to be less open to new experiences, ideas and feelings, because of the poten- tial threats to their fragile emotional state. Convergent with these observations, lower levels of neuroticism are associ- ated with more liberal, curious and open-minded attitudes (Carney et al. 2008; Van Hiel & Mervielde 2004). Strong links between such attitudes and Schwartz’s openness value type (Ashton et al. 2005) suggest that an inverse relation between openness values (see Fig. 1a) and neuroticism is viable. George Zacharopoulos†,∗, Thomas M. Lancaster†,‡,§, Gregory R. Maio† and David E. J. Linden†,‡,§ One dimension contrasts motives to promote the self (self-enhancement) against motives that transcend personal interests (self-transcendence), whereas the other dimension The shared genetic associations between personality traits and human values provide a foundation for expecting that the polygenic association with neuroticism may also relate to 361 Zacharopoulos et al. Figure 1: (a) The circumplex structure of personal values. (b) Plot of hypothesized relationships between three external variables (A, B and C) and the 10 values (SD, self-direction; ST, stimulation; HE, hedonism; AC, achievement; PO, power; SE, security; CO, conformity; TR, tradition; BE, benevolence; UN, universalism). Each dot point could represent a correlation coefﬁcient (modiﬁed from Schwartz 1992). Figure 1: (a) The circumplex structure of personal values. (b) Plot of hypothesized relationships between three external variables (A, B and C) and the 10 values (SD, self-direction; ST, stimulation; HE, hedonism; AC, achievement; PO, power; SE, security; CO, conformity; TR, tradition; BE, benevolence; UN, universalism). Each dot point could represent a correlation coefﬁcient (modiﬁed from Schwartz 1992). Figure 1: (a) The circumplex structure of personal values. (b) Plot of hypothesized relationships between three external variables (A, B and C) and the 10 values (SD, self-direction; ST, stimulation; HE, hedonism; AC, achievement; PO, power; SE, security; CO, conformity; TR, tradition; BE, benevolence; UN, universalism). Each dot point could represent a correlation coefﬁcient (modiﬁed from Schwartz 1992). in a sinusoidal pattern congruent with Schwartz’s circumplex model of values. value orientations. Human values are particularly interesting in connection to neuroticism. A recent meta-analysis of the relations between human values and the big ﬁve traits found reliable trait–value associations, except when looking at neu- roticism (Parks-Leduc et al. 2015). The authors explained this non-association using Cloninger’s (1994) proposition that neu- roticism is more appropriately described as a temperament (i.e. an automatic associative response to emotional stimuli) than as a character trait (i.e. a self-aware volitional concept related to behavioural intentions). This indicates a stronger biological component to neuroticism than to other traits, which, like human values, may be amenable to higher levels of cognitive processing and control. Thus, from this perspec- tive, neuroticism may manifest a genetic component, but lit- tle association with human values. in a sinusoidal pattern congruent with Schwartz’s circumplex model of values. Genes, Brain and Behavior (2016) 15: 361–366 Human values P i i l Participants completed the 56-item Schwartz Value Survey (Schwartz 1992). Participants rated the importance of each of the 56 values as a guiding principle in their lives, using a quasi-bipolar 9-point scale rang- ing from −1 (opposed to my values), 0 (not important), 4 (important) to 7 (of supreme importance). Examples of Schwartz Value Survey items are as follows: ‘equality: equal opportunity for all’ (universal- ism); ‘pleasure: gratiﬁcation of desires’ (hedonism); ‘obedient: dutiful meeting obligations’ (conformity). The average score across the 56 items was then calculated and subtracted from each of the 56 initial raw scores. Schwartz recommends this procedure to help control for superﬂuous individual variations in rating styles (Schwartz 1992). The individual centred item scores were then averaged to form scores for each type of value examined in Schwartz’s model (see Fig. 1a). The internal consistency of these indices was moderate to good (see Table 1). The present research was therefore motivated by the shared genetic variance between human values and person- ality, the existence of a polygenic score for neuroticism, and the ambiguity about neuroticism value relations. We sought to test whether the potential genetic contribution to neu- roticism has similar patterns of the association with human values and the trait on a phenotypic level. To be clear, we were not predicting that values mediate the link between genes and traits or that traits mediate the link between genes and values. In theory, values and traits should recip- rocally inﬂuence each other, as stable individual differences over time, leading to an association that is bidirectional. Our principal aim was to test whether associations with genes emerged for both the trait and values. Moreover, we wished to detect whether any observed associations arose DNA extraction and genotyping Genomic DNA was obtained from saliva using Oragene OG-500 saliva kits. Genotyping was performed using custom genotyping arrays (Illumina HumanCoreExome-24 BeadChip), which contain 570 038 genetic variants (Illumina, Inc., San Diego, CA, USA). Quality con- trol was implemented in PLINK (Purcell et al. 2007) to ensure that the genotypes did not display ambiguous sex, cryptic relatedness (up to third-degree relatives by the identity of descent), genotyping com- pleteness <97% and non-European ethnicity admixture (detected as outliers in iterative EIGENSTRAT analyses of an LD-pruned data set) (Price et al. 2006). The SNPs were excluded where the minor allele frequency was <1%, if the call rate <98% or if the 𝜒2-test for Hardy–Weinberg Equilibrium had a P-value <1 e-04. Individuals’ genotypes were imputed using the pre-phasing/imputation step- wise approach implemented in IMPUTE2/SHAPEIT (Delaneau et al. 2012; Howie et al. 2009) and 1000Genomes (December 2013, release 1000 Genome haplotypes Phase I integrated variant set) as the reference data set. (2) In this eqn (2), K represents the number of correlation coefﬁcients, yk represents the correlation coefﬁcients, yk represents the esti- mated correlation coefﬁcient through the optimization function and yk represents the mean of the correlation coefﬁcients. The denominator is the formula for the variance. Hanel et al. (2016) tested the number of false-positive results for the SFI, using three simulations of m = 100 000 samples each in R. To simulate a random pattern of correlation coefﬁcients, they relied upon two assumptions about the distribution of the correlation coef- ﬁcients. First, they sampled 10 numbers (i.e. number of human val- ues) between −0.5 and 0.5, with k being the number of correlation coefﬁcients (k = 10), assuming a uniform distribution. The numbers −0.5 to 0.5 represent the interval in which most correlation coef- ﬁcients of values with external variables usually fall. Second, they sampled 10 numbers from a normal distribution with ∼N(0, 0.1) and ∼N(0, 0.3). Numbers >\|1\| were restricted to −1 or 1, respectively. For the obtained values of SFI <0.20, the percentages of false positives were below 1% for all the three simulations of 100 000 samples. The percentage of false positives for an SFI <0.20 was 0.49% (i.e. less than ﬁve false-positive results per 1000 comparisons) assuming a nor- mal distribution and 0.76% assuming a uniform distribution. Similarly, assuming normal and uniform distributions, respectively, the false positives were 0.20% and 0.30% for SFI <0.15. y = f (x) = a + b × sin (c × x + d) , (1) Generation of risk proﬁle scores p The PNS was calculated using the method described by the International Schizophrenia Consortium (International Schizophrenia Consortium et al. 2009). The PNS was estimated using publicly available data from the international GWAS (Genetics of Personality Consortium et al. 2015). The SNPs were subsequently pruned for linkage disequilibrium (r2 < 0.2). This method ensured that all SNPs included in the PNS model were fairly independent. The PNSs were calculated using the ‘score’ command in PLINK, which averages the number of risk alleles for each index SNP, weighted by the natural logarithm of the SNP’s odds ratio extracted from the GWAS results (Genetics of Personality Consortium et al. 2015). From the 6 949 612 SNPs, a total of 206 516 quasi-independent SNPs were considered in the PNS (PT < 0.5). We calculated PNS at the liberal P-threshold (PT < 0.5), because it best predicted NS in the GWAS reference data (Genetics of Personality Consortium et al. 2015). There were no outliers in the PNSs, and the scores were normally distributed (Shapiro–Wilk: P > 0.3). The genetics of neuroticism and human values Table 1: Cronbach’s 𝛼for each of the 10 values Table 1: Cronbach’s 𝛼for each of the 10 values Value Number of items Cronbach’s 𝛼 Universalism 7 0.76 Benevolence 9 0.76 Tradition 6 0.63 Conformity 4 0.63 Security 6 0.68 Power 5 0.79 Achievement 6 0.67 Hedonism 2 0.74 Stimulation 3 0.79 Self-direction 6 0.65 Firstly, all four of the parameters (a, b, c and d) of the sinusoidal function were optimized with the R command optim. The parameter a, the y-offset, which moves the function up and down along the ordinate, was restricted to between −1 and 1, as were the correlation coefﬁcients. The same restrictions were applied to parameter b, which determines the differences between the turning points of the sinusoidal function (amplitude). The parameter c, the period of the sine wave, was allowed to range from 85% to 95% of a full sine wave. This restriction was based on the circular model’s assumption that ‘the distances between the values around the circle may not be equal’ (Schwartz et al. 2012). Given that the ﬁrst value type was plotted at x = 1, the parameter d (x-offset), which moves the sinusoidal function along the abscissa, was set to the interval [1 + k/2, 1 −k/2]. Therefore, parameter d was unrestricted because there was no hypothesis regarding the exact starting point of the sine wave for each of the two measures, PNS and NS. To deﬁne a lower and upper bound given these constraints, a method developed by Byrd et al. (1995) was used. We calculated the sum of the squared residuals divided by the variance to estimate the model ﬁt indices for the sinusoidal function. This ﬁt is called the Sinusoidal Fit Index (SFI) (Hanel et al. 2016) and is presented below (eqn 2). et al. 2003; Thompson et al. 2011), and HEXACO’s emotionality dimension is well suited to detecting the links with values (Pozzebon & Ashton 2009). SFI = 1 K −1 K ∑ k=1 (yk −̂yk )2 1 K −1 K ∑ k=1 (yk −yk )2 (2) SFI = 1 K −1 K ∑ k=1 (yk −̂yk )2 1 K −1 K ∑ k=1 (yk −yk )2 (2) Replicating the link between PNS and NS Replicating the link between PNS and NS Our ﬁrst aim was to provide further evidence on the associa- tion between emotionality (NS from HEXACO-PI-R) and PNS. As expected, we obtained a positive association between these variables, r79 = 0.22, P = 0.048 (Fig. 2), replicating the ﬁndings of the personality GWAS (Genetics of Personality Consortium et al. 2015). Personality measure W iﬁd NS i h y We quantiﬁed NS using the 100-item self-reported version of the HEXACO Personality Inventory-Revised (HEXACO-PI-R) (Lee & Ashton 2004). In the HEXACO-PI-R, NS is termed emotionality, and it features subscales for fearfulness, anxiety, dependence and sentimentality. These subscales are combined together as the total emotionality score (𝛼= 0.64). Furthermore, many inﬂuential research programmes have interpreted and labelled neuroticism from the big ﬁve as emotional stability (De Raad et al. 2010; Goldberg 1990; Saucier 1994). It was previously shown that the HEXACO emotional- ity represents an alternative rotation of big ﬁve neuroticism (Ashton et al. 2014) and that they are similar constructs (Ashton et al. 2014; Romero et al. 2015). Furthermore, the emotionality score provides a particularly interesting and important rendition of neuroticism in this context because of its relative emphasis on emotional instability, which leads people to withdraw from the world around them (Angst 362 Genes, Brain and Behavior (2016) 15: 361–366 y = f (x) = a + b × sin (c × x + d) , (1) y = f (x) = a + b × sin (c × x + d) , DNA extraction and genotyping For SFI <0.10, the false positives were 0.05% and 0.08%, and for SFI <0.05, the false positives were 0.005% and 0.007%. Genes, Brain and Behavior (2016) 15: 361–366 Sinusoidal relationship analysis p To test for a sinusoidal pattern of association between values, NS and PNS, we calculated the correlation coefﬁcients of the 10 value types with NS and PNS. The ﬁt of the sinusoidal function presented below (eqn 1) was calculated using the programme R. Structure of values 3b) but the ﬁt to the Figure 3: Correlation coefﬁcients between the 10 value types (x-axis, conformity, tradition, benevolence, universalism, self-direction, stimulation, hedonism, achievement, power and security) and PNS (a) and NS (b). human value benevolence deviated from the sine wave; run- ning the sinusoidal test while excluding benevolence yields a signiﬁcant SFI = 0.08 (Fig. S1). Overall, NS and PNS map onto the human value space in similar, sinusoidal waveforms. Furthermore, in addition to testing the patterns of correlations using the SFI method, we replicated the PNS and NS ﬁnd- ings using two previously established methods, with even more robust results (Boer & Fischer 2013; Roccas et al. 2002) (Appendix S1). Discussion Figure 2: Scatter-plot depicting the positive association between NS and PNS. Both NS (derived from the HEXACO-PI-R, see Material and methods) and PNS were standardized with a z-score transformation. Each dot represents a participant. Figure 2: Scatter-plot depicting the positive association between NS and PNS. Both NS (derived from the HEXACO-PI-R, see Material and methods) and PNS were standardized with a z-score transformation. Each dot represents a participant. Schwartz’s hypothesized circular structure in our sample. This test used two multi-dimensional scaling (MDS) as recommended by Schwartz (Bilsky et al. 2011). The ﬁrst analysis plotted the 56-value items, and the second anal- ysis plotted the 10 higher order values. Both the analyses use the respective correlation matrix to plot the values in a two-dimensional space. The ﬁrst analysis yielded S-Stress = 0.167 and Stress I = 0.274, whereas the second analysis yielded S-Stress = 0.032 and a Stress-I = 0.115. The stress value is an index of how well the data ﬁt the hypothesized conﬁguration; higher stress values signify a poorer conﬁguration. The stress values and the patterns in the MDS (see Table 1) were consistent with the structure hypothesized by Schwartz (1992). In addition, the openness values, self-direction and stimulation, were signiﬁcantly negatively related to NS, but these associations did not reach signiﬁcance when related to the PNS. Figure 3: Correlation coefﬁcients between the 10 value types (x-axis, conformity, tradition, benevolence, universalism, self-direction, stimulation, hedonism, achievement, power and security) and PNS (a) and NS (b). human value benevolence deviated from the sine wave; run- ning the sinusoidal test while excluding benevolence yields a signiﬁcant SFI = 0.08 (Fig. S1). Overall, NS and PNS map onto the human value space in similar, sinusoidal waveforms. Fitting the sinusoidal model to the NS and PNS Fitting the sinusoidal model to the NS and PNS Given our replication of Schwartz’s circular structure in the MDS analyses, we turned to testing whether there are sinu- soidal patterns of association between values and NS and PNS. To address this question, we plotted the correlation coefﬁcients between NS and PNS on the y-axis and each of the 10 lower order values on the x-axis in an order that follows their circular structure. The patterns are shown in Fig. 3. A pattern of sinusoidal association was found between human values and PNS, particularly near the inﬂec- tion points (Fig. 3a), which was signiﬁcant, SFI = 0.19; false positives = 0.6%. Similarly, our analysis of NS show a sinu- soidal association of a similar form (Fig. 3b) but the ﬁt to the sine wave was not reliable, SFI = 29; P > 0.05. Visual inspec- tion of Fig. 3a shows that the correlation between NS and the Structure of values Before testing for a sinusoidal waveform in the pattern of associations between values and NS, and PNS, we validated where x is a vector containing the correlation coefﬁcients of the 10 values with either PNS or NS. 363 Zacharopoulos et al. Figure 3: Correlation coefﬁcients between the 10 value types (x-axis, conformity, tradition, benevolence, universalism, self-direction, stimulation, hedonism, achievement, power and security) and PNS (a) and NS (b). Zacharopoulos et al. Figure 2: Scatter-plot depicting the positive association between NS and PNS. Both NS (derived from the HEXACO-PI-R, see Material and methods) and PNS were standardized with a z-score transformation. Each dot represents a participant. Schwartz’s hypothesized circular structure in our sample. This test used two multi-dimensional scaling (MDS) as recommended by Schwartz (Bilsky et al. 2011). The ﬁrst analysis plotted the 56-value items, and the second anal- ysis plotted the 10 higher order values. Both the analyses use the respective correlation matrix to plot the values in a two-dimensional space. The ﬁrst analysis yielded S-Stress = 0.167 and Stress I = 0.274, whereas the second analysis yielded S-Stress = 0.032 and a Stress-I = 0.115. The stress value is an index of how well the data ﬁt the hypothesized conﬁguration; higher stress values signify a poorer conﬁguration. The stress values and the patterns in the MDS (see Table 1) were consistent with the structure hypothesized by Schwartz (1992). In addition, the openness values, self-direction and stimulation, were signiﬁcantly negatively related to NS, but these associations did not reach signiﬁcance when related to the PNS. Fitting the sinusoidal model to the NS and PNS Given our replication of Schwartz’s circular structure in the MDS analyses, we turned to testing whether there are sinu- soidal patterns of association between values and NS and PNS. To address this question, we plotted the correlation coefﬁcients between NS and PNS on the y-axis and each of the 10 lower order values on the x-axis in an order that follows their circular structure. The patterns are shown in Fig. 3. A pattern of sinusoidal association was found between human values and PNS, particularly near the inﬂec- tion points (Fig. 3a), which was signiﬁcant, SFI = 0.19; false positives = 0.6%. Similarly, our analysis of NS show a sinu- soidal association of a similar form (Fig. Genes, Brain and Behavior (2016) 15: 361–366 Structure of values Furthermore, in addition to testing the patterns of correlations using the SFI method, we replicated the PNS and NS ﬁnd- ings using two previously established methods, with even more robust results (Boer & Fischer 2013; Roccas et al. 2002) (Appendix S1). The genetics of neuroticism and human values Second, the NS variance explained from the PNS was much higher in this study (4%) than in the initial discovery sample (0.6%). A number of factors may account for the larger rela- tion in our study. First, this study measured neuroticism using a single scale in a single homogeneous cohort, whereas the meta-analytic study assessed neuroticism from multiple instruments (even in the same cohort). Second, this study used a single measure of neuroticism with subscales (fear- fulness, anxiety, dependence and sentimentality) that are different and more emotional in focus than in the replica- tion cohort in the meta-analytic study (NEO-FFI’s neuroticism: anxiety, hostility, depression, self-consciousness, impulsive- ness, vulnerability to stress and Amsterdam Biographical Questionnaire). Third, the power of this study merely allows the detection of a moderate effect, and future replication studies may yield a smaller effect; therefore, future research should interpret the current effect size with caution. Despite these possibilities, the current replication of the NS–PNS relation is promising for future research attempting to learn more about this relation and its implications. important dimension of personality, neuroticism. We used empirically robust measures of human values, neuroticism and genetic neuroticism. The results replicated the associ- ation between NS and PNS despite using a different mea- sure of neuroticism than in prior research (i.e. emotionality from HEXACO-PI-R). This result adds to the evidence that the PNS derived by GWAS helps to explain individual varia- tion in neuroticism (Genetics of Personality Consortium et al. 2015). Moreover, it laid the foundation for testing whether human values are linked to both NS and PNS. Results indi- cated that human values were indeed associated with NS and PNS, following the sinusoidal pattern predicted by Schwartz et al. (2012) cross-cultural model. important dimension of personality, neuroticism. We used empirically robust measures of human values, neuroticism and genetic neuroticism. The results replicated the associ- ation between NS and PNS despite using a different mea- sure of neuroticism than in prior research (i.e. emotionality from HEXACO-PI-R). This result adds to the evidence that the PNS derived by GWAS helps to explain individual varia- tion in neuroticism (Genetics of Personality Consortium et al. 2015). Moreover, it laid the foundation for testing whether human values are linked to both NS and PNS. Results indi- cated that human values were indeed associated with NS and PNS, following the sinusoidal pattern predicted by Schwartz et al. (2012) cross-cultural model. References Angst, J., Gamma, A. & Endrass, J. (2003) Risk factors for the bipolar and depression spectra. Acta Psychiatr Scand Suppl, 418, 15–19. Ashton, M.C., Danso, H.A., Maio, G.R., Esses, V.M., Bond, M.H. & Keung, D.K.Y. (2005) Two dimensions of political attitudes and their individual difference correlates: a cross-cultural perspective. Ont Symp P 10, 1–29. Ashton, M.C., Lee, K. & de Vries, R.E. (2014) The HEXACO honesty-humility, agreeableness, and emotionality factors: a review of research and theory. Pers Soc Psychol Rev 18, 139–152. Two other aspects of our results merit further discussion. First, it is informative to contrast the sinusoidal pattern, which is a test of association across all values, with the strength of the correlations with speciﬁc values. This is interesting in part because most of the correlations between speciﬁc val- ues and PNS or NS were weak and unreliable, aside from the signiﬁcant theoretically congruent correlations discussed above (see Table S1). Nonetheless, the sinusoidal ﬁt shows a crucial pattern that is missing from univariate tests that focus on one value at a time. It is possible for individual relations to be weak at the same time as their combined pattern is meaningful and reliable. In the analyses of values, this differ- ence between individual correlations and the net pattern is crucial, because the relative roles of different values are psy- chologically more important and meaningful than the roles of any single value type in isolation, because of the competing implications between values (Rokeach 1973; Schwartz 1992). Bilsky, W., Janik, M. & Schwartz, S.H. (2011) The structural organiza- tion of human values-evidence from three rounds of the European social survey (ESS). J Cross-Cult Psychol 42, 759–776. Boer, D. & Fischer, R. (2013) How and when do personal values guide our attitudes and sociality? explaining cross-cultural variability in attitude-value linkages. Psychol Bull 139, 1113–1147. Byrd, R.H., Lu, P.H., Nocedal, J. & Zhu, C.Y. (1995) A limited memory algorithm for bound constrained optimization. Siam J Sci Comput 16, 1190–1208. Carney, D.R., Jost, J.T., Gosling, S.D. & Potter, J. (2008) The secret lives of liberals and conservatives: personality proﬁles, interaction styles, and the things they leave behind. Polit Psychol 29, 807–840. Cloninger, C.R. (1994) Temperament and personality. Curr Opin Neu- robiol 4, 266–273. De Raad, B., Barelds, D.P., Levert, E., Ostendorf, F., Mlacic, B., Di Blas, L., Hrebickova, M., Szirmak, Z., Szarota, P., Perugini, M., Church, A.T. & Katigbak, M.S. The genetics of neuroticism and human values These ﬁndings fundamentally extend the understanding of human values. Previous twin studies (Keller et al. 1992; Schermer et al. 2008, 2011; Waller et al. 1990) have docu- mented that human values may have a genetic component, but this has occurred without simultaneously pinpointing rel- evant patterns of genes, the pattern of associations with the values and the nature of the common association to the behavioural phenotype for personality. Here, we document a novel sinusoidal relationship between human values and a speciﬁc genetic marker, the PNS – a relationship that was very similar to that found between NS and values. In summary, the present research (1) replicated the prior evidence of a polygenic contribution to neuroticism using a novel measure of the trait, (2) showed an association between speciﬁc genetic components and human values for the ﬁrst time and (3) found a pattern of associations with values that is congruent with Schwartz’s (1992) and Schwartz et al.’s (2012) circular model of values. Together, these results show that it is useful to include value orien- tations as relevant individual differences in polygenic contri- butions to neuroticism-related traits, suggesting that future research should consider values in investigations of polygenic contributions to other traits. Furthermore, as expected, Fig. 3 shows that the sinusoidal waveforms were anchored at one end by negative relations between values promoting stimulation or self-direction on one hand and NS or PNS on the other hand. This pattern ﬁts links between neuroticism and anxiety and depres- sion. As noted earlier, anxiety and depression lead people to withdraw from the world around them (Angst et al. 2003; Thompson et al. 2011). In addition, higher levels of neuroticism are associated with less liberal, curious and open-minded attitudes (Carney et al. 2008; Van Hiel & Mervielde 2004). Neuroticism may contribute to lower open- ness to new experiences, ideas and feelings because of the threats posed by novelty. At the same time, the pattern of withdrawal elicited by lower stimulation and self-direction values may contribute to emotional instability by increasing rumination, perseveration in an isolated environment and self-absorption. Further evidence is needed to explore these possibilities. 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This study was supported by the National Centre for Men- tal Health (NCMH) at Cardiff University, with funds from the National Institute for Social Care and Health Research (NISCHR), Welsh Government, Wales (grant number BR09) and by grant MR/K004360/1 from the Medical Research Council (MRC) and by the MRC Centre for Neuropsychiatric Genetics and Genomics (G0800509). We are grateful to all professionals, patients and volunteers involved with the National Centre for Mental Health (NCMH). Price, A.L., Patterson, N.J., Plenge, R.M., Weinblatt, M.E., Shadick, N.A. & Reich, D. (2006) Principal components analysis corrects for stratiﬁcation in genome-wide association studies. Nat Genet 38, 904–909. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A., Bender, D., Maller, J., Sklar, P., de Bakker, P.I., Daly, M.J. & Sham, P.C. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81, 559–575. Rim, Y. 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Table S1: Pearson correlation coefﬁcients between PNS and NS with the 10 human values (conformity, benevolence, tradition, universalism, self-direction, stimulation, hedonism, achievement, power and security). Rokeach, M. (1973) The Nature of Human Values. Free Press Collier-Macmillan, New York and London. Romero, E., Villar, P. & Lopez-Romero, L. (2015) Assessing six factors in Spain: validation of the HEXACO-100 in relation to the ﬁve factor model and other conceptually relevant criteria. Pers Individ Differ 76, 75–81. Figure S1: Correlation coefﬁcients between the nine value types (x-axis, conformity, tradition, universalism, self-direction, stimulation, hedonism, achievement, power and security) and NS. Saucier, G. (1994) Mini-markers: a brief version of Goldberg’s unipolar big-ﬁve markers. J Pers Assess 63, 506–516. Schermer, J.A., Feather, N.T., Zhu, G. & Martin, N.G. (2008) Pheno- typic, genetic, and environmental properties of the portrait values questionnaire. Twin Res Hum Genet 11, 531–537. Appendix S1: Replication of the sinusoidal ﬁndings of PNS and NS using two previously established methods. Appendix S1: Replication of the sinusoidal ﬁndings of PNS and NS using two previously established methods. 366 Genes, Brain and Behavior (2016) 15: 361–366
https://openalex.org/W2082424449	https://europepmc.org/articles/pmc3662882?pdf=render	English	null	Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations	Frontiers in microbiology	2,013	cc-by	8,361	ORIGINAL RESEARCH ARTICLE bli h d 24 M 2013 published: 24 May 2013 doi: 10.3389/fmicb.2013.00125 Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations Toyotaka Sato 1, Shin-ichi Yokota 2, Ikuo Uchida 3, Torahiko Okubo 1, Masaru Usui 1, Masahiro Kusumoto4, Masato Akiba 4, Nobuhiro Fujii 2 and Yutaka Tamura 1* Masahiro Kusumoto , Masato Akiba , Nobuhiro Fujii and Yutaka Tamura 1 Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu Japan 1 Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary M Ebetsu, Japan boratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary Med betsu Japan , p 2 Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan 3 Dairy Hygiene Research Division, Hokkaido Research Station, National Institute of Animal Health, Sapporo, Japan 4 Bacterial and Parasitic Disease Research Division, Safety Research Team, National Institute of Animal Health, Ibaraki, Japan 2 Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan 3 Dairy Hygiene Research Division, Hokkaido Research Station, National Institute of Animal Health, Sapporo, Japan 4 Bacterial and Parasitic Disease Research Division, Safety Research Team, National Institute of Animal Health, Ibaraki, Japan Fluoroquinolone resistance can cause major clinical problems. Here, we investigated ﬂuoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciproﬂoxacin, levoﬂoxacin, and norﬂoxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciproﬂoxacin concentrations and higher mRNA expression levels of efﬂux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the ﬂuoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to ﬂuoroquinolone in HUE1. Therefore, ﬂuoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB–TolC and other chromosome-encoded efﬂux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring ﬂuoroquinolone resistance in E. coli. Edited by: Edited by: Kunihiko Nishino, Osaka University, Japan Reviewed by: Axel Cloeckaert, Institut National de la Recherche Agronomique, France Junichi Yamagishi, Nihon Pharmaceutical University, Japan Correspondence: Yutaka Tamura, Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, 582 Midorimachi-Bunkyoudai, Ebetsu 069-8501, Japan. e-mail: tamuray@rakuno.ac.jp Kunihiko Nishino, Osaka University, Japan Kunihiko Nishino, Osaka University, Japan Correspondence: Yutaka Tamura, Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, 582 Midorimachi-Bunkyoudai, Ebetsu 069-8501, Japan. e-mail: tamuray@rakuno.ac.jp plasmid-mediated quinolone-resistant determinants (PMQRs), viz., oqxAB and qnrS. In this bacterium, OqxAB is a plasmid- encoded efﬂux pump; however, the gene is present on the chro- mosomal DNA in most Klebsiella pneumoniae and Enterobacter cloacae strains (Bin Kim et al., 2009). The presence of this pump confers resistance to several antimicrobial agents, such as olaquindox (OLA), trimethoprim (TMP), and chloramphenicol (CHL), and decreases bacterial susceptibility to ﬂuoroquinolones (Hansen et al., 2007). QnrS, on the other hand, is a mem- ber of the pentapeptide-repeat protein family that protects DNA gyrase (and probably also topoisomerase IV) from binding to ﬂuoroquinolones, thereby decreasing ﬂuoroquinolone suscepti- bility (Jacoby, 2005). However, acquisition of these PMQRs alone results in only a low level of ﬂuoroquinolone resistance, with MICs that do not exceed the breakpoints for ﬂuoroquinolones (Jacoby, 2005; Hansen et al., 2007). E. coli isolates lacking QRDR mutations in gyrA and parC and showing concomitant acquisi- tion of oqxAB and qnrS have previously been reported in China; however, these isolates did not exceed the breakpoint for CIP (Zhao et al., 2010). Keywords: AcrAB, efﬂux pump, Escherichia coli, ﬂuoroquinolone resistance, oqxAB, qnrS Reviewed by: Axel Cloeckaert, Institut National de la Recherche Agronomique, France Junichi Yamagishi, Nihon Pharmaceutical University Japan www.frontiersin.org BACTERIAL ISOLATES E. coli HUE1 had been isolated from the urinary catheter of a 77-year-old female patient at Hokkaido University Hospital (Sapporo, Japan) in 2007 (Sato et al., 2011). The somatic (O) serotype was determined by the slide agglutination test by using Escherichia coli O antisera (Denka Seiken, Tokyo, Japan), and the ﬂagellar (H) serotype was determined using reference sera obtained from the Statens Serum Institut (Hillerød, Denmark). INTRODUCTION Fluoroquinolones are widely used in the clinical treatment of var- ious bacterial infections, such as urinary tract and blood stream infections caused by Escherichia coli. Many studies have reported the isolation of ﬂuoroquinolone-resistant strains (Peña et al., 1995; Cizman et al., 2001; Sanchez et al., 2012). Fluoroquinolone resistance is mainly caused by point mutations in the quinolone resistance-determining regions (QRDRs) of the DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) subunits (Yoshida et al., 1991; Conrad et al., 1996; Heisig, 1996; Breines et al., 1997). A slight decrease in susceptibil- ity to ﬂuoroquinolones is attributed to a single mutation in gyrA. Secondary mutations in gyrA and additional mutations in parC and/or parE are required to exceed the breakpoint of the ﬂuoro- quinolone MIC (Conrad et al., 1996; Heisig, 1996; Breines et al., 1997). Recently, we reported a ﬂuoroquinolone-resistant E. coli iso- late without QRDR mutations, named HUE1 (Sato et al., 2011). Its MICs for ﬂuoroquinolones, such as ciproﬂoxacin (CIP) and levoﬂoxacin (LVX), exceeded the breakpoints established by the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2011). HUE1 possesses two Our previous ﬁndings suggested that the ﬂuoroquinolone resistance of HUE1, which lacks QRDR mutations, is associated May 2013 \| Volume 4 \| Article 125 \| 1 www.frontiersin.org www.frontiersin.org Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. The presence of oqxA, oqxB, and qnrS was determined by PCR (Sorensen et al., 2003; Cattoir et al., 2007). Full- length nucleotide sequences of oqxAB, qnrS, acrA, acrB, acrR, acrE, acrF, acrS, tolC, soxS, soxR, and rob were determined by PCR and direct sequencing by using the primer pairs listed in Table 1. The nucleotide sequence of marR was determined as previously described (Lindgren et al., 2003). Nucleotide sequences were determined using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA), and sequencing was performed in a 3130 Genetic Analyzer (Life Technologies). The oqxAB nucleotide sequence of HUE1 was submitted to GenBank (accession number AB601773). All gene sequences, except those of oqxAB and qnrS, were compared with those of Escherichia coli strain K12 substrain MG1655, which was deposited in GenBank (accession number U00096), as the reference strain. with not only with the presence of oqxAB and qnrS but also with other ﬂuoroquinolone-resistance mechanism(s) (Sato et al., 2011). In the current study, we investigated the ﬂuoroquinolone- resistance mechanisms of the HUE1 strain. SUSCEPTIBILITY TESTING AND GENETIC ANALYSIS Norﬂoxacin (NOR) was purchased from Sigma-Aldrich (St Louis, MO). Other antibiotics were obtained as described previously (Sato et al., 2011). Susceptibility to ﬂuoroquinolones [CIP, LVX, uriﬂoxacin (URX), sitaﬂoxacin (STX), and NOR], nalidixic acid (NAL), CHL, and TMP was determined by the agar plate dilution method, according to CLSI guidelines (Clinical and Laboratory Standards Institute, 2011). Phe-Arg-β-naphthylamide (PAβN; ﬁnal concentration, 20 mg/L), which is an inhibitor of the resistance-nodulation-division (RND)-type efﬂux pump, was purchased from Sigma-Aldrich. Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy TRANSFORMATION OF PLASMIDS DERIVED FROM HUE1 INTO DH5α Plasmids were isolated from HUE1 as described previously (Kado and Liu, 1981). The plasmids were electroporated into E. coli DH5α (Takara, Shiga, Japan) by using an ECM600 (BTX, San Diego, CA, USA) under the following conditions: voltage, 1.8 kV; capacitance, 25 μF; and resistance, 200 ohms. oqxAB and/or qnrS transformants were screened using Muller-Hinton (MH) agar containing CIP, TMP, and CHL at concentrations ranging from 2- to 8-fold of the respective MIC values. Table 1 \| Sequences of primers used for PCR and DNA sequencing in this study. Gene Forward primer or ﬂuorescent probe (5′–3′) Reverse primer (5′–3′) acrA acrA-1F (caccggcagtttgaggatcg) acrA-1R (gcgcggatcaatctggctta) acrA-2F (gtcgttgctggactgggtca) acrA-2R (atgaacaaaaacagagggtttacg) acrB acrB-1F (tcaatgatgatcgacagtatggct) acrB-1R (ggaacaactggcgagcaaac) acrB-2F (agcggaacgaccagcataac) acrB-2R (gcgggacgtggtcagaatac) acrB-3F (ccagcctggtcaatcagctc) acrB-3R (gcgtgttatggcggaagaag) acrB-4F (cgaataccgccgacagtacc) acrB-4R (ggatgaacccgaatgagctg) acrB-5F (caggattttgccgaactcttca) acrB-5R (ataaccagcaagccgcaagc) acrE acrE-F(atagccgaagttcgcccaca) acrE-R (ctgcgggggtatcggtagtg) acrF acrF-1F (cagtcaggcgattggcgata) acrF-1R (accaccgagccgtcactgtt) acrF-2F (cagcgttaccagggcaacaa) acrF-2R (attttgccgacgctgttggt) acrF-3F (cgctgcttaaacccgtctctg) acrF-3R (cagtcgcggagagccataca) acrF-4F (cgctgggtgggacttacgtt) acrF-4R (ttatcctttaaagcaacggcgga) acrR acrR-F (atcagaacgaccgccagagg) acrR-R (ttattcgttagtggcaggattacga) acrS acrS-F (ttacatgacacttaattcattcgtttga) acrS-R (tgcacatcgctgccttcagt) oqxAB oqxAB-1F (acatttaccggaataaaaat) oqxAB-1R (ggcgaggttttgatagtgga) oqxAB-2F (acggtgtacgtctactttga) oqxAB-2R (gtctcggcaatcactttcg) oqxAB-3F (gcgcgcggagtatcccggcg) oqxAB-3R (ccgcatccttattgttgagc) oqxAB-4F (atcgagatgggttccggtag) oqxAB-4R (taaacggacggaaaatccag) oqxAB-5F (tggcggccctgctgttaaag) oqxAB-5R (gataggtctgcagcgtaccg) oqxAB-6F (ctggacgtgcaggtcgatcg) oqxAB-6R (gataaaggcgatggaggtcat) oqxAB-7F (gagctgtcgaagcagatcct) oqxAB-7R (tgcgacccggtgccggaaat) qnrS qnrSseq-F (ttagtcaggataaacaacaataccca) qnrSseq-R (atggaaacctacaatcatacatatcgg) rob robA-F (catctggacgcccctgcatt) robA-R (agccaatggccccagcatta) soxSR soxSR-F (gcgctattgccagggatggt) soxSR-R (tgtgttgacgtcgggggaaa) tolC tolC-1F (cgggcaggttgtctggctta) tolC-1R (ctggctcaagcgtgcctgta) tolC-2F (gctgcgctgaatgtcgaaaa) tolC-2R (tgcgtggcgtatggattttg) Table 1 \| Sequences of primers used for PCR and DNA sequencing in this study. May 2013 \| Volume 4 \| Article 125 \| 2 Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. ACCUMULATION ASSAYS Intracellular CIP concentrations were assayed using a ﬂuoromet- ric uptake assay (Usui et al., 2009). Forty milligrams of wet cells was treated with CIP (ﬁnal concentration, 10 mg/L) in the pres- ence or absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma-Aldrich; ﬁnal concentration, 150 μM). The ﬂu- orescence of CIP was measured at excitation and emission wavelengths of 277 and 445 nm, respectively, by using an RF- 5000 ﬂuorescence spectrophotometer (Shimadzu, Kyoto, Japan) (Nakaminami et al., 2010). The data shown represent the mean value ± standard deviation values calculated from at least three independent experiments. REAL-TIME REVERSE-TRANSCRIPTION (RT) PCR Overnight cultures were diluted 1:100 in LB broth and grown to the mid-logarithmic phase. RNA was isolated using an RNeasy Mini kit (Qiagen, Hilden, Germany). Gene expression was esti- mated by quantitative real-time reverse-transcription (RT)-PCR by using the probe and primer pairs shown in Table 2. RT-PCR was performed using a QuantiTect Probe RT-PCR kit (Qiagen) in 20 μL reactions containing 2.5 ng of puriﬁed RNA, 0.2 μM of probe, and 0.5 μM of each of the forward and reverse primers. The cycling conditions included reverse transcription at 50◦C for 20 min and PCR involving initial activation at 95◦C for 15 min and 45 cycles each consisting of 1 min at 55◦C and 30 s at 60◦C, in a LightCycler 480 system (Roche, Mannheim, Germany). The E. coli strain AG100 (K-12 argE3 thi-1 rpsL xyl mtl D(gal-uvrB) supE44) (Okusu et al., 1996), which was gifted by Dr. Helen I. Zgurskaya (University of Oklahoma, USA), was used as a con- trol. Expression levels of gapA were used to normalize expression ratios. Data, except for those of oqxB and qnrS1, were calibrated against expression levels in AG100, which were set as 1, to deter- mine fold changes in expression. Data for oqxB and qnrS1 were calibrated to the respective levels in HUE1, which were set as 1. The data shown represent the mean values of three independent experiments. PLASMID CONSTRUCTION Each of the wild-type acrR and wild-type marR DNA segments were ampliﬁed by PCR using DH5α genomic DNA as a tem- plate and primers containing the A1 T7 promoter, consensus Shine-Dalgarno, and HindIII restriction sites (Table 2), which were designed according to a previous report (Edgar et al., 2012). These PCR products were cloned into the HindIII site of pUC19 (wt-acrR or wt-marR), and the plasmids were then transformed into HUE1. Curing of plasmids and generation of spontaneous mutants were performed as previously described, with slight modiﬁcations (Deane and Rawlings, 2004). Brieﬂy, strains were grown in 4 mL LB broth at 40◦C and incubated for periods ranging from a week to a month. Then, clones were picked and grown in MH agar containing sub-MIC concentrations of CIP, TMP, and CHL or no antimicrobials, in order to obtain oqxAB- and/or qnrS-cured clones. Re-introduction of plasmids harboring oqxAB and/or qnrS into plasmid-cured mutants was performed using electropo- ration as described above. The presence of oqxA, oqxB, and qnrS was detected by PCR and Southern hybridization of plasmids, as previously described (Tamamura et al., 2011). Probes for oqxB and qnrS1 were prepared by PCR by using speciﬁc primers, as described previously (Cattoir et al., 2007; Bin Kim et al., 2009). Probe labeling was carried out using a PCR DIG labeling mix (Roche Diagnostics, Tokyo, Japan). CHARACTERISTICS OF HUE1 AND GENETIC ANALYSIS OF OqxAB AND QnrS HUE1 was identiﬁed as ST48 (according to the Max-Planck- Institut für Infektionsbiologie database), phylogenetic group A (Sato et al., 2011), and O125:H37. Its NOR exceeded the break- points established by the CLSI, similarly with CIP and LVX previously tested (Sato et al., 2011). A 4421-bp DNA segment containing oqxAB and a 647-bp DNA segment containing qnrS derived from HUE1 were sequenced. The sequence of oqxAB was 100% identical to that of plasmid pOLA52 (accession number EU370913) in an E. coli isolate obtained from swine in Sweden (Hansen et al., 2004). The sequence of qnrS was 100% identical to that of qnrS1 in Shigella ﬂexneri (accession number AB187515) (Hata et al., 2005). Plasmid proﬁling and Southern blotting anal- ysis showed that oqxAB and qnrS1 were located on 2 independent plasmids, pHFQ1 (>165 kb) and pHFQ2 (>100 kb), respectively (Figure 1, lane 1). STATISTICAL ANALYSIS Statistical signiﬁcance was determined by the Student’s t-test. Differences among more than three groups were determined using the Mann–Whitney U-test. A P-value of 0.05 or less was considered statistically signiﬁcant. CONSTRUCTION OF GENE DELETION MUTANTS Gene disruption was performed by Red/ET recombination by using the Quick and Easy Gene Deletion kit (Gene Bridges GmbH, Heidelberg, Germany) according to the manufacturer’s protocol. The relevant forward and reverse primers are shown in Table 2. Brieﬂy, target gene-speciﬁc minigenes, containing a neomycin/kanamycin- or hygromycin-resistance cassette, were constructed by PCR with primers containing 46 bp of upstream and downstream sequences that contained the start codon or the stop codon (or the last codon before the stop codon) of the tar- get genes, respectively (Table 2). The target genes were replaced with minigenes containing the drug-resistance cassette by using Red/ET recombination. The integration of interrupted genes was veriﬁed by PCR and DNA sequencing. Introduction of pHFQ1 (carrying oqxAB) into DH5α slightly increased the MICs for ﬂuoroquinolones (except for LVX) and NAL, as compared with those for the host strain DH5α (Table 3). Introduction of pHFQ2 (carrying qnrS1) resulted in higher MICs for ﬂuoroquinolones and NAL than those seen for DH5α/pHFQ1. However, introduction of both pHFQ1 and pHFQ2 into DH5α did not increase the MICs beyond the breakpoints for ﬂuoro- quinolone. Deletion of oqxAB or qnrS from the transformants by Red/ET recombination reverted MICs for ﬂuoroquinolones to levels similar to those of DH5α. May 2013 \| Volume 4 \| Article 125 \| 3 www.frontiersin.org www.frontiersin.org Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. Table 2 \| Sequences of real-time RT-PCR probes and primers for RT-PCR or construction of knockout mutants designed in this study. May 2013 \| Volume 4 \| Article 125 \| 5 www.frontiersin.org CONSTRUCTION OF GENE DELETION MUTANTS Gene Forward primer or ﬂuorescent probe (5′–3′) Reverse primer (5′–3′) Purpose acrA acrART-F (ctatcaccctacgctctatcttc)a acrART-R (gcgcgcacgaacatacc)a RT-PCR acrART-P (cgaacccggatcacactct)a,b - RT-PCR acrARed-F (atgaacaaaaacagagggtttacgcctctggcggtcgttctgatgctaattaaccctcactaaagggcg) acrARed-R (agacttggactgttcaggctgagcaccgcttgcggcttgctggttat taatacgactcactatagggctc) Construction of acrA knockout mutant acrB acrBRT-F (gcggtcgtgtgaagaaagttta) acrBRT-R (actcccaacgagaagaggagaa) RT-PCR acrBRT-P (tgaccatcagcagcacgaacataccagt)b - RT-PCR acrBRed-F (atgcctaatttctttatcgatcgcccgatttttgcgtgggtgatcgcaattaaccctcactaaagggcg) acrBRed-R (tcaatgatgatcgacagtatggctgtgctcgatatcttcattcttgc taatacgactcactatagggctc) Construction of acrB knockout mutant acrD acrDRT-F (gcaacgccgaacgctacg) acrDRT-R (cacggtcttccagcggtaag) RT-PCR acrDRT-P (caggaacaggaacaccatgccgccaa)b - RT-PCR acrDREd-F (atggcgaatttctttattgatcgccccatttttgcctgggtgctggcaataattaaccctcactaaagggcg) acrDRed-R (ttattccgggcgcggcttcagcgggaagcggcggcgcaccagca caaagataatacgactcactatagggctc) Construction of acrD knockout mutant acrE acrERT-F (cctcctgccctcctttattctg) acrERT-R (aacggtaacctgcggttcac) RT-PCR acrERT-P (ttctcttctcccttatcgttacaaccggcg)b - RT-PCR acrF acrFRed-F (atggcaaacttttttattcgacgaccgatatttgcatgggtgctggccataattaaccctcactaaagggcg) acrFRed-R (ttatcctttaaagcaacggcggatcaccacaaagaacaccggtacg aagataatacgactcactatagggctc) Construction of acrF knockout mutant acrR T7A1ACRR-F (acttaagcttAAAAGAGTATTGACTTAAAGTCTAACTATAGGATACTTACAGCCATAGGAGGacagct atggcacgaaaaaccaaac)c ACRR-R (ttaagcttcttattcgttagtggcagg) Plasmid construction of wild type-acrR gapA gapART-F (aaaggcgctaacttcgacaa) gapART-R (gaacggtggtcatcagacct) RT-PCR gapART-P (caacgataacttcggcatca)b - RT-PCR marA marART-F (gccgtaagatgacggaaatcg) marART-R (gaaggttcgggtcagagtttg) RT-PCR marART-P (agagtatcggctcgttactttccttcagct)b - RT-PCR marR T7A1MARR-F (acttaagcttAAAAGAGTATTGACTTAAAGTCTAACTATAGGATACTTACAGCCATAGGAGGacagct gtgaaaagtaccagcgatc)c MARR-R (ttaagcttcttacggcaggactttcttaagc) Plasmid construction of wild type-marR mdfA mdfART-F (ccatgtgctgccctggga) mdfART-R (gtcacgaccgagttctttcag) RT-PCR mdfART-P (ttgccgcattggcagcgatctcctt)b - RT-PCR mdfARed-F (atgcaaaataaattagcttccggtgccaggcttggacgtcaggcgttactaattaaccctcactaaagggcg) mdfARed-R (ttacccttcgtgagaatttcccatctgtttatcttttaaaaagataacca taatacgactcactatagggctc) Construction of mdfA knockout mutant (Continued) May 2013 \| Volume 4 \| Article 125 \| 4 Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. Table 2 \| Continued Gene Forward primer or ﬂuorescent probe (5′–3′) Reverse primer (5′–3′) Purpose mdtK mdtKRT-F (gcctgctggtgaacatccc) mdtKRT-R (gcaaggaacatgacccaatacac) RT-PCR mdtKRT-P (tagccacgccacaaccaacgccac)b - RT-PCR mdtKRed-F (gtgcagaagtatatcagtgaagcgcgtctgttattagcattagcaatcccaattaaccctcactaaagggcg) mdtKRed-R (ttagcgggatgctcgttgcagaatgatggctgacggcagacgttgcaggataatac gactcactatagggctc) Construction of mdtK knockout mutant ompC ompCRT-F (aacggtcgtgacgcactg) ompCRT-R (cgatgtaagcagcggtgttc) RT-PCR ompCRT-P (acggcgtcggcggttctatcactt)b - RT-PCR ompF ompFRT-F (gaagctcaacctcttggcaac) ompFRT-R (gccgctggtgtttgtaaatttattag) RT-PCR ompFRT-P (cgggtttcaccgtagttcgctgcca)b - RT-PCR oqxAB oqxBRT-F (tggtggtgcatctgttctcc) oqxBRT-R (catccttcactttcagcgtgg) RT-PCR oqxBRT-P (cgcatatacagcgagtcgtacttcccgc)b - oqxABRed-F (atgagcctgcaaaaaacctggggaaacattcacctgaccgcgctcggcgaattaaccctcactaaagggcg) oqxABRed-R (ctaggcgggcagatcctcctggaccggcttcctgcgggtcaccagtttcctaatac gactcactatagggctc) Construction of oqxAB knockout mutant qnrS qnrSRT-F (aatcatacatatcggcaccacaac) qnrSRT-R (agcacgtcgaaagtcgctg) RT-PCR qnrSRT-P (tgatctcaccttcaccgcttgcacattc)b - RT-PCR qnrSRed-F (atggaaacctacaatcatacatatcggcaccacaacttttcacataaaattaaccctcactaaagggcg) qnrSRed-R (gtcaggataaacaacaatacccagtgcttcgagaatcagttcttgct taatacgactcactatagggct) Construction of qnrS knockout mutant rob robART-F (agtcgaagcggtattgcagc) robART-R (ccaagtggcacttacagagaatg) RT-PCR robART-P (ccagaatcggacgcgcagtcaggc)b - RT-PCR soxS soxSRT-F (cgtcaccgtgcggaacat) soxSRT-R (tgtcccatcagaaaattattcagga) RT-PCR soxSRT-P(cgagcatattgaccagccgcttaacattga)b - RT-PCR tolC tolCRT-F (ggtacgttgaacgagcaggatc) tolCRT-R (ccatcagcaatagcattctgttcc) RT-PCR tolCRT-P (ctggcactgaacaatgcgctgagcaa)b - RT-PCR tolCRed-F (atgaagaaattgctccccattcttatcggcctgagcctttctgggttcagaattaaccctcactaaagggcg) tolCRed-R (tcagttacggaaagggttatgaccgttactggtggtagtgcgtgcggatgtaatacg actcactatagggctc) Construction of tolC knockout mutant aPrimer was designed from nucleotides 818–884 for acrA (corresponding to amino acids 272–294 in AcrA). bFluorescent probe (5′–3′) used in RT-PCR. The underlined sequences are complementary to the kit DNA template, which contains a neomycin/kanamycin- or hygromycin-resistance cassette ﬂanked by FRT recombination sites. cCapital letters show nucleotide sequences of the A1 T7 promoter and Shine-Dalgarno, while “aagctt” in italics indicates the HindIII restriction site. May 2013 \| Volume 4 \| Article 125 \| 5 Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. CONSTRUCTION OF GENE DELETION MUTANTS FIGURE 1 \| Plasmid proﬁle and Southern hybridization of oqxB and qnrS1 in HUE1, its oqxAB- and/or qnrS1-cured strains, and oqxAB−and/or qnrS1−re-introduced strains. (A) Plasmid proﬁle. Electrophoresis was performed at 100 V for 70 min with a 0.8% agarose gel. (B) Southern hybridization with the oqxB probe. (C) Southern hybridization with the qnrS probe. Lane 1, HUE1; lane 2, HUE1A2; lane 3, HUE1A; lane 4, HUE1A-Curoqxqnr; lane 5, HUE1A-Reoqx; lane 6, HUE1A-Reqnr; lane 7, HUE1A-Reoqxqnr; lane 8, HUE1; lane 9, HUE1B-Curoqx; lane 10, HUE1B-Curoqxqnr; lane 11, HUE1B-Reqnr; lane 12, HUE1B-Reoqx; lane 13, HUE1B-Reoqxqnr; R, DNA Molecular Weight MarkII, DIG-labeled: r, BAC-Tracker Supercoiled DNA Ladder. 2, HUE1A2; lane 3, HUE1A; lane 4, HUE1A-Curoqxqnr; lane 5, HUE1A-Reoqx; lane 6, HUE1A-Reqnr; lane 7, HUE1A-Reoqxqnr; lane 8, HUE1; lane 9, HUE1B-Curoqx; lane 10, HUE1B-Curoqxqnr; lane 11, HUE1B-Reqnr; lane 12, HUE1B-Reoqx; lane 13, HUE1B-Reoqxqnr; R, DNA Molecular Weight MarkII, DIG-labeled: r, BAC-Tracker Supercoiled DNA Ladder. FIGURE 1 \| Plasmid proﬁle and Southern hybridization of oqxB and qnrS1 in HUE1, its oqxAB- and/or qnrS1-cured strains, and oqxAB−and/or qnrS1−re-introduced strains. (A) Plasmid proﬁle. Electrophoresis was performed at 100 V for 70 min with a 0.8% agarose gel. (B) Southern hybridization with the oqxB probe. (C) Southern hybridization with the qnrS probe. Lane 1, HUE1; lane CHARACTERIZATION OF MUTANTS DERIVED FROM PLASMID CURING AND PLASMID RE-INTRODUCTION 1280-fold lower than those of HUE1 and were 4- to 16-fold lower than those of HUE1A-Curoqxqnr (Table 3). Similar differences with respect to the MICs of groups A and B were also observed for the mutant series in which pHFQ2 (carrying qnrS1) had been re-introduced. However, most ﬂuoro- quinolone MICs of group B strains in which pHFQ1 (carrying oqxAB) had been re-introduced were only 2- to 4-fold lower than those of mutants in group A. Eleven mutants obtained by curing and re-introduction of the plasmids showed altered ﬂuoroquinolone MICs (Figure 1 and Table 3). These mutants were grouped into two types (groups A and B). In the case of group A, we ﬁrst obtained two strains, named HUE1A and HUE1A2. Although these two mutants still harbored pHFQ1 (carrying oqxAB) and pHFQ2 (carrying qnrS1; Figure 1, lanes 2 and 3), the MICs of NAL and ﬂuoro- quinolones were 2- or 4-fold lower than those of the parental strain, HUE1. Secondary screening of mutants by using HUE1A yielded a mutant, HUE1A-Curoqxqnr, which had lost pHFQ1 and pHFQ2 (Figure 1, lane 4). This mutant had 16- to 64- fold lower MICs for ﬂuoroquinolones than the parental strain, HUE1A. Re-introduction of pHFQ1 and pHFQ2 into HUE1A- Curoqxqnr yielded HUE1A-Reoqxqnr, in which ﬂuoroquinolone MICs recovered to the levels seen for HUE1A, but not to the MICs of the parent strain, HUE1. EFFECTS OF EFFLUX PUMP INHIBITORS AND GENETIC ANALYSIS OF EFFLUX PUMP COMPONENTS IN THE HUE1 STRAIN AND ITS MUTANTS The efﬂux pump inhibitor PAβN reduced the ﬂuoroquinolone MICs of the HUE1 strain from 4- to 16-fold (Table 3). In mutants derived from plasmid curing in groups A and B, the effects of PAβN were less than in the parental strain, HUE1. Remarkably, the ﬂuoroquinolone MICs of three mutants that lost oqxAB in group B (HUE1B-Curoqxqnr, HUE1B-Curoqx, and HUE1B- Reqnr) were barely affected by PAβN. HUE1 exhibited higher mRNA expression of efﬂux pump genes (acrA, acrB, acrE, acrD, mdtK, mdfA, and tolC) and their global activators (soxS, marA, and rob) than the control strains, AG100 and DH5α. In contrast, ompF expression was lower in HUE1 than in the control strains (Table 4). Moreover, In the case of group B, we ﬁrst obtained a mutant, HUE1B- Curoqx, which had lost pHFQ1; a second screening using HUE1B-Curoqx yielded HUE1B-Curoqxqnr, which had lost both pHFQ1 and pHFQ2 (Figure 1, lanes 9 and 10). Interestingly, the ﬂuoroquinolone MICs of HUE1B-Curoqxqnr were 512- and May 2013 \| Volume 4 \| Article 125 \| 6 Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. Table 3 \| Fluoroquinolones and NAL susceptibilities of HUE1, transformants derived from DH5α, and HUE1mutants derived from plasmid curing and reintroduction. EFFECTS OF EFFLUX PUMP INHIBITORS AND GENETIC ANALYSIS OF EFFLUX PUMP COMPONENTS IN THE HUE1 STRAIN AND ITS MUTANTS Strain PCR MIC (mg/L) oqxAB qnrS1 CIP LVX URX STX NOR NAL HUE1 + + 4 (×4)a 8 (×16) 4 (×4) 2 (×16) 16 128 TRANSFORMATION OF pHFQ1 AND pHFQ2 DH5α − − 0.015 0.03 0.015 0.004 0.03 32 DH5α/pHFQ1 + − 0.03 0.03 0.03 0.015 0.06 64 DH5α/pHFQ1(oqxAB::hygr) − − 0.015 0.03 0.015 0.004 0.03 32 DH5α/pHFQ2 − + 0.125 0.5 0.125 0.125 0.25 128 DH5α/pHFQ2(qnrS1::hygr) − − 0.015 0.03 0.015 0.004 0.03 32 DH5α/pHFQ1-pHFQ2 + + 0.25 0.5 0.125 0.25 0.25 >128 MUTANTS OBTAINED FROM PLASMID CURING <Group A> HUE1A and HUE1A2 + + 2 (×2) 2 (×4) 2 (×2) 1 (×8) 8 64 HUE1A-Curoqxqnr − − 0.06 (×2) 0.125 (×4) 0.03 (×1) 0.03 (×4) 0.5 16 HUE1A-Reoqx + − 0.125 (×4) 0.125 (×4) 0.125 (×2) 0.06 (×8) 0.5 32 HUE1A-Reqnr − + 1 (×2) 1 (×4) 1 (×1) 0.5 (×4) 8 64 HUE1A-Reoqxqnr + + 2 (×2) 2 (×4) 2 (×2) 1 (×8) 8 64 <Group B> HUE1B-Curoqx − + 0.25 (×1) 0.125 (×1) 0.125 (×1) 0.03 (×1) 0.25 4 HUE1B-Curoqxqnr − − 0.008 (×1) 0.008 (×1) 0.008 (×1) 0.004 (×1) 0.03 1 HUE1B-Reoqx + − 0.125 (×8) 0.06 (×8) 0.03 (×2) 0.03 (×8) 0.25 16 HUE1B-Reqnr − + 0.125 (×1) 0.125 (×1) 0.125 (×1) 0.03 (×1) 0.25 4 HUE1B-Reoqxqnr + + 1 (×4) 1 (×4) 0.5 (×2) 0.25 (×8) 4 32 aReduction of MIC by PAβN (-fold), rresistance. Table 3 \| Fluoroquinolones and NAL susceptibilities of HUE1, transformants derived from DH5α, and HUE1mutants derived from plasmid curing and reintroduction AL susceptibilities of HUE1, transformants derived from DH5α, and HUE1mutants derived from plasmid Table 3 \| Fluoroquinolones and NAL susceptibilities of HUE1, transformants derived from DH5α, and HUE1mutants derived from plasmid curing and reintroduction. MUTANTS OBTAINED FROM PLASMID CURING aReduction of MIC by PAβN (-fold), rresistance. Table 4 \| mRNA expression levels of global activators, efﬂux pumps, and ompF genes in HUE1, HUE1A, HUE1A-Curoqxqnr, and Table 4 \| mRNA expression levels of global activators, efﬂux pumps, and ompF genes in HUE1, HUE1A, HUE1A-Curoqxqnr, and HUE1B-Curoqqnr. ssion levels of global activators, efﬂux pumps, and ompF genes in HUE1, HUE1A, HUE1A-Curoqxqnr, and Table 4 \| mRNA expression levels of global activators, efﬂux pumps, and ompF genes in HUE1, HUE1A, HUE1A-Curoqxqnr, and HUE1B-Curoqqnr. EFFECTS OF EFFLUX PUMP INHIBITORS AND GENETIC ANALYSIS OF EFFLUX PUMP COMPONENTS IN THE HUE1 STRAIN AND ITS MUTANTS Strain Expression level (relative amount of AG100; in terms of fold change) Global activators Efﬂux pumps Porin soxS marA rob acrA acrB acrE acrD mdtK mdfA tolC ompF HUE1 9.80 147.77 8.17 4.67 6.81 8.70 8.34 6.43 6.76 2.83 0.19 HUE1A 4.82 80.65 4.56 2.89 3.55 4.42 3.93 3.21 3.70 2.10 0.15 HUE1A-Curoqxqnr 4.99 82.95 4.81 3.04 3.61 4.33 4.06 2.99 3.77 2.21 0.15 HUE1B-Curoqxqnr 10.02 154.69 8.55 4.21 5.42 10.52 10.20 7.66 7.85 3.20 0.24 DH5α 1.83 21.84 1.44 1.28 1.29 0.69 0.59 0.76 1.17 1.62 2.78 Expression level (relative amount of AG100; in terms of fold change) HUE1 and its plasmid-cured mutants were wild-type mutants, we found some mutations. acrR was disrupted by the insertion of a transposon, an IS3–IS629 element, in HUE1 and group A HUE1A-Curoqxqnr (Figure 2). HUE1B-Curoqxqnr (group B) had a deletion across acrR (corresponding to amino acids from Met-1 to Leu-73 of AcrR) and acrA (corresponding to amino acids from Met-1 to Asp-106 of AcrA), in addition to the inser- tion of the IS3–IS629 element. In addition, a nucleotide deletion of cytosine at position 223 of marR caused a frameshift in HUE1, HUE1A-Curoqxqnr, and HUE1B-Curoqxqnr. in HUE1A and HUE1A-Curoqxqnr, the mRNA expression of efﬂux pumps and their regulatory genes were approximately half of those in HUE1, while those of HUE1B-Curoqxqnr were similar to those in HUE1. The mRNA expression of qnrS1 and oqxB was not signiﬁcantly different among the HUE1, HUE1A, HUE1A-Reoqxqnr, and HUE1B-Reoqxqnr strains (data not shown). Next, we determined the full DNA sequences of efﬂux pump genes (acrA, acrB, acrE, acrF, and tolC) and their regulatory genes (marR, acrS, soxS, soxR, rob, and acrR). Although most genes in May 2013 \| Volume 4 \| Article 125 \| 7 www.frontiersin.org Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. FIGURE 2 \| Schematic representation of the acrAB−acrR region in HUE1, group A mutants, and group B mutants. (A) Escherichia coli K12 MG1655 (accession no U00096). (B) HUE1 and HUE1A-Curoqxqnr (group A). (C) HUE1B-Curoqxqnr (group B). aa., Amino acids. ∗Position of primer pairs (acrART-F and acrART-R) used in RT-PCR (corresponding to amino acids 272–294 in AcrA). FIGURE 2 \| Schematic representation of the acrAB−acrR region in HUE1, group A mutants, and group B mutants. (A) Escherichia coli K12 MG1655 (accession no U00096). (B) HUE1 and HUE1A-Curoqxqnr (group A). (C) HUE1B-Curoqxqnr (group B). aa., Amino acids. EFFECTS OF EFFLUX PUMP INHIBITORS AND GENETIC ANALYSIS OF EFFLUX PUMP COMPONENTS IN THE HUE1 STRAIN AND ITS MUTANTS ∗Position of primer pairs (acrART-F and acrART-R) used in RT-PCR (corresponding to amino acids 272–294 in AcrA). CHARACTERIZATION OF KNOCKOUT MUTANTS PREPARED BY RED/ET RECOMBINATION resulted in ﬂuoroquinolone MICs that were 2- or 4-fold lower than those for HUE1 (data not shown). HUE1-wt-acrR and HUE1-wt-marR resulted in reduced mRNA expression levels of acrA and acrB, and HUE1-wt-marR also exhibited a signiﬁcant reduction in the expression of marA compared with that for HUE1 (Figure 3). We obtained a series of gene knockout mutants by Red/RT recombination. The ﬂuoroquinolone MICs of HUE1-oqxAB, HUE1-qnrS1, and HUE1-oqxAB qnrS1 were reduced by 2- or 4-fold, 16- or 32-fold, and 32- or 64-fold, as compared to those of HUE1, respectively (Table 5). HUE1-acrA, HUE1-acrB, and HUE1-acrAB demonstrated 4- or 8-fold lower MICs than HUE1. HUE1-acrA oqxAB exhibited 32- or 64-fold lower ﬂuo- roquinolone MICs and HUE1-acrA qnrS1 exhibited 64- or 128- fold lower ﬂuoroquinolone MICs than HUE1. HUE1-oqxAB did not exhibit marked changes in ﬂuoroquinolone MICs com- pared with those of HUE1, while HUE1-acrAB oqxAB demon- strated larger fold changes, ranging from 4- to 16-fold lower, than HUE1-acrAB. Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy INTRACELLULAR FLUOROQUINOLONE CONCENTRATIONS The intracellular CIP concentration in HUE1 was approxi- mately 2.3-fold lower than that in DH5α (p < 0.05; Figure 4). Intracellular CIP concentrations in HUE1A and HUE1A- Curoqxqnr were minimally, albeit signiﬁcantly, higher than those of the parental strain, HUE1 (p < 0.05). In contrast, the intra- cellular CIP concentration in HUE1B-Curoqxqnr was markedly higher than those in HUE1, HUE1A, and HUE1A-Curoqxqnr (p < 0.05), and was similar to those of HUE1-acrA oqxAB, and HUE1-acrAB oqxAB (Figure 4). HUE1-acrA and HUE1- acrAB exhibited signiﬁcantly increased intracellular CIP con- centrations compared with that for HUE1 (p < 0.05). However, the intracellular CIP concentrations of acrD-, acrF-, mdtK-, and mdfA-knockout HUE1 mutants were slightly higher, but not signiﬁcantly different from that for HUE1 (p > 0.05; data not shown). The intracellular CIP concentration in HUE1-oqxAB was also not signiﬁcantly different from that of HUE1; how- ever, HUE1-acrA oqxAB and HUE1-acrAB oqxAB exhibited a clear increase in intracellular CIP levels compared to that in HUE1-acrA or HUE1-acrAB (p < 0.05). HUE1-tolC showed the highest intracellular CIP concentrations (Figure 4), which were not altered by additional knockout of acrAB or oqxAB Knockout of tolC markedly decreased ﬂuoroquinolone MICs by 64- or 128-fold, and the MICs of the tolC knockout mutant (HUE1-tolC) were not altered by additional knockout of acrAB or oqxAB (HUE1-tolC acrAB or HUE1-tolC oqxAB). HUE1- tolC qnrS1 showed the lowest ﬂuoroquinolone MICs, ranging from 512- to 2056-fold lower than those of HUE1. Mutants in which other efﬂux pump-associated genes, acrF, acrD, mdtK, and mdfA, were knocked out showed 2- to 4-fold lower ﬂu- oroquinolone MICs than did HUE1 (Table 5). The mutant derived from HUE1B-Curoqxqnr with knockout acrB (HUE1B- Curoqxqnr-acrB) did not show altered ﬂuoroquinolone MICs, when compared to HUE1B-Curoqxqnr. Transformation of HUE1 with plasmids encoding wild-type acrR or wild-type marR (HUE1-wt-acrR and HUE1-wt-marR) May 2013 \| Volume 4 \| Article 125 \| 8 Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. Table 5 \| Fluoroquinolones susceptibilities of Red/ET recombination mutants derived from HUE1. INTRACELLULAR FLUOROQUINOLONE CONCENTRATIONS Strain MIC (mg/L) MIC reduction (-fold)a CIP LVX URX STX NOR HUE1 4 8 4 2 16 – HUE1- oqxAB 2 2 1 0.5 8 2 or 4 HUE1-qnrS1 0.25 0.25 0.125 0.125 0.5 16 or 32 HUE1-oqxAB qnrS1 0.06 0.125 0.06 0.06 0.5 32 or 64 HUE1-acrA 1 1 0.5 0.25 4 4 or 8 HUE1-acrA oqxAB 0.125 0.125 0.125 0.03 0.25 32 or 64 HUE1-acrA qnrS1 0.06 0.06 0.03 0.03 0.25 64 or 128 HUE1-acrB 1 1 0.5 0.25 4 4 or 8 HUE1-acrAB 1 1 0.5 0.25 4 4 or 8 HUE1-acrAB oqxAB 0.125 0.125 0.125 0.03 0.25 32 or 64 HUE1-acrAB qnrS1 0.06 0.06 0.03 0.03 0.5 32–128, HUE1-tolC 0.06 0.125 0.06 0.015 0.25 64 or 128 HUE1-tolC acrAB 0.06 0.125 0.125 0.03 0.25 32 or 64 HUE1-tolC oqxAB 0.06 0.125 0.125 0.03 0.25 32 or 64 HUE1-tolC qnrS1 0.002 0.004 0.008 0.002 0.015 512–2056 HUE1B-Curoqxqnr 0.008 0.008 0.008 0.004 0.03 512 or 1024 HUE1B-Curoqxqnr-acrB 0.008 0.008 0.008 0.004 0.03 512 or 1024 HUE1-acrF 2 2 2 1 8 2 or 4 HUE1-acrD 2 4 2 1 8 2 HUE1-mdtK 2 4 1 1 8 2 or 4 HUE1-mdfA 2 4 2 1 8 2 or 4 aRelative value of HUE1. Table 5 \| Fluoroquinolones susceptibilities of Red/ET recombination mutants derived from HUE1. known to be the most important chromosomally encoded efﬂux pump related to ﬂuoroquinolone resistance in Escherichia coli (Poole, 2005). During plasmid curing, a series of HUE1 group B mutants showed markedly lower ﬂuoroquinolone MICs and higher intracellular CIP concentrations than did HUE1 and its group A mutants, whereas these mutants had mRNA expression levels of efﬂux pumps similar to those of HUE1. We found that group B mutants possessed deletions across parts of acrA. AcrA is an outer membrane protein that binds to AcrB (which plays a role in ﬂuoroquinolone excretion) and TolC (an outer mem- brane factor) (Kobayashi et al., 2001; Elkins and Nikaido, 2003; Ge et al., 2009). The deleted region of AcrA in group B mutants involves an α-β barrel (53–61 amino acid residues), which forms the AcrB-binding domain, and a part of an α-helical hairpin (99– 172 amino acid residues), which forms the TolC-binding domain (Ge et al., 2009). Fluoroquinolone MICs of the acrA-knockout HUE1 mutant (HUE1-acrA) were similar to those of HUE1B- Reoqxqnr (a group B mutant that possesses oqxAB and qnrS1), HUE1-acrB, and HUE1-acrAB. INTRACELLULAR FLUOROQUINOLONE CONCENTRATIONS Moreover, the intracellular CIP concentration in HUE1B-Curoqxqnr (a group B mutant that had been cured of oqxAB and qnrS1) was not different from that in HUE1B-Curoqxqnr-acrB. These results suggested that reduction of ﬂuoroquinolone MICs in group B strains was caused by functional disruption of AcrAB–TolC, which did not allow cooperation between AcrA and AcrB and TolC, indicat- ing that ﬂuoroquinolone resistance in HUE1 is partially due to AcrAB–TolC. (HUE1-tolC acrAB or HUE1-tolC oqxAB; data not shown). Addition of CCCP resulted in increased intracellular CIP con- centrations in all strains, with levels ranging from 21.8 ± 2.3 to 25.8 ± 5.2 ng/mg wet cells; there were no statistical differences (p > 0.05, data not shown). www.frontiersin.org DISCUSSION k h It is known that QRDR mutations are required for the ﬂu- oroquinolone MICs to exceed the breakpoints (Jacoby, 2005; Hansen et al., 2007; Cesaro et al., 2008; Zhao et al., 2010). To our knowledge, a ﬂuoroquinolone-resistant strain without QRDR mutations has been reported only in a laboratory-derived resistant mutant strain of Acinetobacter baumannii that had been selected by in vitro ﬂuoroquinolone exposure experiments; how- ever, the resistance mechanism of this strain remains unclear (Chopra and Galande, 2011). In the current study, we demon- strated the ﬂuoroquinolone-resistance mechanisms in an E. coli clinical isolate, HUE1, which lacks mutations in QRDRs (Sato et al., 2011). HUE1 possessed two PMQRs on different plasmids, pHFQ1 (harboring oqxAB) and pHFQ2 (harboring qnrS1). Transformation experiments using DH5α as a host clearly showed that oqxAB and qnrS1 did in fact contribute to ﬂuoro- quinolone resistance. However, transformants containing both plasmids still did not exceed the MIC breakpoints. It has been suggested that some efﬂux pumps are associ- ated with ﬂuoroquinolone resistance in HUE1. AcrAB-TolC is May 2013 \| Volume 4 \| Article 125 \| 9 www.frontiersin.org www.frontiersin.org Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. FIGURE 3 \| mRNA expression levels of marA, acrA, and acrB in HUE1 and its acrR or marR transformants. The values indicate the expression levels of indicated genes relative to those in HUE1 (fold-changes). White, HUE1; gray, HUE1-pUC19; diagonal line, HUE1-wt-acrR (HUE1 strain transformed wild-type of acrR); black, HUE1-wt-marR (HUE1 strain transformed wild-type marR). ∗p < 0.05 compared to HUE1. Data represent the mean ± standard deviation values calculated from at least three independent experiments. FIGURE 4 \| Intracellular concentrations of CIP in HUE1 and its mutants obtained from plasmid curing and Red/ET recombination. Data represent the mean ± standard deviation values of the results from three independent experiments. ∗p < 0.05 compared to HUE1. †p < 0.05 between HUE1-acrA and HUE1-acrA oqxAB and between HUE1-acrAB and HUE1-acrAB oqxAB, respectively. A AB T lC i i t ll d b l ti t (i l b l ti t f A AB (K t l 2008) I t d ti transformed wild-type of acrR); black, HUE1-wt-marR (HUE1 strain transformed wild-type marR). ∗p < 0.05 compared to HUE1. Data represent the mean ± standard deviation values calculated from at least three independent experiments. FIGURE 3 \| mRNA expression levels of marA, acrA, and acrB in HUE1 and its acrR or marR transformants. DISCUSSION k h This will require further investigation. AcrAB and OqxAB. Although OqxAB minimally contributed to ﬂuoroquinolone resistance in HUE1, it may largely contribute to supplementation of AcrAB functions. TolC is also required for the function of several efﬂux pumps (other than AcrAB), such as AcrEF and AcrD, in S. enterica serovar Typhimurium (Horiyama et al., 2010). Therefore, overexpression of TolC should also be an important element in the ﬂuoroquinolone-resistance mechanism in HUE1 cells, as veriﬁed by our ﬁnding of the greatest reduction in ﬂuoroquinolone MICs by knockout of tolC. Other chromosomally encoded efﬂux pumps, such as AcrEF– TolC and AcrD, belonging to the RND family, MdtK (also known as YdhE or NorE), belonging to the multidrug and toxic com- pound extrusion family, and MdfA, belonging to the major facilitator superfamily, also excrete ﬂuoroquinolones (Nishino and Yamaguchi, 2001; Poole, 2005; Nishino et al., 2006). The mRNA expression of the related genes were also higher in HUE1, and HUE1 knockout mutants of these genes exhibited reduced ﬂuoroquinolone MICs compared to HUE1, although the inﬂu- ence of these knockouts was weaker than that of AcrAB. These results indicated that the ﬂuoroquinolone-resistance mechanism in HUE1 was most likely associated with the overexpression of AcrAB–TolC, with minor contributions of other chromosomally encoded efﬂux pumps, in addition to 2 PMQRs, viz., oqxAB and qnrS1. In conclusion, this study is the ﬁrst to reveal the existence of a ﬂuoroquinolone-resistance mechanism that is mediated with- out QRDR mutations in the E. coli clinical isolate, HUE1. This mechanism involved the concomitant presence of oqxAB and qnrS1 and was associated with the overexpression of AcrAB, other chromosomally encoded efﬂux pumps, and TolC. HUE1 was identiﬁed as phylogenic group A−O125:H37−ST48. This clonal group has also been isolated from humans and animals world- wide (Jorgensen et al., 2010; Bortolaia et al., 2011; Croxall et al., 2011). However, the susceptibilities of ST48 strains to ﬂuoro- quinolone compounds vary and have not yet been deﬁned in all cases. Therefore, further epidemiological and molecular biology analyses of the ST48 lineage are required. OqxAB belongs to the RND-type efﬂux pump family, as does AcrAB (Hansen et al., 2004). Although OqxAB did not cause marked changes in ﬂuoroquinolone MICs and intracellular CIP concentrations, we observed more distinct changes following acrAB deletion. OqxAB requires TolC to excrete AMP, CHL, and OLA, as does AcrAB (Hansen et al., 2004). REFERENCES Susceptibility Testing. CLSI M100– S21. Wayne, PA: Clinical and Laboratory Standards Institute. Edgar, R., Friedman, N., Molshanski- Mor, S., and Qimron, U. (2012). Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. Appl. Environ. Microbiol. 78, 744–751. ESBL-producing enterobacterial isolates. J. Antimicrob. Chemother. 60, 394–397. Susceptibility Testing. CLSI M100– S21. Wayne, PA: Clinical and Laboratory Standards Institute. ESBL-producing enterobacterial isolates. J. Antimicrob. Chemother. 60, 394–397. Bin Kim, H., Wang, M. H., Park, C. H., Kim, E. C., Jacoby, G. A., and Hooper, D. C. (2009). oqxAB encoding a multidrug efﬂux pump in human clinical isolates of enter- obacteriaceae. Antimicrobial Agents Chemother. 53, 3582–3584. Cesaro, A., Bettoni, R. R., Lascols, C., Mérens, A., Soussy, C. J., and Cambau, E. (2008). Low selec- tion of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes. J. Antimicrob. Chemother. 61, 1007–1015. Conrad, S., Oethinger, M., Kaifel, K., Klotz, G., Marre, R., and Kern, W. V. (1996). GyrA mutations in high- level ﬂuoroquinolone-resistant clinical isolates of Escherichia coli. J. Antimicrob. Chemother. 38, 443–455. Elkins, C. A., and Nikaido, H. (2003). Chimeric analysis of AcrA func- tion reveals the importance of its C-terminal domain in its interac- tion with the AcrB multidrug efﬂux pump. J. Bacteriol. 185, 5349–5356. Bortolaia, V., Larsen, J., Damborg, P., and Guardabassi, L. (2011). Potential pathogenicity and host range of extended-spectrum beta- lactamase-producing Escherichia coli isolates from healthy poul- try. Appl. Environ. Microbiol. 77, 5830–5833. Croxall, G., Hale, J., Weston, V., Manning, G., Cheetham, P., Achtman, M., et al. (2011). Molecular epidemiology of extrain- testinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospi- tal care settings. J. Antimicrob. Chemother. 66, 2501–2508. Chopra, S., and Galande, A. (2011). A ﬂuoroquinolone- resistant Acinetobacter baumannii without the quinolone resistance- determining region mutations. J. Antimicrob. Chemother. 66, 2668–2670. Ge, Q., Yamada, Y., and Zgurskaya, H. (2009). The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efﬂux pump AcrAB-TolC. J. Bacteriol. 191, 4365–4371. Breines, D. M., Ouabdesselam, S., Ng, E. Y., Tankovic, J., Shah, S., Soussy, C. J., et al. (1997). Quinolone resis- tance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoi- somerase IV. Antimicrob. Agents Chemother. 41, 175–179. DISCUSSION k h The values indicate the expression levels of indicated genes relative to those in HUE1 (fold-changes). White, HUE1; gray, HUE1-pUC19; diagonal line, HUE1-wt-acrR (HUE1 strain FIGURE 4 \| Intracellular concentrations of CIP in HUE1 and its mutants obtained from plasmid curing and Red/ET recombination. Data represent the mean ± standard deviation values of the results from three independent experiments. ∗p < 0.05 compared to HUE1. †p < 0.05 between HUE1-acrA and HUE1-acrA oqxAB and between HUE1-acrAB and HUE1-acrAB oqxAB, respectively. FIGURE 4 \| Intracellular concentrations of CIP in HUE1 and its mutants obtained from plasmid curing and Red/ET recombination. Data represent the mean ± standard deviation values of the results from three independent experiments. ∗p < 0.05 compared to HUE1. †p < 0.05 between HUE1-acrA and HUE1-acrA oqxAB and between HUE1-acrAB and HUE1-acrAB oqxAB, respectively. FIGURE 4 \| Intracellular concentrations of CIP in HUE1 and its mutants obtained from plasmid curing and Red/ET recombination. Data represent the mean ± standard deviation values of the results from three independent experiments. ∗p < 0.05 compared to HUE1. †p < 0.05 between HUE1-acrA and HUE1-acrA oqxAB and between HUE1-acrAB and HUE1-acrAB oqxAB, respectively. global activators of AcrAB (Keeney et al., 2008). Introduction of wild-type acrR or wild-type marR to HUE1 actually reduced acrA and acrB mRNA expression and reduced ﬂuoroquinolone MICs. These data suggested that overexpression of AcrAB–TolC in HUE1 was mediated by the concomitant disruptions of AcrR and MarR. However, other mechanisms are also responsible for the overexpression of AcrAB–TolC and other chromosomally AcrAB-TolC expression is controlled by several activators (i.e., soxS, marA, and rob) and repressors (i.e., acrR, marR, and soxR) (White et al., 1997; Poole, 2005; Keeney et al., 2008). HUE1 exhib- ited higher expression of acrA, acrB, tolC and these activators. In addition, HUE1 had concomitant disruption of the acrR and marR repressors. AcrR is a local repressor of AcrAB (Wang et al., 2001), and MarR is a repressor of MarA, which is one of the May 2013 \| Volume 4 \| Article 125 \| 10 Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy Fluoroquinolone resistance in non-QRDR-mutated E. coli Sato et al. encoded efﬂux pumps, since the group A series of HUE1 mutants was found to have only approximately half of the mRNA expres- sion levels of chromosomal efﬂux pump genes, including those encoding AcrAB, despite having the same disruptions in acrR and marR as HUE1. DISCUSSION k h In this study, knockout of tolC markedly decreased ﬂuoroquinolone MICs and intracellu- lar CIP concentrations and negated the effects of both acrAB and oqxAB. This ﬁnding indicated that OqxAB, in conjunction with TolC, was also involved in mediating decreased ﬂuoroquinolone susceptibility. TolC is therefore implicated in the functions of both ACKNOWLEDGMENTS The authors thank Hirotsugu Akizawa (Hokkaido University Hospital) for providing E. coli clinical isolates. This work was sup- ported in part by a Grant-in-Aid from the Japanese Ministry of Health, Labour, and Welfare (H24-Shokuhin-Ippan-008) and a grant from the Program for Developing the Supporting System for Upgrading Education and Research from the Japan Ministry of Education, Culture, Sports, Science, and Technology. REFERENCES REFERENCES Cizman, M., Orazem, A., Krizan- Hergouth, V., and Kolman, J. (2001). Correlation between increased consumption of ﬂu- oroquinolones in outpatients and resistance of Escherichia coli from urinary tract infections. J. Antimicrob. Chemother. 47, 502. Hansen, L. H., Jensen, L. B., Sorensen, H. I., and Sorensen, S. J. (2007). Substrate speciﬁcity of the OqxAB multidrug resistance pump in Escherichia coli and selected enteric bacteria. J. Antimicrob. Chemother. 60, 145–147. 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Received: 23 March 2013; accepted: 02 May 2013; published online: 24 May 2013. Received: 23 March 2013; accepted: 02 May 2013; published online: 24 May 2013. Citation: Sato T, Yokota S-i, Uchida I, Okubo T, Usui M, Kusumoto M, Akiba M, Fujii N and Tamura Y (2013) Fluoroquinolone resistance mech- anisms in an Escherichia coli isolate, HUE1, without quinolone resistance- determining region mutations. Front. Microbiol. 4:125. doi: 10.3389/fmicb. 2013.00125 Jacoby, G. A. (2005). Mechanisms of resistance to quinolones. Clin. Infect. Dis. 41, S120–S126. Nishino, K., and Yamaguchi, A. (2001). Analysis of a complete library of putative drug transporter genes in Escherichia coli. J. Bacteriol. 183, 5803–5812. Citation: Sato T, Yokota S-i, Uchida I, Okubo T, Usui M, Kusumoto M, Usui, M., Uchiyama, M., Iwanaka, M., Nagai, H., Yamamoto, Y., and Asai, T. (2009). Intracellular concentrations of enroﬂoxacin in quinolone-resistant Salmonella enterica subspecies enterica serovar Choleraesuis. Int. J. 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Copyright © 2013 Sato, Yokota, Uchida, Okubo, Usui, Kusumoto, Akiba, Fujii and Tamura. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. White, D. G., Goldman, J. D., Demple, B., and Levy, S. B. (1997). Role of the acrAB locus in organic sol- vent tolerance mediated by expres- sion of marA, soxS, or robA in Escherichia coli. J. Bacteriol. 179, 6122–6126. Poole, K. (2005). Efﬂux-mediated anti- microbial resistance. J. Antimicrob. Chemother. 56, 20–51. Kobayashi, K., Tsukagoshi, N., and Aono, R. (2001). Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by Sanchez, G. V., Master, R. N., Karlowsky, J. A., and Bordon, J. M. (2012). In vitro antimicrobial May 2013 \| Volume 4 \| Article 125 \| 12 Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy Frontiers in Microbiology \| Antimicrobials, Resistance and Chemotherapy
https://openalex.org/W3117518483	https://mel.cgiar.org/reporting/download/hash/7ff076e1f2d6a82342a7fa2a44f25e79	English	null	The Global Durum Wheat Panel (GDP): An International Platform to Identify and Exchange Beneficial Alleles	Frontiers in plant science	2,020	cc-by	19,435	Edited by: Soren K. Rasmussen, University of Copenhagen, Denmark Edited by: Soren K. Rasmussen, University of Copenhagen, Denmark Reviewed by: Monica Rodriguez, University of Sassari, Italy Benjamin Kilian, Global Crop Diversity Trust, Germany Correspondence: Filippo M. Bassi f.bassi@cgiar.org †These authors have contributed equally to this work 1 Council for Agricultural Research and Economics-Research Centre for Genomics and Bioinformatics, Fiorenzuola d’Arda, Italy, 2 Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy, 3 Council for Agricultural Research and Economics-Research Centre for Cereal and Industrial Crops, Foggia, Italy, 4 Council for Agricultural Research and Economics-Research Centre for Cereal and Industrial Crops, Bergamo, Italy, 5 Cereal Crops Research Unit, Edward T. Schafer Agricultural Research Center, United States Department of Agriculture, Agricultural Research Service, Fargo, ND, United States, 6 Agriculture Victoria, Agribio, Centre for AgriBiosciences, Bundoora, VIC, Australia, 7 School of Applied Systems Biology, La Trobe University, Bundoora, VIC, Australia, 8 School of Agriculture, Food and Wine, Faculty of Sciences, Waite Research Institute, The University of Adelaide, Adelaide, SA, Australia, 9 Department of Field Crops, Faculty of Agriculture, Çukurova University, Adana, Turkey, 10 Centro de Recursos Naturales Renovables de la Zona Semiárida, Departamento de Agronomía, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas, Bahía Blanca, Argentina, 11 Department of Plant Pathology, University of Minnesota, St. Paul, MN, United States, 12 Swift Current Research and Development Centre, Agriculture and Agri-Food Canada, Swift Current, SK, Canada, 13 International Center for Agricultural Research in the Dry Areas, Beirut, Lebanon, 14 State Plant Breeding Institute, University of Hohenheim, Stuttgart, Germany, 15 Plant Sciences and Crop Development Center, University of Saskatchewan, Saskatoon, SK, Canada, 16 International Maize and Wheat Improvement Center, Texcoco de Mora, Mexico Reviewed by: Monica Rodriguez, University of Sassari, Italy Benjamin Kilian, Global Crop Diversity Trust, Germany Correspondence: Filippo M. Bassi f.bassi@cgiar.org †These authors have contributed equally to this work Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 05 June 2020 Accepted: 24 November 2020 Published: 21 December 2020 Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Representative, broad and diverse collections are a primary resource to dissect genetic diversity and meet pre-breeding and breeding goals through the identiﬁcation of beneﬁcial alleles for target traits. ORIGINAL RESEARCH published: 21 December 2020 doi: 10.3389/fpls.2020.569905 The Global Durum Wheat Panel (GDP): An International Platform to Identify and Exchange Beneﬁcial Alleles Elisabetta Mazzucotelli1†, Giuseppe Sciara2†, Anna M. Mastrangelo3,4, Francesca Desiderio1, Steven S. Xu5, Justin Faris5, Matthew J. Hayden6,7, Penny J. Tricker8, Hakan Ozkan9, Viviana Echenique10, Brian J. Steffenson11, Ron Knox12, Abdoul A. Niane13, Sripada M. Udupa13, Friedrich C. H. Longin14, Daniela Marone3, Giuseppe Petruzzino3, Simona Corneti2, Danara Ormanbekova2, Curtis Pozniak15, Pablo F. Roncallo10, Diane Mather8, Jason A. Able8, Ahmed Amri13, Hans Braun15, Karim Ammar16, Michael Baum13, Luigi Cattivelli1, Marco Maccaferri2, Roberto Tuberosa2 and Filippo M. Bassi13* From 2,500 tetraploid wheat accessions obtained through an international collaborative effort, a Global Durum wheat Panel (GDP) of 1,011 genotypes was assembled that captured 94–97% of the original diversity. The GDP consists of a wide representation of Triticum turgidum ssp. durum modern germplasm and landraces, along with a selection of emmer and primitive tetraploid wheats to maximize diversity. GDP accessions were genotyped using the wheat iSelect 90K SNP array. Among modern durum accessions, breeding programs from Italy, France and Central Asia provided the highest level of genetic diversity, with only a moderate decrease in genetic diversity observed across nearly 50 years of breeding (1970–2018). Further, the breeding programs from Europe had the largest sets of unique alleles. LD was lower in the landraces (0.4 Mbp) than in modern germplasm (1.8 Mbp) at r2 = 0.5. ADMIXTURE analysis of modern germplasm deﬁned a minimum of 13 distinct genetic clusters (k), which could be traced to the breeding program of origin. Chromosome regions putatively subjected to strong selection pressure were identiﬁed from ﬁxation index (Fst) and diversity reduction index (DRI) metrics in pairwise comparisons among Received: 05 June 2020 Accepted: 24 November 2020 Published: 21 December 2020 INTRODUCTION by Nazareno Strampelli in 1910; Scarascia Mugnozza, 2005; Dexter, 2008; Royo et al., 2009; Taranto et al., 2020) and the “best × best” strategy traditionally used by breeders to drive the genetic gain (Hoisington et al., 1999; Maccaferri et al., 2003; van Ginkel and Ortiz, 2018) are the two main causes of this phenomenon. Genetic erosion of the durum wheat cultivated gene-pool in comparison with wild relatives and landraces has been reported, analogously to other crop species (Tanksley and McCouch, 1997; Gur and Zamir, 2004; Raman et al., 2010; Royo et al., 2010; Laidò et al., 2013; Kabbaj et al., 2017; Maccaferri et al., 2019), and it represents a real concern for breeders as it might lead to a lack of novel beneﬁcial alleles for selection, yield stagnation, and/or increased susceptibility to biotic and abiotic stresses. Therefore, breeders are devoting increasing resources and eﬀort to identify beneﬁcial alleles and traits from novel germplasm sources to reinvigorate their programs. Indeed, pre- breeding activities have been pursued by international programs at ICARDA (Zaïm et al., 2017; Bassi et al., 2019; Robbana et al., 2019; El Haddad et al., 2020) and CIMMYT (Singh et al., 2018; Ledesma-Ramírez et al., 2019), and by national research institutes to introgress beneﬁcial alleles from landraces and wild relatives, in parallel to international initiatives which aim to identify, collect, conserve and use the wild cousins of some of the most important food crops, as the CWR project “Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives1. Population structure and genetic diversity have been studied in several modern and landrace collections of durum wheat. Many studies have focused on panels from a restricted country/area such as landraces from Southern Italy (Marzario et al., 2018), Iran (Talebi and Fayaz, 2016), Spain (Giraldo et al., 2016), Tunisia (Robbana et al., 2019; Slim et al., 2019), Turkey and Syria (Baloch et al., 2017), Palestine, Jordan and Israel (Abu-Zaitoun et al., 2018), or speciﬁc breeding programs (N’Diaye et al., 2018). Others have considered durum wheat collections of wider origin encompassing a few hundred entries. Among the earliest studies reporting on assembling international and diverse panels of mainly elite durum lines and cultivars, Maccaferri et al. (2005, 2006, 2010, 2011), Reimer et al. (2008) and Laidò et al. (2013) all reported on the genome-wide molecular diversity and LD-decay rate estimated with SSR and DArTTM markers. Citation: Mazzucotelli E, Sciara G, Mastrangelo AM, Desiderio F, Xu SS, Faris J, Hayden MJ, Tricker PJ, Ozkan H, Echenique V, Steffenson BJ, Knox R, Niane AA, Udupa SM, Longin FCH, Marone D, Petruzzino G, Corneti S, Ormanbekova D, Pozniak C, Roncallo PF, Mather D, Able JA, Amri A, Braun H, Ammar K, Baum M, Cattivelli L, Maccaferri M, Tuberosa R and Bassi FM (2020) The Global Durum Wheat Panel (GDP): An International Platform to Identify and Exchange Beneﬁcial Alleles. Front. Plant Sci. 11:569905. doi: 10.3389/fpls.2020.569905 December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 1 Mazzucotelli et al. Genetic Diversity and Allele History decades of release and breeding programs. Clusters of putative selection sweeps (PSW) were identiﬁed as co-localized with major loci controlling phenology (Ppd and Vrn), plant height (Rht) and quality (gliadins and glutenins), underlining the role of the corresponding genes as driving elements in modern breeding. Public seed availability and deep genetic characterization of the GDP make this collection a unique and ideal resource to identify and map useful genetic diversity at loci of interest to any breeding program. Keywords: durum wheat, genetic diversity, selection sweep, breeding history, wheat initiative INTRODUCTION More recently, germplasm collections have been characterized with the Illumina iSelect 90K SNP (Maccaferri et al., 2016; Mangini et al., 2018; Saccomanno et al., 2018) and Durum wheat [Triticum turgidum L. ssp. durum (Desf.) Husn.] is the 10th most important crop worldwide with an annual production of over 40 million tons (Sall et al., 2019). It provides the raw material for semolina, pasta, couscous, burghul and several other dishes of the Mediterranean tradition (Oliveira et al., 2012). Durum wheat evolved from domesticated emmer wheat, T. turgidum ssp. dicoccum (Schrank ex Schübl.) Thell., which originated from wild emmer wheat, T. turgidum ssp. dicoccoides (Körn. ex Asch. & Graebn.) Thell. in the Fertile Crescent approximately 10,000 years ago (Ozkan et al., 2002; Dubcovsky and Dvorak, 2007). Thus, three distinct phases can be identiﬁed in the human-driven tetraploid wheat evolution process: (i) domestication (from wild to domesticated emmer wheat), (ii) continued evolution under domestication (from domesticated emmer wheat to durum wheat landraces) and (iii) improvements achieved by modern breeding (from landraces to modern durum wheat varieties) (Maccaferri et al., 2019). As a consequence of this evolution, four mega-germplasm groups of tetraploid wheat can be deﬁned: tetraploid wild relatives, tetraploid primitive wheats (domesticated and cultivated), durum wheat landraces and modern durum wheat varieties. During the second evolution phase, the transition from the domesticated form of emmer to durum landraces underwent strong selection pressure by ancient farmers (Tanksley and McCouch, 1997). Modern breeding has accelerated this process by artiﬁcially crossing “best by best” and selecting for “the best” with impressive genetic gains being realized, resulting in the development of improved varieties accumulating beneﬁcial alleles (Slafer et al., 1994; Borrelli and Trono, 2016; van Ginkel and Ortiz, 2018). Genetic gain is typically quantiﬁed as the slope of the regression between yield and year of release of varieties. A genetic gain of 0.3–1.2% per year has been recorded for durum wheat over the last century in diﬀerent growing regions (e.g., Giunta et al., 2007; Royo et al., 2008; Clarke et al., 2010; Bassi and Nachit, 2019; Mondal et al., 2020) and often associated with variations in morpho-physiological traits, such as a shift toward earlier ﬂowering and a reduced plant height, with a corresponding increase in harvest index (e.g., De Vita et al., 2007; Royo et al., 2007; Isidro et al., 2011; Bassi and Nachit, 2019). 1https://www.cwrdiversity.org/ Frontiers in Plant Science \| www.frontiersin.org INTRODUCTION subjected to GWAS for response to diseases, root morphology, canopy traits related to phenology, photosynthesis and grain yield potential (e.g., Maccaferri et al., 2010, 2016; Canè et al., 2014; Condorelli et al., 2018). Similarly, Kabbaj et al. (2017) used a mixed set of modern lines and landraces to deﬁne the genetic diversity and origin of modern durum wheat as well as to identify loci controlling resistance to insect pests and tolerance to heat stress (Bassi et al., 2019; El Hassouni et al., 2019). The largest study to date considered a collection of 429 USDA-ARS durum entries including cultivars and landraces from 64 countries. This collection was analyzed with 6,538 polymorphic SNPs (Chao et al., 2017) from the Illumina iSelect wheat 9K array (Cavanagh et al., 2013). More recently, a deeper study of genetic diversity was carried out for the Tetraploid wheat Global Collection (TGC) consisting of 1,856 single-seed puriﬁed gene bank entries chosen to comprehensively explore the diversity in tetraploid wheat from durum landraces through domesticated and wild emmer (Wang et al., 2014) in combination with the availability of the reference genome assembly of the cultivar ‘Svevo’ (Maccaferri et al., 2019). g y Genetic diversity is not necessarily considered as relevant per se. Rather, with advances in genetics, genomics and functional genomics (Tuberosa and Pozniak, 2014), researchers and breeders are increasingly targeting speciﬁc genomic regions known to be relevant, with the objective to improve the exploitable and useful diversity (Kabbaj et al., 2017; N’Diaye et al., 2018). Accordingly, developing a detailed knowledge at the molecular level of historical loss of diversity events, together with the identiﬁcation of successful allelic combinations progressively accumulated over repeated breeding cycles, are instrumental for a more eﬀective management of breeding programs (Pfeiﬀer et al., 2001). With this aim, the international durum wheat research community met in Bologna, Italy, in 2015 under the umbrella of the Expert Working Group on Durum Wheat Genomics and Breeding, as part of the Wheat Initiative2, to take joint action toward the identiﬁcation of beneﬁcial alleles and to make them available for breeding programs and pre-breeding eﬀorts. The result of this international call to action is presented here under the name of the Global Durum wheat Panel (GDP). INTRODUCTION This panel was designed with the aim of capturing most of the readily exploitable genetic diversity, sharing it freely to facilitate research discoveries, and ultimately providing a rapid mean to exchange useful alleles worldwide. This article describes the germplasm composition and genetic structure of the GDP to provide the basic knowledge needed to support its international phenotypic characterization and exploitation. From this ﬁrst multiplication, a total of 762 entries produced enough seed for distribution to 28 collaborators under the name of GDP version 1 (GDPv1-19), which substantially included all T. durum lines (modern, EPO, and landraces germplasm) (Supplementary Table S3). In the 2018–2019 season, a second and ﬁnal multiplication cycle was conducted to produce enough seed of 976 entries to generate sets of 50 seeds per entry, ready to sow by 21 requesting partners. These sets were distributed under the name of GDP version 2 (GDPv2-20) (Supplementary Table S3). Unfortunately, some entries were lost during multiplication due to excessive susceptibility to yellow rust races in Lebanon. Additional sets remain available for request and distribution under SMTA at this link: http: //indms.icarda.org/. Furthermore, 42 additional entries were included in GDPv2-20, mostly representing recently released European varieties and T. durum lines carrying introgressions of Fhb1 developed by Boku University (Prat et al., 2017; Supplementary Table S3). INTRODUCTION However, the positive yield trend has often been reached at the cost of eroding genetic diversity within elite gene pools (Fernie et al., 2006; Bassi and Nachit, 2019). The limited number of landraces that were used as founder lines of the modern gene pool (e.g., the ﬁrst modern durum breeding program spearheaded December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 2 Genetic Diversity and Allele History Mazzucotelli et al. (SMTA, Noriega et al., 2019) to allow full exploitation for breeding and research. This initial set of germplasm was deﬁned as the Durum Wheat Reference Collection (DWRC, Supplementary Table S2) and grown in the 2015–2016 season at the ICARDA experimental farm in Terbol, Lebanon. The DWRC included 1,541 T. turgidum ssp. durum modern breeding accessions (cultivars, varieties and elite lines) from 49 countries/programs, an evolutionary population set from INRA France of 180 entries (Evolutionary Pre-breeding pOpulation, EPO, David et al., 2014), 416 T. turgidum ssp. durum landraces obtained from 48 countries, and 366 wild and primitive tetraploids from 37 countries (T. turgidum ssp. dicoccoides and dicoccum, turgidum, turanicum, polonicum, carthlicum, respectively). Each entry was planted in two rows of 2 m in length under supplemental irrigation. Fungicide and fertilizer were provided in-season, following optimal local management practices. From each plot a single tiller was selected and tagged at ﬂowering based on spike size, phenology and shape to be representative of most plants within the same plot. From this tiller, a leaf sample was collected for initial molecular screening. At maturity, the spike of the tagged tiller was harvested and used for advancement. In the 2016–2017 season at the same ﬁeld station, 10 seeds from each spike were planted in rows of 0.5 m in length. Irrigation and chemical treatments were used to maximize productivity. Using the initial molecular data, a subset of approximately 1,000 entries were selected and deﬁned as the Global Durum wheat Panel (GDP). The whole row was bulk-harvested and used for further advancement. In the 2017–2018 season, each entry was planted in plots of 6 m2 at the American University of Beirut (AUB) experimental farm in Lebanon. Fungicide, irrigation and fertilizer were applied in order to maximize productivity. Plots were visually inspected for homogeneity and oﬀ-types were manually rouged. 2www.wheatinitiative.org Plant Materials A total of 2,503 accessions of tetraploid wheat were obtained from 25 worldwide partners representing institutions, universities, gene banks and private companies (Supplementary Table S1), all exchanged under the Standard Material Transfer Agreement DNA Extraction and Genotyping The initial molecular screening of the DWRC was performed by sending one leaf from each selected tiller to LGC Genomics (United Kingdom) for DNA extraction and subsequent analyses. Ninety-four KASP R⃝markers (Supplementary Table S2) were December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 3 Genetic Diversity and Allele History Mazzucotelli et al. selected because evenly distributed along the genome and highly polymorphism (Kabbaj et al., 2017), including markers tagging important loci: PpdA1, VrnA1, and RhtB1. Accessions with more than 50% missing data were discarded, as well as markers which were monomorphic or detected multiple loci (gene calls with multiple allelic classes and heterozygous calls at high frequency). decade of release (ﬁve decades considered: ’70–’80, ’81–’90, ’91–’00, ’01–’10, and ’11–’18). Because the year of release was not available for elite lines included in the GDP, the year in which the cross was performed was used to estimate the year of release by adding 10 years. Polymorphic SNP datasets were selected according to the set ﬁltering for minor allele frequency (MAF) > 5% and pruning at r2 < 0.99. Lines selected to be part of the GDP were genotyped using the Illumina iSelect 90K SNP array technology (Wang et al., 2014) at the USDA-ARS Small Grain Genotyping Laboratory, Fargo, ND, United States. A pool of three seeds originating from the single spike selected in 2015–2016 were sown in Jiﬀy pots; 10 days old leaves were collected and DNA extracted using the NucleoSpin Plant II kit (Macherey-Nagel) according to manufacturer’s instructions. The raw data (Theta/R) from single genotyping experiments was exported from GenomeStudio software (Illumina Ltd.) and jointly analyzed for cluster assignment and genotype calling using a custom script as described in Maccaferri et al. (2019). The script parameters were d = 3, to call samples only within three standard deviations from a known cluster position, and r = 0.8, minimum conﬁdence score that the sample belonged to the cluster to which it was assigned versus the next closest cluster. Stepwise data curation was conducted on polymorphic SNP markers. First, markers with minor alleles present in fewer than three genotypes were discarded. Second, the remaining markers were ﬁltered to retain SNPs with a unique map position in the available genetic maps (Maccaferri et al., 2015, 2019), and with the marker sequences aligned to a single position along the Svevo reference genome RefSeq V1.0 (Maccaferri et al., 2019). DNA Extraction and Genotyping After merging adjacent peaks, the index distribution (Jordan et al., 2015) was re- calculated and the 95th percentile was chosen as the index- speciﬁc signiﬁcance threshold. DNA Extraction and Genotyping Third, those markers showing multiple hits along the genome were checked for linkage disequilibrium (LD) against the hypothetical nearby mapped markers, and assigned a unique position based on the highest r2 (above a 0.3 threshold) with the putatively contiguous markers. SNP imputation was performed using Beagle 5 software using default parameters (Browning et al., 2018). The imputation accuracy was measured at 98.6% by running 1,000 replicates of randomly masked 1% of the called genotypes (Nothnagel et al., 2009; Hancock et al., 2012). Using the software PLINK (Chang et al., 2015), redundant markers were pruned based on genome wide linkage disequilibrium set at r2 = 0.99 and merged into one unique SNP call. Moreover, three additional pruned hapmaps were produced selecting a single SNP among those with r2 of 0.8, 0.5 and 0.3 to run the population structure analysis. Genetic diversity among and within populations was calculated by AMOVA, ﬁxation index (Fst, Wright, 1965) and the polymorphism information content (PIC, Botstein et al., 1980). The within populations total number of polymorphic loci (N), Nei’s gene diversity (Nei, 1973), and mean number of pairwise diﬀerences were calculated, and signiﬁcance was determined based on LSD at P < 0.05. Population diﬀerentiation was assessed based on Nei’s genetic distance (Nei, 1972) and population pairwise Fst. All values were derived using the Arlequin 3.5 software (Excoﬃer and Lischer, 2010), and signiﬁcance levels for variance components and Fst statistics were estimated based on 10,000 and 1,000 permutations, respectively. p p y Furthermore, single locus analyses of genetic diversity across the whole genome were conducted to identify genomic regions putatively aﬀected by human-driven selection sweeps. Signals of putative selection sweeps were assessed using a hapmap pruned for r2 < 0.99 calculating two diﬀerent indices: Fst was estimated by Arlequin 3.5 software, and the diversity reduction index (DRI) was calculated using the modiﬁed ROD formula presented in Maccaferri et al. (2019). To reduce spurious signals due to diﬀerent coalescence time between SNPs, the raw single SNP-based results were smoothed by averaging with a sliding window of 15 SNPs with a one-marker step. Signiﬁcance of selection signals was assessed in a two-step procedure. In the ﬁrst step, signal peaks falling in the top 10% percentile of the distribution were identiﬁed. Additional neighboring signals were merged into the one representing the highest value, considering as neighbors loci falling within a physical distance lower than the LD. Population Structure Analysis and Selection of the GDP Collection A preliminary population stratiﬁcation analysis was carried out on the DWRC panel using a curated set of 88 KASP(R) markers. The GDP set was then re-stratiﬁed using the Illumina 90k SNP genotyping data and three possible pruned hap- maps (r2 set at 0.3, 0.5, and 0.8) were considered in order to optimize the trade-oﬀbetween uniformity of genomic sampling and informativeness. Based on the analysis results, the pruned SNP-set at r2 = 0.5 was used for all subsequent population structure analyses. For both the DWRC and GDP, the population structure was estimated by the model-based likelihood method ADMIXTURE optimized using the block relaxation algorithm and the quasi-Newton convergence acceleration method and q = 3 secants (Alexander et al., 2009), as well as by means of Ward’s clustering of Nei’s genetic distances, using the Frontiers in Plant Science \| www.frontiersin.org Identiﬁcation and Clustering of Putative Selection Sweep (PSW) Signals poppr v. 2.8.3 and adegenet packages of R (Jombart, 2008; Kamvar et al., 2014; R Core Team, 2016). For both methods, the sub-population membership was deﬁned for k values increasing from 2 to 20. The parameters used to deﬁne the optimal number of clusters were ADMIXTURE’s cross-validated error rate and minimum group size. Lines with strong admixture were deﬁned as those showing less than 30% identity (membership) with any ancestry in the model-based likelihood analysis. Because the GDP is a selected sub-set of the initial DWRC panel, the population stratiﬁcation was ﬁrst used to deﬁne the most representative DWRC entries to be included in the GDP, and secondly to deﬁne what degree of genetic diversity was lost because of the sub- sampling process. Pairwise similarity estimated as identity-by- state (IBS) was also calculated for the DWRC population to ﬁlter for duplicated/highly similar entries using TASSEL5 software (Bradbury et al., 2007). To select the subset of DWRC entries that composed the GDP the following procedures were followed. First, genotypes representing historical founders, parents of mapping populations, or known germplasm carrying interesting alleles/phenotypic traits were included, while the name and pedigree were inspected and compared to the similarities deﬁned at the molecular level (IBS-GS matrix) to discard duplicated entries with >0.95 similarity (only one entry was retained per group). The remaining entries were classiﬁed into six groups, ﬁve of which were deﬁned by genetic structure at k of 5, and one extra split to incorporate the EPO set, which was clearly diﬀerentiated from the other groups. The GDP collection was then assembled through a stratiﬁed-sampling method, therefore choosing representative entries from each main Ward’s cluster and sub-clusters, depending on each subgroup/subspecies being considered and chosen in order to maximize the number of sub-clusters being considered for GDP sampling. Genotypes with low average genetic similarity to other entries (rare haplotypes) were also chosen. The genetic diversity level present in the two collections was compared to conﬁrm that no major genetic diversity losses occurred after sampling the GDP from the DWRC. The Shannon-Wiener’s diversity index, Nei’s expected heterozygosity, allelic evenness (Shannon, 1948; Nei, 1978; Smith and Wilson, 1996), MAF, and the site frequency spectrum (SFS) distribution were assessed at the locus level both in the DWRC and GDP based on the 88 KASP markers. Diversity indexes analyses were conducted using the “locus_table” and “poppr” function of the poppr R package (Kamvar et al., 2014). Genetic Diversity Within the GDP and Putative Signal of Selection Sweeps Genetic diversity and population diﬀerentiation within the GDP, both at the genome-wide and at the single-locus level, were assessed within and between populations deﬁned according to passport data provided by contributors or retrieved from GRIS (Genetic Resources Information System for Wheat and Triticale) through www.wheatpedigree.net. Accessions of wild emmer, primitive cultivated sub-species, and durum landraces were classiﬁed on the basis of the country of collection, whereas modern durum germplasm (cultivars, varieties and elite lines) were grouped based on the breeding program of origin and December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 4 Mazzucotelli et al. Genetic Diversity and Allele History Panel The original DWRC was comprised of 2,503 accessions that were genotyped with 94 KASP(R) markers (Supplementary Table S2). The curation process yielded a ﬁnal set of 2,493 accessions (99.1%), each with 88 (93.6%) reliable KASP(R) marker proﬁles. Population structure assessed by ADMIXTURE (Supplementary Figure S1) highlighted three subsets at k = 3: (i) a group including T. turgidum spp. dicoccum and dicoccoides, (ii) a second group including modern durum wheat germplasm and (iii) a third group comprising modern North American germplasm together with most durum landraces and accessions of the primitives T. turgidum spp. turgidum, turanicum and polonicum as durum-related sub-species. At k = 4 the North American modern germplasm was separated from landraces and the mentioned primitive subspecies. Finally, at k = 5 the group of the modern durum wheat germplasm was further subdivided in two groups: the ﬁrst one tracing its ancestry to the CIMMYT breeding program, and the second one composed of the Southern European germplasm and those entries with ancestry from the ICARDA breeding program. The structure of the population was conﬁrmed using bootstrapped Ward’s clustering (Supplementary Figure S2). A total of 398 genotypes represented identical entries contributed by multiple partners. The remaining entries were divided into six groups: ﬁve deﬁned by genetic structure at k = 5 and one additional group to incorporate the EPO set. When each of these subsets was subjected to population structure assessment based on Ward’s clustering, the sub- clustering concurred with the clustering computed on the whole DWRC and a detailed picture of group diﬀerentiation based on geographic origin was revealed. The entries to be included in the GDP were then identiﬁed based on the Ward’s clustering using a stratiﬁed-sampling method. Following the criteria deﬁned in Material and Methods, three groups of durum wheat modern germplasm were selected (Supplementary Figure S3): (i) CIMMYT- and ICARDA-derived genetic materials, and modern semi-dwarf and vernalization-insensitive Identiﬁcation and Clustering of Putative Selection Sweep (PSW) Signals Detection of putative selection sweep (PSW) signals was based on genome-wide Fst and DRI metrics calculated for modern vs. landraces and for pairwise groups of entries classiﬁed by decade or breeding program. PSW clusters were deﬁned as two signiﬁcant signals on the same chromosomal region in a single pair/comparison or among pairs/comparisons. Moreover, signals also partially overlapping were grouped into one cluster. The catalog of PSW was integrated with data from the literature that included major genes cloned in wheat, known QTL and the comprehensive catalog (a.k.a. QTLome) deﬁned in Maccaferri et al. (2019). LD Decay k lines mostly adapted to the Mediterranean environment for a total of 288 genotypes; (ii) 96 elite semi-dwarf durum wheat lines with photoperiod and/or vernalization sensitivity mainly developed in Canada, France, Italy, and Central Europe; (iii) 96 non-semi-dwarf durum wheat lines of diﬀerent origins. Three additional groups were selected to incorporate more genetic diversity including; (iv) 96 EPO lines (Supplementary Figure S4); (v) 192 durum wheat landraces representing the geographical distribution of the original collection (Supplementary Figure S5); and (vi) a ﬁnal group including domesticated emmer lines (96, Supplementary Figure S6), wild emmer accessions and other tetraploid primitives (96, Supplementary Figures S7, S8, respectively). A seventh group of 42 entries including recently registered European varieties and durum lines carrying Fhb1 introgressions developed at the Boku University (Austria) was also included. The ﬁnal GDP selection consisted of 1,028 accessions, 976 of which were multiplied in suﬃcient quantity and quality for seed re-distribution by ICARDA, while 42 among European varieties and accessions with Fhb1 introgressions are available from University of Bologna and Boku University, respectively (Supplementary Table S3) for a total of 1,018 entries available as seed stocks. Figure 1 shows the geographic origin of the GDP accessions. lines mostly adapted to the Mediterranean environment for a total of 288 genotypes; (ii) 96 elite semi-dwarf durum wheat lines with photoperiod and/or vernalization sensitivity mainly developed in Canada, France, Italy, and Central Europe; (iii) 96 non-semi-dwarf durum wheat lines of diﬀerent origins. Three additional groups were selected to incorporate more genetic diversity including; (iv) 96 EPO lines (Supplementary Figure S4); (v) 192 durum wheat landraces representing the geographical distribution of the original collection (Supplementary Figure S5); and (vi) a ﬁnal group including domesticated emmer lines (96, Supplementary Figure S6), wild emmer accessions and other tetraploid primitives (96, Supplementary Figures S7, S8, respectively). A seventh group of 42 entries including recently registered European varieties and durum lines carrying Fhb1 introgressions developed at the Boku University (Austria) was also included. The ﬁnal GDP selection consisted of 1,028 accessions, 976 of which were multiplied in suﬃcient quantity and quality for seed re-distribution by ICARDA, while 42 among European varieties and accessions with Fhb1 introgressions are available from University of Bologna and Boku University, respectively (Supplementary Table S3) for a total of 1,018 entries available as seed stocks. Figure 1 shows the geographic origin of the GDP accessions. LD Decay k that the sampling process that originated the GDP caused a 3– 6% loss of the initial DWRC diversity. The SFS (Supplementary Figure S10) showed that the distribution of the allele frequencies in the GDP is comparable to that observed in the initial DWRC, except for an appreciable decrease in three rare allele frequency classes (MAF: 0.05–0.10, 0.10–0.15, and 0.35–0.40) and a corresponding increase for three high frequency classes (MAF: 0.15–0.20, 0.30–0.35, and 0.45–0.50). 3https://wheat.pw.usda.gov/GG3/global_durum_genomic_resources 4https://wheat.triticeaetoolbox.org/breeders_toolbox/protocol/158 LD Decay k Pairwise marker correlations (r2 values) were calculated on the SNP dataset of the GDP for each chromosome using TASSEL5 (Bradbury et al., 2007). LD decay curves were ﬁtted using the non-linear model described in Rexroad and Vallejo (2009). Critical parameters of marker distances at r2 = 0.3 and 0.5 were extrapolated from the ﬁtted regression curves. The r2 of unlinked markers (background noise) was estimated as the 95th quantile of r2 values of markers on diﬀerent chromosomes (unlinked set). To estimate the local LD value along chromosomes, each marker LD was calculated using the mean r2 with the 50 nearest markers, and then smoothed as one value using the step-sliding window. December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 5 Genetic Diversity and Allele History Mazzucotelli et al. FIGURE 1 \| Distribution of the geographic origin of the GDP accessions used for genetic diversity analysis. Countries of origin are grouped as follows: Central Europe: Austria, Hungary, Ukraine, Sweden, Poland, United Kingdom, and Germany; Balkans: Serbia, Bosnia, and Herzegovina, Bulgaria, Romania, Greece, and Crete; North Africa: Egypt, Libya, Algeria, Tunisia, and Morocco; West Asia: Turkey, Syria, Lebanon, Israel, Jordan, Iran, Iraq, Armenia, Azerbaijan, Georgia, Oman, Yemen, and Saudi Arabia; Central Asia: Kazakhstan, Afghanistan, Russia, Uzbekistan, and China; Horn of Africa: Ethiopia, Eritrea, and Kenya. FIGURE 1 \| Distribution of the geographic origin of the GDP accessions used for genetic diversity analysis. Countries of origin are grouped as follows: Central Europe: Austria, Hungary, Ukraine, Sweden, Poland, United Kingdom, and Germany; Balkans: Serbia, Bosnia, and Herzegovina, Bulgaria, Romania, Greece, and Crete; North Africa: Egypt, Libya, Algeria, Tunisia, and Morocco; West Asia: Turkey, Syria, Lebanon, Israel, Jordan, Iran, Iraq, Armenia, Azerbaijan, Georgia, Oman, Yemen, and Saudi Arabia; Central Asia: Kazakhstan, Afghanistan, Russia, Uzbekistan, and China; Horn of Africa: Ethiopia, Eritrea, and Kenya. FIGURE 1 \| Distribution of the geographic origin of the GDP accessions used for genetic diversity analysis. Countries of origin are grouped as follows: Central Europe: Austria, Hungary, Ukraine, Sweden, Poland, United Kingdom, and Germany; Balkans: Serbia, Bosnia, and Herzegovina, Bulgaria, Romania, Greece, and Crete; North Africa: Egypt, Libya, Algeria, Tunisia, and Morocco; West Asia: Turkey, Syria, Lebanon, Israel, Jordan, Iran, Iraq, Armenia, Azerbaijan, Georgia, Oman, Yemen, and Saudi Arabia; Central Asia: Kazakhstan, Afghanistan, Russia, Uzbekistan, and China; Horn of Africa: Ethiopia, Eritrea, and Kenya. Frontiers in Plant Science \| www.frontiersin.org Deep Genotyping of the Global Durum Wheat Panel (GDP) turgidum sub-species accessions as landraces and grouped according to the country of origin. The EPO population was considered as a separate group based on its highly distinct genetic structure. The primary objective was to describe the pattern of genetic diversity across the history of durum wheat evolution and breeding so these groups composed as above described were considered: (i) modern germplasm, (ii) landraces and (iii) emmer (T. turgidum ssp. dicoccum) accessions, for a total of 861 genotypes. AMOVA highlighted a moderate level (23%) of genetic variance distinguishing the three groups (Table 2A), with a larger portion still existing within groups (77%). Reduction of overall diversity was observed in modern lines with respect to both T. turgidum ssp. dicoccum and landraces. Durum landraces showed a level of genetic diversity even higher than that of T. turgidum ssp. dicoccum accessions included in the GDP, perhaps due to ascertainment bias associated to the type of genotyping array used for the analysis, originally developed to maximize polymorphism among modern bread and durum breeding lines. However, in pairwise diﬀerentiation analysis Fst value was higher in the comparison landraces vs. dicoccum (Fst = 0.2688) with respect to the comparison landraces vs. modern lines (Fst = 0.1378) (Figure 2A). The EPO population, which was bred by INRA based on a composite cross to introduce diversity from wild and primitive accessions of T. turgidum subspecies, showed a relatively high level of diversity (David et al., 2014). Considering the all durum dataset (885 entries and 8,802 polymorphic SNPs), AMOVA results across the three main groups (modern lines, landraces and EPO accessions) showed that the highest proportion of molecular variance (86.94%) was observed within clusters rather than among clusters (13.06%) (Table 2B). Landraces showed the highest value of Nei’s genetic diversity (0.358), followed by modern germplasm (0.292) and EPO (0.288) (Table 2B). As to among- population comparisons, the highest diﬀerentiation was found for landrace vs. modern comparisons (Fst = 0.127), while an Fst of 0.1 was calculated for the EPO vs. modern comparison (Figure 2B). This result is also conﬁrmed by comparable values of PIC and Fst calculated for landraces (0.282 and 0.101, respectively, Table 2C) and modern lines (0.278 and 0.117, respectively, Table 2E). along the chromosomes was higher in proximal and distal portions compared to pericentromeric regions (Supplementary Figure S11B), and the opposite for the interlocus distances (Supplementary Figure S11C). Deep Genotyping of the Global Durum Wheat Panel (GDP) Genotyping of the GDP with the iSelect 90K wheat SNP array generated 42,520 polymorphic SNPs. After several quality ﬁltering steps, a total of 16,633 SNP markers were retained and imputed for missing data. Both datasets are available at the repositories GrainGenes3 and T3/Wheat4. The tetraploid genome was thus probed by a mean of 1,188 SNP markers per chromosome with an average density of 1.7 SNPs per Mbp or 6.3 SNPs per cM (Table 1). Almost one third (4,119) of the consecutive SNPs were located within 0.5 Kbp of each other, possibly due to the redundancy of the Illumina 90K SNP design, and 4,938 SNPs were located at various interlocus distances between 1 and 100 Kbp. The remaining 7,259 SNPs mapped at distances from >0.1 to 5 Mbp, and only 302 SNPs mapped at distances >5 Mbp (Supplementary Figure S11A). The genome coverage calculated as a percent of the physical genome length probed by SNP markers was almost complete with an average of 0.998% (Table 1). The marker density To assess the extent of the genetic diversity loss in the sampling process from the DWRC to GDP, diﬀerent indices were calculated based on the KASP data for the two panels. Locus level correlations between DWRC and GDP values resulted in Pearson’s coeﬃcients of 0.94 for the MAF, 0.95 for allelic evenness, 0.96 for expected heterozygosity and 0.97 for Shannon- Wiener’s diversity index (Supplementary Figure S9), indicating December 2020 \| Volume 11 \| Article 569905 6 Genetic Diversity and Allele History Mazzucotelli et al. TABLE 1 \| Genome coverage by the SNP marker dataset expressed for each chromosome and genome. Chromosome N◦SNP Mean SNP/Mb Chromosome coverage (%) 1A 1,138 1.9 0.998 1B 1,638 2.4 0.997 2A 1,096 1.4 0.999 2B 1,800 2.3 0.997 3A 979 1.3 0.999 3B 1,250 1.5 0.999 4A 750 1.0 0.996 4B 863 1.3 0.998 5A 962 1.4 0.997 5B 1,419 2.0 0.999 6A 974 1.6 0.998 6B 1,267 1.8 0.993 7A 1,248 1.7 0.998 7B 1,249 1.7 0.997 Genome A 7,147 1.5 0.998 Genome B 9,486 1.9 0.997 Total 16,633 1.7 0.997 Mean 1,188 1.7 0.998 TABLE 1 \| Genome coverage by the SNP marker dataset expressed for each chromosome and genome. durum like T. turgidum ssp. turgidum, turanicum and polonicum (Maccaferri et al., 2019). Therefore, the genetic diversity analyses reported hereafter were carried out including all durum- related T. Deep Genotyping of the Global Durum Wheat Panel (GDP) After excluding six accessions due to failed genotyping, ﬁltering carried out at the accession level based on IBS_GS matrix (Supplementary Table S4) allowed for the identiﬁcation of 10 accessions whose genotypic data were not relevant (misclassiﬁed accessions or contaminated DNA) that were discarded from further analysis. High-density genotyping data are therefore available for a ﬁnal set of 1,011 accessions, while for a total of 1001 accessions both seed stock and genotypic data are provided (Supplementary Table S3). Frontiers in Plant Science \| www.frontiersin.org Genetic Diversity Analysis TABLE 2 \| AMOVA and gene diversity for ﬁve germplasm sub-sets deﬁned according to passport data: (A) GDP without the wild accessions, with grouping based on historical selection steps: T. dicoccum accessions, T. durum germplasm sub-sets landraces, T. durum germplasm sub-sets cultivars; (B) all T. durum germplasm sub-sets; groups are EPO, T. durum germplasm sub-sets landraces, modern lines; (C) all landraces grouped according to country of origin; (D) all T. durum germplasm sub-sets modern lines, classiﬁed according to decade of release; (E) all T. durum germplasm sub-sets modern lines, classiﬁed based on breeding program. TABLE 2 \| AMOVA and gene diversity for ﬁve germplasm sub-sets deﬁned according to passport data: (A) GDP without the wild accessions, with grouping based on historical selection steps: T. dicoccum accessions, T. durum germplasm sub-sets landraces, T. durum germplasm sub-sets cultivars; (B) all T. durum germplasm sub-sets; groups are EPO, T. durum germplasm sub-sets landraces, modern lines; (C) all landraces grouped according to country of origin; (D) all T. durum germplasm sub-sets modern lines, classiﬁed according to decade of release; (E) all T. durum germplasm sub-sets modern lines, classiﬁed based on breeding program. g g g sub-sets; groups are EPO, T. durum germplasm sub-sets landraces, modern lines; (C) all landraces grouped according to country of origin; (D) all T. durum germplasm sub-sets modern lines, classiﬁed according to decade of release; (E) all T. durum germplasm sub-sets modern lines, classiﬁed based on breeding program. (A) Source of variation d.f. Sum of squares Variance components Percentage of variation Among populations 2 222,822.13 443.9 22.98 Within populations 859 1,278,090.16 1,487.88 77.02 Total 861 1,500,912.3 1,931.79 Fst 0.23 T. durum groups N◦accessions N◦polymorphic loci over 10173 Nei’s gene diversity Mean number of pairwise differences LANDRACE 286 10,154 0.332 3,375.08 EMMER 103 9,901 0.317 3,220.49 MODERN 473 10,010 0.264 2,681.76 Mean value 0.304 3,092.45 Lsd (p = 0.0005) 0.002 17.7 (B) Source of variation d.f. Sum of squares Variance components Percentage of variation Among populations 2 103,704.61 207.33 13.06 Within populations 852 1,175,524.24 1,379.72 86.94 Total 854 1,279,228.85 1,587.05 Fst 0.13 PIC 0.273 range (0.09–0.375) T. durum groups N◦accessions N◦polymorphic loci over 8802 Nei’s gene diversity Mean number of pairwise differences LANDRACE 286 8,796 0.358 3,151.85 MODERN 473 8,781 0.292 2,567.05 EPO 96 8,213 0.288 2,538.16 Mean value 0.313 2,752.35 Lsd (p = 0.0005) 0.002 17.6 (C) Source of variation d.f. Genetic Diversity Analysis Genotyping data allowed to characterize the GDP for genetic diversity and diﬀerentiation within and among groups deﬁned on the base of passport data (Supplementary Table S3). GDP entries were classiﬁed according to the following criteria. The introduction of the semi-dwarf RhtB1b allele from CIMMYT durum lines (Motzo and Giunta, 2007; Ortiz et al., 2007) represents the origin of the post green revolution germplasm, so all entries generated from crosses carried out after 1970 were considered as modern germplasm. North American varieties and breeding materials released after 1970 were also included in the modern set, even though these did not carry the RhtB1b allele, which is not beneﬁcial in the northern semi- arid prairie environment. All durum lines pre-dating 1970 were considered as landraces, although in a few cases these were obtained through breeding selection of populations or voluntary hybridization among landraces. Notably, the characterization of genetic diversity could not clearly distinguish T. turgidum spp. durum landraces from other T. turgidum sub-species related to Durum landraces (282) were grouped into 14 sub-populations according to the country of origin. This clustering process accounted only for 10.1% of the variance, while the vast majority of diversity still remained unclustered within sub-populations (Table 2C). Nei’s gene diversity values ranged from 0.280 (United States–Canada) to 0.374 (Turkey–Transcaucasian). To analyze the changes in diversity within the modern germplasm over time and across breeding groups, the totality of 473 cultivars and elite lines were divided into sub-groups based on two diﬀerent criteria: (i) decade of release from 1970 to 2018; and (ii) country of registration/release, which roughly deﬁnes the main groups of breeding programs. Frontiers in Plant Science \| www.frontiersin.org December 2020 \| Volume 11 \| Article 569905 7 Genetic Diversity and Allele History Mazzucotelli et al. TABLE 2 \| AMOVA and gene diversity for ﬁve germplasm sub-sets deﬁned according to passport data: (A) GDP without the wild accessions, with grouping based on historical selection steps: T. dicoccum accessions, T. durum germplasm sub-sets landraces, T. durum germplasm sub-sets cultivars; (B) all T. durum germplasm sub-sets; groups are EPO, T. durum germplasm sub-sets landraces, modern lines; (C) all landraces grouped according to country of origin; (D) all T. durum germplasm sub-sets modern lines, classiﬁed according to decade of release; (E) all T. durum germplasm sub-sets modern lines, classiﬁed based on breeding program. Genetic Diversity Analysis Sum of squares Variance components Percentage of variation Among populations 13 62,147.3 169.12 10.1 Within populations 270 403,266.13 1,504.72 89.9 Total 281 465,413.43 1,673.84 Fst 0.101 PIC 0.282 range (0.09–0.375) Landrace group N◦accessions N◦polymorphic loci over 9414 Nei’s gene diversity Mean number of pairwise differences Turkey-Transcaucasian 29 9,253 0.374 3,521.67 Central Asia 18 9,035 0.356 3,348.15 Arabian Peninsula 9 7,790 0.349 3,285.50 Iberian Peninsula 21 9,048 0.346 3,253.83 Central Europe 18 8,547 0.341 3,206.41 South Asia 6 6,640 0.329 3,094.53 Greece 16 8,539 0.327 3,082.52 Italy 34 8,656 0.303 2,874.58 Ethiopia 26 8,174 0.302 2,843.20 North Africa 47 9,059 0.301 2,839.57 Argentina 5 6,107 0.300 2,829.40 (Continued) December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 8 Genetic Diversity and Allele History Mazzucotelli et al. TABLE 2 \| Continued Landrace group N◦accessions N◦polymorphicloci over 9414 Nei’s gene diversity Mean number of pairwise differences Levant 46 8,496 0.289 2,722.53 North America 5 5,340 0.280 2,640.00 Australia 2 3,136 0.333 3,136.00 Mean value 0.323 3,048.42 Lsd* (p = 0.05) 0.005 48.1 Lsd* (p = 0.001) 0.008 75.8 (D) Source of variation d.f. Sum of squares Variance components Percentage of variation Among populations 4 13,737.04 29.36 2.95 Within populations 444 429,609.05 967.59 97.05 Total 448 443,346.08 996.95 Fst 0.029 Breeding decade N◦lines N◦polymorphicloci over 5685 Nei’s gene diversity Mean number of pairwise differences 70–80 19 5,334 0.357 2,027.88 81–90 62 5,668 0.364 2,069.90 91–00 93 5,679 0.348 1,979.88 01–10 132 5,681 0.337 1,914.88 11–18 143 5,675 0.326 1,855.32 Mean value 0.346 1,969.57 Lsd (p = 0.0005) 0.003 33.2 Breeding group 70–80 81–90 91–00 01–10 11–18 Total Australia 0 1 2 5 4 12 Central Asia 2 1 3 2 1 9 Central Europe 0 1 4 2 12 19 CIMMYT 3 2 5 6 29 45 Spain 3 4 10 6 1 24 Ethiopia 0 0 1 1 3 5 France 0 7 12 13 7 39 ICARDA 0 8 12 45 40 105 Italy 9 12 22 23 14 80 North America 2 8 13 5 5 33 South America 0 7 1 7 10 25 South Mediterranean 0 11 8 17 17 53 Total 19 62 93 132 143 (E) Source of variation d.f. (Continued) Genetic Diversity Analysis Sum of squares Variance components Percentage of variation Among populations 11 60,189.98 121.56 11.67 Within populations 460 423,162.29 919.92 88.33 Total 471 483,352.27 1,041.48 Fst 0.117 PIC 0.278 range (0.09–0.375) (Continued) N◦accessions N◦polymorphicloci over 9414 Nei’s gene diversity Mean number of pairwise differences (Continued) December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 9 Genetic Diversity and Allele History Mazzucotelli et al. TABLE 2 \| Continued Breeding group N◦lines N◦polymorphic loci over 5918 Nei’s gene diversity Mean number of pairwise differences Italy 80 5,885 0.343 2,031.16 Central Asia 14 5,463 0.339 2,006.78 France 39 5,768 0.339 2,006.03 South America 25 5,535 0.335 1,984.11 Spain 27 5,591 0.332 1,963.08 Central Europe 25 5,466 0.321 1,902.33 South Mediterranean 53 5,776 0.312 1,848.12 Ethiopia 8 4,253 0.297 1,755.71 North America 33 5,316 0.296 1,749.61 ICARDA 110 5,813 0.294 1,741.78 CIMMYT 46 5,046 0.256 1,513.59 Australia 12 4,208 0.255 1,506.68 Mean value 0.310 1,834.08 Lsd (p = 0.05) 0.005 29.7 Lsd (p = 0.001) 0.008 46.9 Geographic area of origin has been assigned to GDP landraces based on country where they have been sampled, as follows: North America: Canada + United States; Arabian Peninsula: Oman, Yemen, and Saudi Arabia; Central Europe: Bulgaria, Serbia, Poland, Ukraine, Hungary, Romania, United Kingdom, Germany, Sweden, Bosnia, and Herzegovina; Iberian peninsula: Spain and Portugal; Levantine: Iran, Iraq, Jordan, Syria, and Israel; North Africa: Egypt, Tunisia, Algeria, Morocco, Libya, and Malt; Turkey-Transcaucasia: Turkey, Armenia, Azerbaijan, and Georgia; South Asia: Pakistan and India; Central Asia: Kazakhstan, Russia, and Afghanistan. GDP modern lines have been assigned to breeding program based on the country where lines have been developed, as follows: South Mediterranean: Egypt, Algeria, Tunisia, Syria, Lebanon, Jordan, and Morocco; North America: Canada + United States (including Desert durum); Central Europe: Austria, Serbia, Hungary, Ukraine, and Bulgaria; Central Asia: Kazakhstan and Uzbekistan. For each summary, the ﬁrst table reports results of AMOVA, the second table contains computations about gene diversity within groups/populations. AMOVA was signiﬁcant at p < 10−6 upon 10,000 permutations. LSD for within group gene diversity was calculated at the indicated p-value considering n = the least number of group genotypes, except for the landrace group where n = 5 was chosen. For decade of release, a third table reports the composition of each decade group with respect to the breeding programs. Genetic Diversity Analysis Thus, ﬁve decades (’70–’80, ’81–’90, ’91–’00, ’01–’10, ’11– ’18) and 12 breeding program groups (Australia, North America, Central Europe, Central Asia, France, Italy, South America, Spain, South Mediterranean, Ethiopia, ICARDA, CIMMYT) (Supplementary Table S3) were considered. For temporal groups (decades), AMOVA analysis revealed a very low, even if statistically signiﬁcant, percentage of variation among groups (2.95%, Table 2D), attributing the near totality of variance to individuals within groups. Nei’s gene diversity showed a constant decreasing trend starting from the decade (’81–’90) to the most recently released (2011–2018), with limited but signiﬁcant variation. The mean number of pairwise diﬀerences within a decade (Figure 2C), and pairwise Fst among groups conﬁrmed the trend; the highest diﬀerence in Fst values was observed in the comparison between the ’70–’80s and the 2011–2018 decades, conﬁrming a progressive and generalized shift toward the enrichment of fewer successful haplotypes during breeding history (Figure 2C). (0.335) (Table 2E). As for among-population comparisons, the Italian modern group showed generally lower pairwise Fst values as compared to all the other groups, with relatively higher values against the Northern programs and lower values against the other Mediterranean groups (Figure 2D). A reverse pattern of diﬀerentiation was evident for the French breeding programs, showing stronger similarities with the Northern programs. Low Fst values were calculated for pairwise comparisons among Central Europe, North America and Central Asia programs. Likewise, both CIMMYT and ICARDA showed the highest Fst values in the comparison with these breeding groups and the lowest Fst values with the Mediterranean groups. Between them, ICARDA and CYMMIT showed a Fst = 0.09. Analogously, low Fst values evidenced known interactions of international breeding programs with national programs, like ICARDA vs. Ethiopia and North African countries. The Australian breeding program appeared to stand as a separate group. Genetic Diversity Analysis Geographic area of origin has been assigned to GDP landraces based on country where they have been sampled, as follows: North America: Canada + United States; Arabian Peninsula: Oman, Yemen, and Saudi Arabia; Central Europe: Bulgaria, Serbia, Poland, Ukraine, Hungary, Romania, United Kingdom, Germany, Sweden, Bosnia, and Herzegovina; Iberian peninsula: Spain and Portugal; Levantine: Iran, Iraq, Jordan, Syria, and Israel; North Africa: Egypt, Tunisia, Algeria, Morocco, Libya, and Malt; Turkey-Transcaucasia: Turkey, Armenia, Azerbaijan, and Georgia; South Asia: Pakistan and India; Central Asia: Kazakhstan, Russia, and Afghanistan. GDP modern lines have been assigned to breeding program based on the country where lines have been developed, as follows: South Mediterranean: Egypt, Algeria, Tunisia, Syria, Lebanon, Jordan, and Morocco; North America: Canada + United States (including Desert durum); Central Europe: Austria, Serbia, Hungary, Ukraine, and Bulgaria; Central Asia: Kazakhstan and Uzbekistan. For each summary, the ﬁrst table reports results of AMOVA, the second table contains computations about gene diversity within groups/populations. AMOVA was signiﬁcant at p < 10−6 upon 10,000 permutations. LSD for within group gene diversity was calculated at the indicated p-value considering n = the least number of group genotypes, except for the landrace group where n = 5 was chosen. For decade of release, a third table reports the composition of each decade group with respect to the breeding programs. Thus, ﬁve decades (’70–’80, ’81–’90, ’91–’00, ’01–’10, ’11– ’18) and 12 breeding program groups (Australia, North America, Central Europe, Central Asia, France, Italy, South America, Spain, South Mediterranean, Ethiopia, ICARDA, CIMMYT) (Supplementary Table S3) were considered. For temporal groups (decades), AMOVA analysis revealed a very low, even if statistically signiﬁcant, percentage of variation among groups (2.95%, Table 2D), attributing the near totality of variance to individuals within groups. Nei’s gene diversity showed a constant decreasing trend starting from the decade (’81–’90) to the most recently released (2011–2018), with limited but signiﬁcant variation. The mean number of pairwise diﬀerences within a decade (Figure 2C), and pairwise Fst among groups conﬁrmed the trend; the highest diﬀerence in Fst values was observed in the comparison between the ’70–’80s and the 2011–2018 decades, conﬁrming a progressive and generalized shift toward the enrichment of fewer successful haplotypes during breeding history (Figure 2C). Genetic Diversity Analysis TABLE 2 \| Continued Breeding group N◦lines N◦polymorphic loci over 5918 Nei’s gene diversity Mean number of pairwise differences Italy 80 5,885 0.343 2,031.16 Central Asia 14 5,463 0.339 2,006.78 France 39 5,768 0.339 2,006.03 South America 25 5,535 0.335 1,984.11 Spain 27 5,591 0.332 1,963.08 Central Europe 25 5,466 0.321 1,902.33 South Mediterranean 53 5,776 0.312 1,848.12 Ethiopia 8 4,253 0.297 1,755.71 North America 33 5,316 0.296 1,749.61 ICARDA 110 5,813 0.294 1,741.78 CIMMYT 46 5,046 0.256 1,513.59 Australia 12 4,208 0.255 1,506.68 Mean value 0.310 1,834.08 Lsd (p = 0.05) 0.005 29.7 Lsd (p = 0.001) 0.008 46.9 Geographic area of origin has been assigned to GDP landraces based on country where they have been sampled, as follows: North America: Canada + United States; Arabian Peninsula: Oman, Yemen, and Saudi Arabia; Central Europe: Bulgaria, Serbia, Poland, Ukraine, Hungary, Romania, United Kingdom, Germany, Sweden, Bosnia, and Herzegovina; Iberian peninsula: Spain and Portugal; Levantine: Iran, Iraq, Jordan, Syria, and Israel; North Africa: Egypt, Tunisia, Algeria, Morocco, Libya, and Malt; Turkey-Transcaucasia: Turkey, Armenia, Azerbaijan, and Georgia; South Asia: Pakistan and India; Central Asia: Kazakhstan, Russia, and Afghanistan. GDP modern lines have been assigned to breeding program based on the country where lines have been developed, as follows: South Mediterranean: Egypt, Algeria, Tunisia, Syria, Lebanon, Jordan, and Morocco; North America: Canada + United States (including Desert durum); Central Europe: Austria, Serbia, Hungary, Ukraine, and Bulgaria; Central Asia: Kazakhstan and Uzbekistan. For each summary, the ﬁrst table reports results of AMOVA, the second table contains computations about gene diversity within groups/populations. AMOVA was signiﬁcant at p < 10−6 upon 10,000 permutations. LSD for within group gene diversity was calculated at the indicated p-value considering n = the least number of group genotypes, except for the landrace group where n = 5 was chosen. For decade of release, a third table reports the composition of each decade group with respect to the breeding programs. LD Decay G id durum groups of EPO, landraces, modern lines; (C) T. durum modern lines classiﬁed according to decade of release; (D) T. durum modern lines classiﬁed based on breeding program. In each matrix, above diagonal elements (shades of green) contain the average number of pairwise differences, while below diagonal elements (shades of blue) report pairwise Fst values. Diagonal elements (shades of red) contain gene diversity within groups calculated as mean number of pairwise differences. Signiﬁcance was assessed upon 1000 permutations. All values are signiﬁcant at p < 0.001, except values marked with which were signiﬁcant at p < 0.01, or values in italics that were not signiﬁcant. Relative Neighbor-Joining phylogenetic tree based on Nei’s distance are also reported for panels (C,D). reports LD calculated for each chromosome and for modern and landraces, independently. of the breeding groups except Australia, South America, and Ethiopia, ranging between 39 and 100% of the unique alleles. It was interesting to note that for CIMMYT and ICARDA, 100% of unique alleles were also rare, similarly to Italy (99%). Among the remaining unique alleles, none was a frequent allele in the target breeding group, and most (64%) had frequency from 5 to 10%. However, 53 SNPs showed higher frequency, suggesting a role in a speciﬁc breeding target or for adaptation to the corresponding environmental conditions. LD Decay G id The last analysis considered the modern germplasm, clustered according to breeding groups. AMOVA attributed the highest proportion of molecular variance (88.33%, Table 2E) to individuals within breeding programs, while variation between populations accounted for the remaining portion (11.67%). Moderate levels of diversity were observed for Australia and CIMMYT showing the lowest values (0.255 and 0.256, respectively), followed by ICARDA (0.294), North America (0.296), and Ethiopia (0.297), up to highest values calculated for Italy (0.343), Central Asia and France (0.339), and South America Genome-wide LD decay was calculated for the two major T. turgidum ssp. durum groups of the GDP collection: modern and landraces. As expected, LD was lower in landraces than in modern lines (Figure 3). The critical r2 values of 0.3 and 0.5 were reached at a distance of 0.9–0.4 Mbp in landraces, and at distances of 4.2–1.8 Mbp in modern. Overall, 95% of unlinked markers showed a r2 value <0.09 in landraces and 0.04 in modern. These r2 values corresponded to distances of 4.2 Mbp in landraces and of 42.3 Mbp in modern. Supplementary Figure S12 December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 10 Genetic Diversity and Allele History Mazzucotelli et al. FIGURE 2 \| Population differentiation calculated as pairwise Fst and average number of pairwise differences between groups/populations deﬁned according to passport data for: (A) evolution from domesticated emmer, to landraces, to modern lines; (B) all T. durum groups of EPO, landraces, modern lines; (C) T. durum modern lines classiﬁed according to decade of release; (D) T. durum modern lines classiﬁed based on breeding program. In each matrix, above diagonal elements (shades of green) contain the average number of pairwise differences, while below diagonal elements (shades of blue) report pairwise Fst values. Diagonal elements (shades of red) contain gene diversity within groups calculated as mean number of pairwise differences. Signiﬁcance was assessed upon 1000 permutations. All values are signiﬁcant at p < 0.001, except values marked with which were signiﬁcant at p < 0.01, or values in italics that were not signiﬁcant. Relative Neighbor-Joining phylogenetic tree based on Nei’s distance are also reported for panels (C,D). FIGURE 2 \| Population differentiation calculated as pairwise Fst and average number of pairwise differences between groups/populations deﬁned according to passport data for: (A) evolution from domesticated emmer, to landraces, to modern lines; (B) all T. Detection of Putative Selection Signals in Durum Wheat Groups Considering the durum modern germplasm and its whole MAF-unﬁltered SNP dataset of 16,633 SNPs, 889 unique breeding program-speciﬁc alleles were found (5.4% of the total, Supplementary Table S5). “Unique” is used to deﬁne a minor allele that occurs only in the germplasm of one breeding program and not in any other. The groups with the largest set of unique alleles were Central Europe, Central Asia, and Italy, with 289, 208, and 102 unique alleles, respectively (Table 3). Ethiopia and Australia were characterized by the lowest number of unique alleles with 13 and 9, respectively. It was then possible to identify rare alleles (with MAF less than 0.05) within the group of unique alleles. In particular, rare unique alleles were observed in all Fixation of loci controlling traits of interest by intense selection during the breeding process may result in steep increases in allele frequency, reduced variation (reported as a selective sweep), and therefore divergence in allele frequency in the proximity of the selected loci. Low-resolution genomic scans can be used to identify regions containing loci and causative genes with a putative major inﬂuence on breeding processes. Scans for PSW between modern and landraces (Supplementary Table S6) identiﬁed 53 PSW clusters, based on Fst only (24) or December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 11 Genetic Diversity and Allele History Mazzucotelli et al. FIGURE 3 \| Genome wide linkage disequilibrium (LD) decay in respect to physical distance in the two main groups of the GDP collection: (A) modern germplasm, (B) landraces. FIGURE 3 \| Genome wide linkage disequilibrium (LD) decay in respect to physical distance in the two main groups of the GDP collection: (A) modern germplasm, (B) landraces. ge disequilibrium (LD) decay in respect to physical distance in the two main groups of the GDP collection: (A) modern germplasm, decade comparisons (32, 10, and 2 PSW clusters, respectively), while 18 PSW clusters were detected in a single comparison. PSW clusters physical size extended from 11 Mbp for cluster Cls- chr3B.1 to 386 Mbp for Cls-chr6A.4, with an average of 52 Mbp (Supplementary Table S7). As expected, the largest clusters were predominantly located in centromeric and peri-centromeric regions. Promising putative candidate genes were found to co- locate with nine PSW clusters (Supplementary Table S7). on both indices, Fst and DRI (8). Detection of Putative Selection Signals in Durum Wheat Groups Most clusters (73%) extended for less than 50 Mbp, but three extended for >150 Mbp. All chromosomes were found to carry PSW clusters, with chromosome 1B being the most targeted by breeders’ selection. Promising putative candidate genes were found to co-locate with eleven PSW clusters, for instance the genes Rht1-B and Ppd- A1 on chromosomes 4B and 2A, respectively (Supplementary Table S6). Considering four subsequent decades of release, 62 putative signal clusters were highlighted across all six pairwise comparisons between the four decades (Supplementary Table S7). Chromosome 2B showed the highest number (9) of PSW clusters, whereas only two clusters per chromosome were identiﬁed on chromosomes 4A, 4B and 5B. Considering the ﬁve decades comparisons separately, 92 putative signals were found for DRI, 74 for Fst, and 46 were conﬁrmed by both methods. The signals were distributed across the four comparisons: 30 were found for the ’70–’80 vs. ’81–’90 decades, 33 for both the comparisons ’81–’90 vs. ’91–’00 and ’91–’00 vs. ’01–’10, and 24 for ’01–’10 vs. ’11–’18. Most clusters were identiﬁed for two diﬀerent y Further pairwise comparisons were carried out for breeding groups that contributed more than 30 entries to the GDP (Figure 4). This investigation included modern T. durum genotypes from CIMMYT, ICARDA, Italy, France and North America, for a total of 10 pairwise comparisons. In total, 126 PSW clusters were identiﬁed (Supplementary Table S8), 59 of them supported by both indices, 40 based on DRI only, and 28 by Fst only. PSW cluster size ranged between 11 and 468 Mbp, with an average of 45.7 Mbp, and most clusters (81%) extending for less than 50 Mbp. Clusters were found in two or more comparisons (54), and only ﬁve were pair-speciﬁc. For 19 clusters December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 12 Genetic Diversity and Allele History Mazzucotelli et al. TABLE 3 \| Unique alleles in the different breeding groups. Detection of Putative Selection Signals in Durum Wheat Groups ICARDA and the Italian breeding programs had the highest numbers, 105 and 110, respectively. Considering pair-speciﬁc PSW clusters, CIMMYT and French groups showed the lowest number of speciﬁc PSW clusters (9), while Italy and ICARDA presented 12 and 11, respectively, and North America showed the highest number of speciﬁc PSW clusters (18). a possible correspondence with a putative candidate gene could be proposed. The North American breeding group had the lowest number of PSW clusters (79), followed by CIMMYT with 88 clusters and the French breeding program with 100 PSW clusters. ICARDA and the Italian breeding programs had the highest numbers, 105 and 110, respectively. Considering pair-speciﬁc PSW clusters, CIMMYT and French groups showed the lowest number of speciﬁc PSW clusters (9), while Italy and ICARDA presented 12 and 11, respectively, and North America showed the highest number of speciﬁc PSW clusters (18). The modern durum germplasm was ﬁrst split at k = 4 separating photoperiod sensitive accessions from northern countries (North America, France, Austria and the EPO entries) and Mediterranean-adapted photoperiod insensitive accessions. K = 10 was the minimum k value at which both Ward’s clustering and ADMIXTURE clearly separated the modern durum entries originating from the two main CGIAR (CIMMYT and ICARDA) breeding programs. At k = 13, modern durum entries were already divided in four sub-sets corresponding to French origin and EPO (subp. 1), CIMMYT (subp. 2), ICARDA (subp. 3), North American and Austrian (subp. 4). At k = 18 the group containing mainly CIMMYT durum wheat modern lines was further split in three sub-groups: the ﬁrst one contained CIMMYT and other modern lines with diﬀerent origins, the second one included CIMMYT and Egyptian germplasm, and the third one only modern germplasm from the Mediterranean countries. Only at k = 20 was the EPO set split into two groups. Detection of Putative Selection Signals in Durum Wheat Groups Breeding groups N◦of unique alleles N◦of unique alleles with MAF > 5% in the target group Average frequency of the unique alleles in the target group Central Europe 289 122 0.11 Central Asia 208 208 0.07 Italy 102 1 0.03 ICARDA 60 0 0.01 North Africa 57 2 0.02 North America 49 9 0.04 France 46 9 0.03 South America 23 14 0.09 Spain 22 5 0.05 CIMMYT 21 0 0.02 Ethiopia 13 13 0.14 Australia 9 9 0.16 Tot 899 392 0.06 TABLE 3 \| Unique alleles in the different breeding groups. domesticated emmers from the Fertile Crescent together with Ethiopian durum and T. turgidum ssp. carthlicum entries. At k = 20, emmer accessions were further split between central Asian domesticated emmer (subp. 11), European domesticated emmer (subp. 12) and wild emmer (subp. 13). At k = 2, the second mega-cluster included most T. turgidum ssp. durum (96%), T. turgidum ssp. turanicum and most of T. turgidum ssp. polonicum (67%). Separation between durum modern and landraces started at k = 3. At k = 6, durum landraces and primitive tetraploids were split into two main groups: Asian and North African landraces. Further meaningful landrace sub-groups were split at higher k values. The group including mainly Ethiopian accessions was split in two sub- groups: the ﬁrst one contained accessions of T. turgidum spp. carthlicum, polonicum and durum landraces, while the second one was mainly T. turgidum ssp. dicoccum accessions, which might represent the founder group of Ethiopian durums. Durum landraces and primitive tetraploids were grouped into subpopulations as follows: Central Mediterranean landraces (subp. 5), a mixed group of other Mediterranean landraces and old Italian cultivars such as the breeding germplasm founder Cappelli, and (subp. 6) more recent Italian cultivars directly related to landraces (subp. 7), Ethiopian durum landraces and emmers plus T. turgidum ssp. carthlicum (subp. 8), Central Asia durum landraces and all T. turgidum ssp. turanicum (subp. 10). Notably, sub-population 9 included a group of ICARDA founder cultivars belonging to the Om Rabi set, which were derived from crossing the Syrian landrace Haurani to the CIMMYT cultivar Jori (Kabbaj et al., 2017). a possible correspondence with a putative candidate gene could be proposed. The North American breeding group had the lowest number of PSW clusters (79), followed by CIMMYT with 88 clusters and the French breeding program with 100 PSW clusters. GDP Stratiﬁcation Analysis Population stratiﬁcation was conducted based on both Ward’s clustering and admixture sub-population membership from k = 2 up to k = 20 based on the SNP dataset pruned at r2 = 0.5. Results of these analysis are shown in Figure 4C while Supplementary Table S9 reports sub-population memberships for each genotype and K value based on the two analyses. Applying SNP pruning with r2 = 0.8 outperformed the other two in terms of cross- validated group assignment (Figure 4A), although pruning at r2 = 0.5 provided comparable results. Grouping statistics, in particular the minimum group size (Figure 4B), stabilized at k > 11, despite the fact that cross-validated assignment error steadily decreased at higher k values (Figure 4A) and meaningful diﬀerences were still observed up to k values of 20. At k = 2, most accessions of T. turgidum spp. dicoccum (98%), dicoccoides (98%), carthlicum (92%) and turgidum (77%) clustered together (reported as dark yellow Q membership bars in Figure 4C), separated from all the durum wheat entries (reported as dark blue Q membership bars in Figure 4C). Notably, a small group of 33 (4%) of landraces from Ethiopia and the Arabian Peninsula clustered in the former group, showing appreciable genetic kinship with emmer from the Fertile Crescent. At k = 5, the emmer group was split in two main branches, one grouping wild emmer together with European and Fertile Crescent domesticated emmers, and the second having The GDP phylogenetic tree estimated through Neighbor- Joining clustering for all accessions is reported in Figure 5 and Supplementary Table S9. Bootstrap values indicating branches’ consistency are reported in detail in Supplementary Figure S13. Overall, good correlation was observed between population stratiﬁcation analysis performed through admixture and the position on the Neighbor-Joining tree. Three main branches were grouped: (i) wild and domesticated emmers and T. turgidum ssp. carthlicum, (ii) durum landraces including the founders of modern germplasm and (iii) modern durums. Among durum landraces, one of the two sub-branches included December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 13 Genetic Diversity and Allele History Mazzucotelli et al. GDP Stratiﬁcation Analysis FIGURE 4 \| ADMIXTURE’s grouping statistics: (A) cross validation error rate, and (B) minimum group size, from k = 2 to k = 20 for three LD pruned SNP datasets (r2 = 0.3, r2 = 0.5, r2 = 0.8); (C) population structure of the GDP collection based on Ward’s clustering and ADMIXTURE (SNP dataset at r2 = 0.5); membership from k = 2 to k = 20. North African/Southern European landraces and pioneering durum cultivars obtained from landrace selection and landrace intercrossing, such as Senatore Cappelli (selection from a landrace) and Capeiti8 (cross between Cappelli and a Syrian landrace selection). The second group included durum landraces from West Asia including Haurani, well-known as the most FIGURE 4 \| ADMIXTURE’s grouping statistics: (A) cross validation error rate, and (B) minimum group size, from k = 2 to k = 20 for three LD pruned SNP datasets (r2 = 0.3, r2 = 0.5, r2 = 0.8); (C) population structure of the GDP collection based on Ward’s clustering and ADMIXTURE (SNP dataset at r2 = 0.5); membership from k = 2 to k = 20. FIGURE 4 \| ADMIXTURE’s grouping statistics: (A) cross validation error rate, and (B) minimum group size, from k = 2 to k = 20 for three LD pruned SNP datasets (r2 = 0.3, r2 = 0.5, r2 = 0.8); (C) population structure of the GDP collection based on Ward’s clustering and ADMIXTURE (SNP dataset at r2 = 0.5); membership from k 2 to k 20 North African/Southern European landraces and pioneering durum cultivars obtained from landrace selection and landrace intercrossing, such as Senatore Cappelli (selection from a landrace) and Capeiti8 (cross between Cappelli and a Syrian landrace selection). The second group included durum landraces from West Asia including Haurani, well-known as the most December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 14 Genetic Diversity and Allele History Mazzucotelli et al. FIGURE 5 \| Neighbor joining tree of the GDP collection and comparison between NJ and ADMIXTURE model-based ancestry grouping methods. Details on accessions included in each clade are reported in Supplementary Table S9. FIGURE 5 \| Neighbor joining tree of the GDP collection and comparison between NJ and ADMIXTURE model-based ancestry grouping methods. Details on accessions included in each clade are reported in Supplementary Table S9. GDP Stratiﬁcation Analysis the second step, 1,011 entries were selected from the DWRC to capture most of this diversity (94–97%), with the strongest reduction aﬀecting some rare alleles. widely cultivated landrace population in its area of origin, showing developmental and morphological traits relevant for adaptation to low water availability and high temperatures, widely exploited by the ICARDA durum program since its inception (Elings and Nachit, 1991; Pagnotta et al., 2005). Another small group of interest is that composed of Central- Asian durum landraces that were included phylogenetically within the emmer clade. This group was found to lie between the main emmer clades and the modern durum, supporting a possible role of its members as founders of the Northern breeding programs (Paulsen and Shroyer, 2008). In the GDP, the mean PIC values of 0.27 for landraces and 0.28 for modern lines and ranging from 0.09 to 0.38 (Table 2B) indicated a generally higher or similar level of genetic diversity captured within the GDP compared to previously studied collections. Recent studies reported PIC values of 0.26 for durum modern germplasm (Chao et al., 2017), 0.19 for a set of both landraces and modern lines (Ren et al., 2012), and 0.18 in a collection of 168 durum wheat accessions of diﬀerent origins (Roncallo et al., 2019). Analogously, AMOVA on clusters within GDP based on geography and breeding program of origin showed that only 13% of the total genetic variance could be captured among groups, while most diversity remained among individuals within clusters. These results concur with those reported by Soriano et al. (2016) with 172 landraces from 21 countries, by Roncallo et al. (2019) with a panel of 168 durum accessions and by N’Diaye et al. (2018) with a panel of Canadian durum cultivars where only 10% of variation was captured among groups. Other studies considering similar panels reported capturing over 30% of the total genetic variance by clustering germplasm based Genetic Diversity and Population Structure in GDP and Breeding Groups The GDP builds on several studies that have investigated the diversity and phylogeny of durum wheat by assembling these into one panel. The two-step approach deployed here started by gathering entries representing nearly all genetic diversity studies ever conducted for durum wheat within the DWRC. In December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 15 Genetic Diversity and Allele History Mazzucotelli et al. on kinship matrix, but using relatively higher k values (Kabbaj et al., 2017; Robbana et al., 2019). Our study aimed primarily at evaluating the historical diversity based on passport information, rather than on clusters derived from population structure. It is therefore evident that the passport information alone, while of great historical interest, is unable to capture the true genetic diversity of durum wheat worldwide. AMOVA on stratiﬁed groups may reveal much more variance among sub-populations, as indeed reported by other authors (Kabbaj et al., 2017; Roncallo et al., 2019). The moderate diversiﬁcation among breeding groups (11.67% of the total variance) and very little among decades of release (2.95% of the total variance) revealed by AMOVA on the 473 modern durum wheat accessions (Tables 2D,E) was probably due to the wide and frequent exchange of parents among durum breeders worldwide. This was clearly evidenced in the Italian breeding programs, characterized by an overall higher level of diversity and lower diﬀerentiation against most of the other breeding programs, thus reﬂecting the necessity to breed for the many diﬀerent agro-ecological zones that exist in Italy (Fischer et al., 2012). Overall, the results presented here suggest that good genetic diversity remains available within the breeding groups for direct exploitation, and there is even greater potential when considering exchanges between breeding groups. Current varieties exhibit increased yield potential, spike fertility, pasta quality and are resistant to widespread diseases such as rusts. The process of selection has evidently resulted in “signatures” being incorporated into the durum wheat genome, speciﬁc to each breeder’s targets and selection procedures, as well as shared preferences across breeding programs. The large set of unique alleles in the germplasm of historical breeding groups from Central Europe, Central Asia and Italy appear as a function of the longer eﬀort to improve adaptation compared to more recent breeding groups. Genetic Diversity and Population Structure in GDP and Breeding Groups CIMMYT and North America groups, respectively (clusters Cls_clv-chr2A.1 and Cls_clv-chr2A.1, Cls- chr2B.1; Supplementary Table S8), indicating a generalized selection strategy to ﬁne tune the photoperiod insensitive alleles The EPO is an evolutionary durum wheat pre-breeding population obtained through initial crossing of modern French varieties with various tetraploid wheat subspecies (David et al., 2014). When compared to landraces and modern durum lines, EPO lines showed the same level of genetic diversity in terms of mean number of pairwise diﬀerences and expected heterozygosity of modern lines, indicating that the genetic background of EPO lines is relatively homogeneous while being enriched in exotic alleles. Substantial agreement between NJ, ADMIXTURE and Ward’s clustering indicated a complex, still well-deﬁned stratiﬁcation of the population, driven by historical, geographical and environmental factors. Phylogenetic analysis (Figure 5) highlighted three well-deﬁned landrace groups of geographically distinct origin, holding a pivotal role as founders of diﬀerent breeding programs. These included landraces from North Africa, West Asia and Central Asia as founders of modern breeding, in particular of ICARDA and Italy (Kabbaj et al., 2017; Soriano et al., 2018), while Central Asian landraces have played a critical role in the foundation of the North American modern durum germplasm via the early introduction from Russia and Turkey by Mennonite immigrants (Moon, 2008; Paulsen and Shroyer, 2008). The identiﬁcation of these founders concurs with the results reported by Kabbaj et al. (2017), Maccaferri et al. (2019), and Taranto et al. (2020), supporting the validity of the phylogeny studies conducted for the GDP. Genetic Diversity and Population Structure in GDP and Breeding Groups The large set of unique alleles, a high proportion of which were rare in Central Europe (58%) and Italy (99%), is consistent with extended selection for a particular environment. Studies aiming to describe allele ﬁxation and genetic diversity are of great importance to guide breeders in planning their crosses and introgressions (Kabbaj et al., 2017; Taranto et al., 2020). In this regard, unique alleles can be seen as strategic targets for capturing exploitable genetic variability when linked to important traits. important traits. The inﬂuence of selection on the genome was reﬂected in the diversity reduction index (DRI) and Fst metrics. Overall putative selection signals were found throughout the entire genome, including the centromeric regions. The average signal size of 50 Mbp suggested strong selection pressure. Several PSW clusters identiﬁed in this study co- located with known loci relevant to durum wheat breeding, thus demonstrating the predictive validity of the genome- wide search method. Expected signals associated with the transition from landraces to modern were related to the control of traits strongly selected in the post Green Revolution period causing the almost complete ﬁxation of such loci in the modern subpopulations. As an example, Cls-chr4B.2 included the widely used Rht1-B (Khush, 2001; Evenson and Gollin, 2003; Borojevic and Borojevic, 2005). This locus has also been identiﬁed as a putative signal of selection when comparing the ’70–’80 and ’81–’90 decades (Cls-chr4B.1, Supplementary Table S7) as well as when contrasting North American germplasm (tall cultivars) vs. Italy/France (semi- dwarf), and ICARDA (mix tall and semi-dwarf) vs. Italy (all semi-dwarf) breeding programs. Phenology is also a trait under strong and constant selection pressure, supported by the PSW cluster in the landraces vs. modern germplasm (Supplementary Table S6) that co-located with the photoperiod insensitive gene Ppd-A1 (Beales et al., 2007; Maccaferri et al., 2008; Wilhelm et al., 2009; Bentley et al., 2011). The signal marked the transition from landraces to modern cultivars since the photoperiod insensitive allele was widely and positively selected, as already reported by Motzo and Giunta (2007). Following the Green Revolution, selection for photoperiod insensitivity continued as shown by the inclusion of both PPD homeologs on chromosomes 2A and 2B in cluster signals. PSW signals for the Ppd-A1 and Ppd-B1 regions were identiﬁed from comparisons of the Italian, French and ICARDA breeding groups vs. Putative Signature of Selection Across the Breeding History and the Breeding Groups The phytoene synthase, Psy-B1, a major gene responsible for yellow pigment content in the wheat grain and a common target of modern durum breeding for semolina color is a strong candidate (Pozniak et al., 2007). A signal for this locus emerged from the comparison of landraces vs. modern lines and North America (Cls-chr7B.12) vs. French and ICARDA breeding groups (Supplementary Tables S6, S9). The signal also appeared for three decade pairwise comparisons (Supplementary Table S7). suggesting a common historical selection for yellowness based on a number of co-located QTL clusters (Roncallo et al., 2012; Giraldo et al., 2016; Colasuonno et al., 2019) associated to speciﬁc Psy-B1 alleles (reviewed in Colasuonno et al., 2019). y pp y PSW clusters could also be related to selection for increased spike fertility and grain yield potential, particularly in the landrace to modern comparisons (Supplementary Table S6). This is the case of Cls-chr3B.2 and Cls-chr7A.2 whose intervals include the determinant of grain weight identiﬁed in bread wheat TaCKX6 (cytokinin oxidase/dehydrogenase, Zhang et al., 2012) and TaTGW-7A (Hu et al., 2016), respectively. Additionally, Cls-chr2A.4 and Cls-chr2B.3 overlapped with the recently cloned gene related to ﬂoret fertility GNI-A1 (Sakuma et al., 2019), while in some comparisons among breeding groups (Supplementary Table S8) a PSW cluster (Cls-chr2A.3) overlaps with TaSus2 (sucrose synthase), a main driver of starch accumulation in wheat found to be associated with strong changes in haplotype frequency in bread wheat (Hou et al., 2014). Considering nitrogen metabolism and grain protein content, an important quality trait for durum wheat, the landraces vs. modern contrast co-located Cls-chr2A.5 and Cls- chr2B.5 with genes encoding for glutamine synthase GS2- 2A and GS2-2B (Supplementary Table S8). Both these genes play a key role in high protein content (Gadaleta et al., 2011). Clusters could be related to selection for quality of grain proteins as shown by Cls-chr1B.4 and Cls-chr6A.1 overlapping with genes for glutenins (Glu-B1, Xu et al., 2008) and gliadins (Gli-6A, Gu et al., 2004), respectively. In particular, Cls-chr6A.1 was detected for landraces vs. modern and for three breeding programs pairwise comparisons (i.e., ICARDA, CIMMYT and Italy vs. North America and France) (Supplementary Tables S6, S9), while Cls-chr1A.1 was identiﬁed in three decade pairwise comparisons and in ICARDA vs. CIMMYT (Supplementary Tables S7, S9). Putative Signature of Selection Across the Breeding History and the Breeding Groups Intense breeding in the past decades led to the development of superior cultivars for a broad range of edaphic environments. December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 16 Genetic Diversity and Allele History Mazzucotelli et al. to match the ideal phenology for the targeted environment (Maccaferri et al., 2008). (PSW found for decade and breeding program pairwise comparisons, co-locating with Glu-A3 and gliadins), Cls-chr1B.4 (Glu-B1), Cls-chr6A.1, Cls-chr6A.2 and Cls-chr6A.3, with the last three PSW partially overlapping and co-locating with Gli-6A (Supplementary Tables S7, S9). (PSW found for decade and breeding program pairwise comparisons, co-locating with Glu-A3 and gliadins), Cls-chr1B.4 (Glu-B1), Cls-chr6A.1, Cls-chr6A.2 and Cls-chr6A.3, with the last three PSW partially overlapping and co-locating with Gli-6A (Supplementary Tables S7, S9). Another important class of genes known to have undergone strong selective pressure in bread wheat are the VRN. In contrast to PPD, the PSW signal for VRN loci was much weaker in the GDP. For instance, no PSW cluster included Vrn1-5A (Yan et al., 2003), while Vrn3-7A (Yan et al., 2006) generated PSW signals in both A and B sub-genomes. For example, Cls-chr7A.4 was identiﬁed in the North American group vs. ICARDA, CIMMYT and Italy; Cls-chr7A.5 was identiﬁed for the comparisons of CIMMYT vs. ICARDA and Italy; and Cls-chr7B.1 corresponded to Vrn3-7B for the comparisons of CIMMYT vs. France and Italy (Supplementary Table S8). Mild vernalization requirements are still present in modern cultivars for the Mediterranean areas where wheat is cultivated as a fall-sown cereal, and distinctions at these loci might depend on the breeder’s target of extending or reducing the overall cycle in diﬀerent agro- ecologies. Lastly, among the earliness per se genes, ELF3-A1 (Zikhali et al., 2016) appears the most likely candidate for the PSW cluster Cls-chr1A.8, which diﬀerentiated both France and North America modern germplasm when comparing ICARDA and Italy (Supplementary Table S8). Lastly, presence of gene candidates was observed for three strong PSW clusters that occurred in chromosome 7B (Cls-chr7B.3, centromeric and Cls-chr7B.12, distal) and in chromosome 5B (Cls-chr5B.5) and that are putatively related to grain quality. The two signals in chromosome 7B were associated to a strong QTL for grain yellow pigment content (reviewed in Colasuonno et al., 2019). Putative Signature of Selection Across the Breeding History and the Breeding Groups The co-localization between PSW clusters and glutenin and gliadin alleles is not unexpected given the inﬂuence of these genes on pasta quality, which is a major target of selection. Convincingly, three chromosomes, 1A, 1B, and 6A, involved in seed storage proteins were represented in the PSW clusters: Cls-chr1A.1 A recent study Taranto et al. (2020), aiming to deﬁne PSW among Italian cultivars and landraces also identiﬁed several of the selection sweeps proposed here, including the major loci controlling phenology and quality characteristics. In summary, the report of PSW clusters in this manuscript is a ﬁrst attempt to carry out such analysis across breeding programs from diﬀerent countries. Although the causative genes of the PSW clusters remain to be veriﬁed, several plausible candidates have been proposed. The GDP provides then an unprecedented opportunity for international collaborations to more eﬀectively harness and exploit the diversity identiﬁed here. 5http://indms.icarda.org/ Frontiers in Plant Science \| www.frontiersin.org ACKNOWLEDGMENTS The authors wish to thank Mr. Abu Nakad Rukoz, M.me Nada Saghbini, and M.me Hoda Abou Younes for maintaining, multiplying, and distributing the pure seeds of the GDP collection in Lebanon. Recognition goes also to the several germplasm donors that supported this international initiative. DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories: GrainGenes https://wheat.pw.usda.gov/GG3/ global_durum_genomic_resources, and T3/Wheat https://wheat. triticeaetoolbox.org/breeders_toolbox/protocol/158. AUTHOR CONTRIBUTIONS FB, RT, MM, LC, JA, KA, and SX designed this initiative. EM, DM, SC, SX, JF, and MH produced the genotypic data and all authors supported the genotyping. EM, GS, AM, FD, GP, MM, RT, LC, and FB analyzed the data. EM, GS, AM, MM, and FB developed the ﬁrst draft. All authors reviewed and approved the ﬁnal version of this manuscript. CONCLUSION In the present study, a very large and diverse durum wheat panel referred to as the GDP has been assembled and made publicly available to drive further discovery and deployment of beneﬁcial alleles. The GDP is maintained and distributed by ICARDA Genbank5 under Terms and Conditions of SMTA. The genotypic datasets (both raw data and upon quality ﬁltering and imputing) can be found in the online repositories GrainGenes (see text footnote 3), and T3/Wheat (see text footnote 4). The genetic characterization of this panel increases the knowledge of genetic relationships and population structure of worldwide durum wheat, while facilitating the identiﬁcation of the optimal sources of genetic diversity for a given target locus. The entire durum community is now empowered to use this panel to discover novel and useful alleles via GWAS. Finally, since the GDP is an open resource available to the whole community, the discovery of useful alleles can be immediately incorporated December 2020 \| Volume 11 \| Article 569905 Frontiers in Plant Science \| www.frontiersin.org 17 Genetic Diversity and Allele History Mazzucotelli et al. Agriculture and Agri-Food of Canada, Saskatchewan Wheat Development commission, SeCan, and the Saskatchewan Ministry of Agriculture; data analysis was partially supported by: PRIMA2019 CEREALMED “Enhancing diversity in Mediterranean cereal farming systems,” H2020 InnoVar Project “Next generation variety testing for improved cropping on European farmland,” APSOV-UNIBO 2020–2022 Research Agreement “Identiﬁcation of loci and markers of agronomic interest in wheat” and MIPAAF Italy Systemic-1063 “An integrated approach to the challenge of sustainable food systems: adaptive and mitigatory strategies to address climate change and malnutrition.” Several partners dedicated time and eﬀort in kind to ensure the good outcome of this initiative. in breeding activities irrespective of the country or research group that makes the discovery. This is particularly true now that a number of genomic resources are available for wheat, including the reference sequence of the durum wheat genome (Maccaferri et al., 2019). We believe that this international eﬀort is a great example of how a whole community can come together to support breeders in their eﬀorts to adapt and develop more resilient durum wheat varieties able to withstand climate change and ensure a great future for this important crop. FUNDING The work of the Global Durum wheat Panel was ﬁnancially made possible by several international and national donors: the Wheat Initiative – Expert Working Group in Durum Wheat Genomics and Breeding supported the meetings and interactions of the durum wheat research community; CRP WHEAT (CIMMYT) supported the genotyping with KASP of the DWRC collection; “Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives,” which is supported by the Government of Norway, managed by the Global Crop Diversity Trust with the Millennium Seed Bank of the Royal Botanic Gardens, Kew supported the ﬁeld work for seed puriﬁcation and multiplication; CRP WHEAT CoA 3.2 supported the seed distribution to partners; import of seeds and respect of quarantine procedure was supported by USDA-ARS; the high resolution genotyping work was conducted under: H2020-MSCA-RISE 2015 EXPOSEED (ID: 691109) “Exploring the molecular control of seed yield in crops, PICT-2015-1401 ANPCyT- Argentina “Análisis de la estructura del genoma y mapeo por asociación para caracteres de calidad y rendimiento en trigo candeal,” Genome Canada – Genome Prairie (Saskatchewan Ministry of Agriculture), FAO/ITPGRFA (W3B-PR-21 Morocco), Premier’s Research and Industry Fund (Government of South Australia – IRGP15), GRDC (DAN00163), USDA-ARSUSDA-ARS (3060-21000-038-00D), Lieberman-Okinow Endowment at University of Minnesota, Grains Research and Development Corporation (GRDC) and the University of Adelaide, International Funding Initiative of Figure S1 \| Population structure of the DWRC collection based on ADMIXTURE analysis. Figure S2 \| Population structure of the DWRC collection based on bootstrapped Ward’s clustering. Figure S3 \| Bootstrapped Ward’s clustering of the DWRC subgroup of T. durum cultivars, varieties and élite lines. Figure S4 \| Bootstrapped Ward’s clustering of DWRC subgroup EPO. Figure S5 \| Bootstrapped Ward’s clustering of DWRC subgroup of T. durum landraces. Figure S6 \| Bootstrapped Ward’s clustering of DWRC subgroup of T. dicoccum accessions. Figure S7 \| Bootstrapped Ward’s clustering of DWRC subgroup of T. dicoccoides accessions. Figure S8 \| Bootstrapped Ward’s clustering of DWRC subgroup of T. turgidum subspecies carthlicum, aethiopicum, polonicum, turanicum, turgidum. Figure S9 \| Sampling effect on genetic diversity between DWRC and GDP: correlation of diversity indexes between GDP and DWRC for Shannon-Wiener index, expected heterozygosity, evenness, and minor allele frequency. Figure S10 \| Site frequency spectrum of loci in GDP and DWRC. Figure S11 \| Distribution of the SNPs along the chromosome and inter SNP distances. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2020. 569905/full#supplementary-material REFERENCES Canè, M. A., Maccaferri, M., Nazemi, G., Salvi, S., Francia, R., Colalongo, C., et al. (2014). Association mapping for root architectural traits in durum wheat seedlings as related to agronomic performance. Mol. Breed. 34, 1629–1645. doi: 10.1007/s11032-014-0177-1 Abu-Zaitoun, S. Y., Chandrasekhar, K., Assili, S., Shtaya, M. J., Jamous, R. M., Mallah, O. B., et al. (2018). Unlocking the genetic diversity within a middle-east panel of durum wheat landraces for adaptation to semi-arid climate. Agronomy 8:233. doi: 10.3390/agronomy8100233 Cavanagh, C. R., Chao, S., Wang, S., Huang, B. E., Stephen, S., Kiani, S., et al. (2013). 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Bioinformatics 23, 2633–2635. doi: 10.1093/ bioinformatics/btm308 Dubcovsky, J., and Dvorak, J. (2007). candidate gene; (B) signiﬁcant values for each metrics (Fst, DRI) for each SNP sliding window. candidate gene; (B) signiﬁcant values for each metrics (Fst, DRI) for each SNP sliding window. Figure S12 \| Local LD of T. durum landraces and modern lines presented for each chromosome. Figure S13 \| Bootstrap neighbor joining phylogenetic tree of GDP. Table S7 \| PSWs for modern, between different decades: (A) list of clusters of PSWs between different decades, with position on the Svevo reference genome, metrics detecting PSWs in each comparison, and the candidate gene; (B) signiﬁcant values for each metrics (Fst, DRI) for each SNP sliding window in each comparison. Table S1 \| List of private companies, institutions, international organizations which contributed tetraploid wheat germplasm to the initial DWRC. Table S2 \| DWRC: (A) list of accessions constituting the DWRC; (B) scoring of DWRC accessions based on KASP marker set; (C) KASP markers list used for the DWRC genotyping. Table S8 \| PSWs for modern, between different breeding groups: (A) list of clusters of PSWs between different breeding programs, with position on the Svevo reference genome, metrics detecting PSWs for each comparison, and the candidate gene; (B) signiﬁcant values for each metrics (Fst, DRI) for each SNP sliding window in each comparison. Table S3 \| List of accession constituting the GDP, with passport data. The categories based on passport data used to classify accessions for the diversity analyses are also reported, as well as available data about ﬂowering habit and allele status at some known genes (Rht, Ppd, Cdu, Vrn, etc.). Table S9 \| Stratiﬁcation analysis of GDP: (A) grouping on the base of the main model-based ancestry estimation and neighbor joining tree position. Accessions are sorted on the base of their position on the NJ tree of Figure 5 and colors correspond to those of groups highlighted in the same Figure 5; (B) Ward’s clustering of GDP from K2 to K20; (C) membership value for each GDP accession at K = 13 based on ADMIXTURE ancestry estimation. Table S4 \| Genetic distance matrix of the GDP. Table S5 \| List of unique alleles within the breeding groups of the GDP. 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Cereal Sci. 47, 394–398. doi: 10.1016/j.jcs.2007.05.002 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Yan, L., Fu, D., Li, C., Blechl, A., Tranquilli, G., Bonafede, M., et al. (2006). The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc. Natl. Acad. Sci. U.S.A. 103, 19581–19586. doi: 10.1073/pnas.0607142103 The reviewer MR declared a past co-authorship with several of the authors DM and FL to the handling editor. Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima, T., and Dubcovsky, J. (2003). Positional cloning of the wheat vernalization gene VRN1. Proc. Natl. Acad. Sci. 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https://openalex.org/W1566309247	https://europepmc.org/articles/pmc2363489?pdf=render	English	null	Osteopontin is required for full expression of the transformed phenotype by the ras oncogene	British journal of cancer	2,000	cc-by	9,677	Y Wu, DT Denhardt and SR Rittling1 The protein was originally characterized as transformation-associated due to its increased expression in transformed cell lines (Senger et al, 1983): indeed, a wide variety of transformed cells in culture express much higher levels of the protein than their normal counterparts (Senger et al, 1988). These observations have been extended in that among transformed cells, the highest levels of expression were found in the most metastatic cell lines (Chambers et al, 1992a). OPN is particularly highly expressed in ras-transformed cells (Chambers et al, 1990), and ras has a direct effect on OPN transcription, via a ras-stimulated transcription factor that enhances transcription of the OPN gene (Guo et al, 1995). OPN is also highly expressed in tumors in vivo. In papillomas and carcinomas induced by DMBA/TPA initiation/promotion in mouse skin, OPN expression levels correlate with tumor stage (Craig et al, 1990). In mammary tumors arising in transgenic mice expressing ras and/or myc specifically in mammary gland, OPN expression is dramatically increased at both the message and protein level over levels in the corresponding normal mammary gland (Rittling and Novick, 1997). OPN is typically overexpressed in human tumors (Brown et al, 1994; Bellahcène and Castronovo, 1995; Hirota et al, 1995; Tuck et al, 1998), although the source of the protein in these malignancies can be either tumor cells or tumor-associated macrophages (Brown et al, 1994; Casson et al, 1997) The secreted phosphoprotein osteopontin (OPN), ubiquitously expressed in body fluids, binds with high affinity to integrins of the αv class (Liaw et al, 1995; Hu et al, 1995a; 1995b) including αvβ3, αvβ1, and α vβ5. Interaction of the protein with several β1 integrins has also been reported recently (Smith et al, 1996; Denda et al, 1998; Bayless et al, 1998), as well as with a non-integrin cell surface receptor, CD-44 (Weber et al, 1996; Katagiri et al, 1999). In vivo, OPN expression is increased in a variety of pathologies (reviewed in Rittling and Denhardt, 1999), where it may act as a macrophage chemoattractant (Singh et al, 1990; Giachelli et al, 1998), or as a repressor of induced nitric oxide synthase (iNOS) levels (Hwang et al, 1994; Rollo et al, 1996): this activity may be especially relevant in pathologies involving ischaemia, in which nitric oxide (NO) production is thought to be an important medi- ator of tissue damage (Goligorsky et al, 1997; Noiri et al, 1999). Y Wu, DT Denhardt and SR Rittling1 Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA Summary The secreted phosphoprotein osteopontin (OPN) is strongly associated with the process of neoplastic transformation, based both on its pattern of expression in vivo and in vitro and on functional analyses. We have used 3T3 cells derived from wildtype and OPN-deficient mice and transformed by transfection with oncogenic ras to assess the role of OPN in transformation in vitro and in tumorigenesis in vivo. There was no effect of an absence of OPN on the ability of the cells to undergo immortalization or to form morphologically transformed foci following ras transfection. Wildtype and OPN-deficient cell lines were established from such foci, and lines with similar ras mRNA levels selected for further analysis. Ras-transformed cell lines from both wildtype and OPN-deficient mice could form colonies in soft agar indicating that this process can occur in the absence of OPN. However, the ability of the OPN-deficient cell lines to form colonies was reduced as compared to wildtype cell lines. Tumorigenesis in syngeneic and nude mice was assessed for a subset of cell lines that formed colonies efficiently in soft agar. Cell lines unable to make OPN formed tumors in these mice much more slowly than wildtype cells, despite similar growth of the cells on plastic and in soft agar. Taken together, these results indicate that maximal transformation by ras requires OPN expression, and implicate increased OPN expression as an important effector of the transforming activity of the ras oncogene. © 2000 Cancer Research Campaign Keywords: tumorigenesis, anchorage-independence, ras, integrins, immortalization 1987; Chambers et al, 1993; Senger et al, 1994), and stimulates migration of several cell types, including endothelial cells (Senger et al, 1996) and smooth muscle cells (Liaw et al, 1995). Changes in cell adhesion properties are hallmarks of the processes of tumorigenesis and metastasis. Indeed, normal cells depend on interaction with the extracellular matrix for growth, and the loss of this dependence is the aspect of cell transformation in vitro most closely correlated with tumorigenicity (Freedman and Shin, 1974). The integrin family of cell surface receptors mediates attachment of cells to the extracellular matrix (reviewed in Hynes, 1992; Juliano, 1994), and alterations in integrin binding or signaling properties are being increasingly recognized as important in tumorigenesis (Ruoslahti, 1997; Clezardin, 1998). There is extensive evidence derived from observations made both in vitro and in vivo linking elevated osteopontin expression and transformation/tumorigenesis. British Journal of Cancer (2000) 83(2), 156–163 © 2000 Cancer Research Campaign British Journal of Cancer (2000) 83(2), 156–163 © 2000 Cancer Research Campaign © 2000 Cancer Research Campaign doi: 10.1054/ bjoc.2000.1200, available online at http://www.idealibrary.com on doi: 10.1054/ bjoc.2000.1200, available online at http://www.idealibrary.com on Received 28 October 1999 Accepted 17 February 2000 Correspondence to: SR Rittling MATERIALS AND METHODS RNA preparation, analysis, and probes were as described (Rittling and Novick, 1997). Western blotting for OPN was as in (Rittling et al, 1998b) and (Rittling and Feng, 1998), using antiserum 732, developed in the OPN-deficient mice against mouse osteopontin (Kowalski et al, unpublished). For quantification of ras protein levels, cells were scraped into RIPA buffer with protein inhibitors, and the protein concentration determined. Equal amounts of protein (25–35 µg) were separated on 12% SDS-PAGE and blotted. The blots were reacted with anti-H-ras antibody F235 (Santa Cruz Biotechnology, non-cross reactive with N-ras and K-ras) at 1 µg ml–1. Transfection and selection already transformed cells indicated that the protein is important in transformation. Thus, transfection of different antisense OPN constructs or ribozymes into transformed, tumorigenic cells resulted in impaired anchorage-independent growth, as well as tumor-forming ability in vivo (Gardner et al, 1994; Su et al, 1995; Feng et al, 1995; Behrend et al, 1994). Even more compelling are results showing that transfection of OPN expression constructs into cells producing benign tumors converts them to a malignant phenotype (Oates et al, 1996; Chen et al, 1997). Plasmid DNA (pSV-Ha-ras: 20 µg per 100 mm dish, 10 µg per 60 mm dish) was introduced into cells by the calcium phosphate precipitation method, followed by glycerol shock (Malyankar et al, 1994). Foci, identified as clumps of piled-up cells against a background monolayer, were picked after incubation for 2 weeks with medium containing 3% or 1% FBS, to select for cells with reduced growth factor requirements. Transfection efficiency was assessed by cotransfection with a β-galactosidase expression construct. In order to more clearly define the role OPN plays in transfor- mation in vitro and tumorigenesis in vivo, we have examined these processes in cells derived from mice with a targeted disruption of the OPN gene (Rittling et al, 1998). The results presented here indicate that while anchorage-independent growth and tumor formation can occur in the absence of OPN, these phenotypes, in particular tumor formation in vivo, are substantially enhanced in wildtype, OPN-expressing cells as compared to OPN deficient ones. Thus, OPN is required for full expression of the transformed phenotype. Mice Wildtype and OPN-deficient mice were housed and bred sepa- rately under specific pathogen-free conditions as described (Rittling et al, 1998). Embryos were obtained from homozygous females, either wildtype or OPN-deficient, that had been mated to males of the same genotype. Mice used for embryo collection were in the 129 × C57BL/6 F2 genetic background. Growth curves Cells were plated in 12-well dishes at 2.7 × 104 cells per well. Each day for 6 days, cells were removed from triplicate wells and counted, and the results plotted. Cell number at 3 days after plating, towards the end of the exponential growth phase, is reported. Osteopontin is required for maximal transformation 157 Osteopontin is required for maximal transformation 157 Preparation and immortalization of mouse embryo fibroblasts Mouse embryo fibroblast (MEF) cells were prepared as described (Rittling, 1996). Briefly, mouse embryos were removed from the uterus of 12–16-day pregnant mice, minced finely with scissors and digested with 0.125% trypsin. The cells obtained were plated on 150 mm tissue culture dishes at a density of 1 × 107 in DMEM (Gibco, Grand Island NY, USA) supplemented with 10% FBS (Gemini Bio-Products, Calabasas, CA, USA). For the first and second passages, cells were plated on 100 mm tissue culture dishes at a density of 1 × 106; in later passages, cells were plated at 3T3 density (8 × 105) on 100 mm dishes. For immortalization, cells were passed every three days and plated at 8 × 105 on 100 mm dishes (Rittling, 1996). The numbers of cells plated (No), and the numbers of cells after three days of growth (N) were recorded, and the ratio (N/No) was calculated as a growth-rate indicator. Cells were considered to be immortalized when the ratio N/No became higher than 3.0. Tumor formation Subconfluent, rapidly growing cells were collected and suspended in PBS at 1 × 106 cells per ml, and 0.5 ml of this cell suspension was injected subcutaneously in the upper dorsal region of 3–4 syngeneic (129 × C57Bl/6 F1, wildtype) and/or nude mice. Tumor size was measured with calipers every second day until the tumors reached 20% of the weight of the animal. Tumor volume was calculated according to the following formula: 4/3π (l-1) (w-1)2, the volume of an oblate ellipse, where l is the long dimension of the tumor, and w is the width, or short dimension, in mm; 1 mm was subtracted from each measurement to compensate for the Colony formation in soft agar Anchorage-independent growth assays were performed using 6- well plates (Freedman and Shin, 1974). Experiments were done in triplicate. Each well was coated with 2 ml of a base layer containing 0.6% agar, 1 × DMEM, 10% FBS. Subconfluent cells were collected in 10 ml of medium, counted and suspended in DMEM containing 10% FBS, 0.3% agar and 1 ml of the mixture containing 104 cells was plated over the 2 ml base layer in each well (three wells total for each cell line). After 14 days incubation with twice-a-week feeding (1 ml each time per dish of DMEM, 0.3% agar and 10% FBS), colonies (consisting of about 20 cells or more) in 10 high-power fields per dish were counted, and total colonies per dish calculated. Y Wu, DT Denhardt and SR Rittling1 In vitro, the protein has cell attachment activity (Somerman et al, A series of functional studies links OPN causally to tumori- genesis. Experiments in which OPN mRNA level was reduced in 156 Tetracycline-inducible cell lines OPN-deficient 3T3 cells were transfected sequentially with pTet- Off (pUHD-15-1) (Gossen and Bujard, 1992), and with OPN under the control of the Tet operator, and the clones expressing the highest level of OPN selected for further analysis. The resulting clonal lines were transfected with pSV-Ha-ras, and foci were picked. Transformed cells from these foci were plated in soft agar at 105 cells per 35 mm well, in the presence or absence of 2 µg ml–1 tetracycline. British Journal of Cancer (2000) 83(2), 156–163 © 2000 Cancer Research Campaign 158 Y Wu et al 158 Y Wu et al +/+ –/– E F C D A B G H Figure 2 Comparison of focus-forming ability between ras-transfected wildtype and OPN-deficient spontaneously immortalized cells. 3T3 cells were transfected with 20 µg pSV-Ha-ras per 100 mm dish using a calcium phosphate precipitation method. After growth at 37°C for 2 weeks in DMEM plus 3% FBS, the cells were fixed with methanol and stained with Giemsa. A and B: Mock transfection; A, C, E, G: wildtype 3T3 lines; B, D, F, H: OPN-deficient 3T3 lines. C–H represent focus formation in six independently derived 3T3 lines. 1016 1015 1014 1013 1012 1011 1010 109 108 107 106 0 20 40 60 80 Days in culture Cumulative cell number Figure 1 Immortalization rate of the MEFs derived from wildtype and OPN-deficient mice. Shown is the cumulative cell number in the population plotted as a function of days in culture. Closed symbols represent cells derived from wildtype mice, and the open symbols represent cells derived from the OPN-deficient mice. Each line represents immortalization of cells from a single mouse. 1016 1015 1014 1013 1012 1011 1010 109 108 107 106 0 20 40 60 80 Days in culture Cumulative cell number B A Cumulative cell number C Figure 1 Immortalization rate of the MEFs derived from wildtype and OPN-deficient mice. Shown is the cumulative cell number in the population plotted as a function of days in culture. Closed symbols represent cells derived from wildtype mice, and the open symbols represent cells derived from the OPN-deficient mice. Each line represents immortalization of cells from a single mouse. E thickness of the skin. In cases where results from only two mice are shown, a third animal in the experiment was sacrificed prior to 20 days, usually because the tumor had grown beyond 20% of the weight of the animal. Tetracycline-inducible cell lines For metastasis, 105 cells in 0.1 ml of PBS were injected via the lateral tail vein of syngeneic F1 mice, and the mice were sacrificed after 4 weeks. Lungs were excised, fixed and cut into 1–2 mm slices prior to embedding. Several lung slices were embedded in a single block, and tumor size in the resultant H+E stained sections determined by measurement of the projected image. Mice were housed in microisolator cages in an AALAC- approved animal facility, and all procedures were approved by the Rutgers University Institutional Review Board – Animal Care and Facilities Committee. G H Figure 2 Comparison of focus-forming ability between ras-transfected wildtype and OPN-deficient spontaneously immortalized cells. 3T3 cells were transfected with 20 µg pSV-Ha-ras per 100 mm dish using a calcium phosphate precipitation method. After growth at 37°C for 2 weeks in DMEM plus 3% FBS, the cells were fixed with methanol and stained with Giemsa. A and B: Mock transfection; A, C, E, G: wildtype 3T3 lines; B, D, F, H: OPN-deficient 3T3 lines. C–H represent focus formation in six independently derived 3T3 lines. Establishment of immortal and transformed cell lines The average number of foci formed was 85 ± 26.5 for WT cell lines and 87 ± 27.2 for OPN –/– lines. lines. Figure 4 illustrates these data for a single set of cell lines: two additional sets were similarly analysed. Following normaliza- tion to β-actin mRNA levels, neither ras nor cathepsin L mRNA levels were found to differ significantly between the wildtype and OPN-deficient clones. For each immortal cell line, 10–20 foci were picked, and expanded. RNA was prepared from the resulting transformed cell lines while they were in exponential growth 3 days after plating, and analyzed for ras mRNA levels by northern blotting. This screening process was undertaken to select for transformed clones that expressed comparable levels of ras mRNA, to minimize differences between clones due to differential expression of the transfected ras allele. Thus, in each set of screened clones, wild- type and OPN-deficient clones that expressed similar levels of ras mRNA were selected and expanded for further analysis. From six independent immortal cell lines (three of each genotype) a total of 19 wildtype and 17 OPN-deficient ras-transformed cell lines were selected for further analysis. The ability of the transformed cell lines to grow under anchorage-independent conditions was tested by seeding 104 cells in 35 mm wells in 0.3% agar in complete medium with 10% FCS. After 2 weeks, colonies containing more than 20 cells were counted. The OPN-deficient cells were able to form colonies in soft agar under these conditions, and in several cell lines colonies were formed with as high efficiency as the wildtype cell lines (Figure 4), and the morphology of the colonies was similar (data not shown). However, when the number of colonies formed per 104 cells plated was averaged over all the cell lines analyzed (19 wildtype and 17 OPN –/–), the wildtype cell lines were found to form nearly twice as many colonies as the OPN-deficient cells (Figure 5), despite considerable variability in the numbers of colonies formed by the individual cell lines. To confirm this observation, ras transformed OPN –/– cell lines were developed that expressed OPN under the control of a tetracycline-repressible promoter (Gossen and Bujard, 1992). In these cells OPN expression is reduced about 10-fold in the presence of tetracycline. Establishment of immortal and transformed cell lines 601 389 785 154 1181 1071 777 609 543 0 0 462 Figure 4 ras, cathepsin L, and OPN mRNA levels and colony formation in soft agar of a series of wildtype (+/+) and OPN-deficient (–/–) transformed cell lines. Cell lines with comparable ras levels were selected from the initial screen and expanded. RNA was prepared after 3 days’ growth, and growth in soft agar was determined as described in Methods. Lanes labelled PAP2 are RNA isolated from the metastatic, ras transformed NIH 3T3 cell line PAP2 (Chambers et al, 1990). Lanes 2 and 3 (3T3) contain RNA prepared from the parental 3T3 cells used for transfection. The same blot was probed for ras (top panel), cathepsin L (middle panel), and OPN (lower panel). The experiment shown is representative of three such experiments. The number of colonies formed per 104 cells plated in soft agar for each cell line is shown at the bottom of the figure. Figure 4 ras, cathepsin L, and OPN mRNA levels and colony formation in soft agar of a series of wildtype (+/+) and OPN-deficient (–/–) transformed cell lines. Cell lines with comparable ras levels were selected from the initial screen and expanded. RNA was prepared after 3 days’ growth, and growth in soft agar was determined as described in Methods. Lanes labelled PAP2 are RNA isolated from the metastatic, ras transformed NIH 3T3 cell line PAP2 (Chambers et al, 1990). Lanes 2 and 3 (3T3) contain RNA prepared from the parental 3T3 cells used for transfection. The same blot was probed for ras (top panel), cathepsin L (middle panel), and OPN (lower panel). The experiment shown is representative of three such experiments. The number of colonies formed per 104 cells plated in soft agar for each cell line is shown at the bottom of the figure. such spontaneously derived immortal cell lines represent fairly homogeneous populations (Rittling, 1996). These immortal cell lines were transfected with an activated ras construct (pSV-Ha-ras), and grown in the presence of 3%, or in some cases 1%, fetal bovine serum for 2 weeks, to select for cells with reduced serum requirements. Morphologically transformed foci formed in every case with similar efficiencies (Figure 2, Table 1), and there was no apparent effect of a lack of OPN on focus formation. Establishment of immortal and transformed cell lines Primary embryo fibroblasts were prepared from 13–15-day preg- nant females. Initial experiments in which these primary cells were transfected with a mutant ras construct together with an activated p53 construct indicated that in both wildtype and OPN-deficient mice, the rate of focus formation was too low to be useful. Therefore, spontaneously immortalized cell lines were derived by continuous passage according to the 3T3 protocol. Three indepen- dent mouse embryo fibroblast (MEF) cultures from both wildtype and OPN-deficient mice were obtained, and passed to obtain immortal cell lines. While there is considerable variability in the rate at which different cell lines pass through crisis (Rittling, 1996), there was no consistent difference between the wildtype and OPN-deficient cells (Figure 1), nor was there any consistent difference in the average growth rate of the wildtype and OPN- deficient cell lines (data not shown). Previous work has shown that © 2000 Cancer Research Campaign Table 1 Focus formation in wildtype (+/+) and OPN-deficient (–/–) immortal cell lines after ras transfection. 3T3 cell lines were plated at 5 × 105 cells per 100 mm dish, and transfected with an activated ras expression construct. Two weeks following transfection, plates were fixed and stained, and the total foci in one representative 100 mm dish were counted. Transfection efficiency did not vary significantly between the transfections. Results from three independent experiments with six different 3T3 lines are shown. Experiment Number of foci Number of foci (+/+) (–/–) 1 84 118 2 59 67 3 112 76 Table 1 Focus formation in wildtype (+/+) and OPN-deficient (–/–) immortal cell lines after ras transfection. 3T3 cell lines were plated at 5 × 105 cells per 100 mm dish, and transfected with an activated ras expression construct. Two weeks following transfection, plates were fixed and stained, and the total foci in one representative 100 mm dish were counted. Transfection efficiency did not vary significantly between the transfections. Results from three independent experiments with six different 3T3 lines are shown. © 2000 Cancer Research Campaign Experiment Number of foci Number of foci (+/+) (–/–) 1 84 118 2 59 67 3 112 76 British Journal of Cancer (2000) 83(2), 156–163 Osteopontin is required for maximal transformation 159 OPN MEF 3T3 T –/– MEF 3T3 T +/+ Figure 3 OPN levels secreted by primary, immortal and transformed cell lines. Media conditioned by confluent cells for 18 h were collected. Establishment of immortal and transformed cell lines Growth of these cells in soft agar, but not on plastic, was reduced in the presence of tetracycline by about 20–30% (Figure 5), confirming that OPN under certain circum- stances can facilitate anchorage-independent growth of cells. OPN protein levels in the primary, immortal, and transformed cells were monitored by western blotting of media conditioned by the different cells. While the primary wildtype cells secrete readily detectable OPN, the level is increased about 10-fold in the corre- sponding immortal cells, and increased still further, another seven- fold, after ras transfection (Figure 3). No OPN was detected in media conditioned by OPN-deficient cells. While the increase in OPN secretion after ras transformation was expected, and confirms that ras is active in these transformed cells, these results indicate that immortalization can also increase the amount of OPN secreted by cells. Establishment of immortal and transformed cell lines 10 µl of each medium was used in a western blot with mouse antiserum 732 directed against mouse OPN. A representative set of cells is shown. For each set, medium was collected from primary cells (MEF lanes), immortal cells (3T3 lanes) and transformed cells (T lanes). Similar results were obtained with two other sets of cell lines. OPN MEF 3T3 T –/– MEF 3T3 T +/+ ras CL OPN +/+ –/– PAP2 3T3(+/+) 3T3(–/–) 601 389 785 154 1181 1071 777 609 543 0 0 462 No. of colonies per 104 cells in soft agar Figure 4 ras, cathepsin L, and OPN mRNA levels and colony formation in soft agar of a series of wildtype (+/+) and OPN-deficient (–/–) transformed cell lines. Cell lines with comparable ras levels were selected from the initial screen and expanded. RNA was prepared after 3 days’ growth, and growth in soft agar was determined as described in Methods. Lanes labelled PAP2 are RNA isolated from the metastatic, ras transformed NIH 3T3 cell line PAP2 (Chambers et al, 1990). Lanes 2 and 3 (3T3) contain RNA prepared from the parental 3T3 cells used for transfection. The same blot was probed for ras (top panel), cathepsin L (middle panel), and OPN (lower panel). The experiment shown is representative of three such experiments. The number of colonies formed per 104 cells plated in soft agar for each cell line is shown at the bottom of the figure. Figure 3 OPN levels secreted by primary, immortal and transformed cell lines. Media conditioned by confluent cells for 18 h were collected. 10 µl of each medium was used in a western blot with mouse antiserum 732 directed against mouse OPN. A representative set of cells is shown. For each set, medium was collected from primary cells (MEF lanes), immortal cells (3T3 lanes) and transformed cells (T lanes). Similar results were obtained with two other sets of cell lines. Figure 3 OPN levels secreted by primary, immortal and transformed cell lines. Media conditioned by confluent cells for 18 h were collected. 10 µl of each medium was used in a western blot with mouse antiserum 732 directed against mouse OPN. A representative set of cells is shown. For each set, medium was collected from primary cells (MEF lanes), immortal cells (3T3 lanes) and transformed cells (T lanes). Similar results were obtained with two other sets of cell lines. © 2000 Cancer Research Campaign British Journal of Cancer (2000) 83(2), 156–163 Analysis of transformation in vitro +/+ = average colony formation per 104 cells of 19 wildtype cell lines; –/– = average colony formation per 104 cells of 15 OPN-deficient cells; –Tet = colony formation of 105 OPNTet cells in the absence of Tet (expressing OPN); +Tet = colony formation of 105 OPNTet cells in the presence of Tet (reduced OPN expression). Shown is the mean +/– SEM, P < 0.05. 4000 3000 2000 1000 0 0 5 10 15 20 25 30 35 Days after injection Tumour volume (mm3) Days after injection that could form colonies efficiently in soft agar and that had similar ras mRNA and protein levels (Figure 6, Table 2). These cells were collected, resuspended in PBS and injected subcutaneously into nude or syngeneic mice. The mice used for these injections were wildtype as there are at present no syngeneic OPN-deficient hosts for these 129 × C57Bl/6 F2 cells. Every second day after the tumors became palpable, the tumor size was measured with calipers. In every case the wildtype cells formed tumors significantly earlier than did the OPN-deficient cell lines (Figure 7) despite comparable growth of the cells in vitro both on plastic and in soft agar (Table 2). Similar results were obtained with both syngeneic and nude mice (Table 2). The main defect observed in the OPN –/– cells was in the lag-time before tumor formation could be detected: once the tumors were formed, the growth rate of the OPN –/– and wildtype tumors was similar (Figure 7). Thus OPN is required for efficient initiation of tumor growth in vivo in this system. Figure 7 Growth of tumours in nude mice after injection of wildtype cell line 279-3-7 (solid symbols) or OPN-deficient cell line 247-3-8 (open symbols). 5 × 105 cells in PBS were injected subcutaneously into the dorsal area of 3–4 mice. Tumour size was measured every other day, and tumour volume calculated as described in Methods. Each line represents results from a single mouse. Results for a representative pair of cell lines are shown – other cell lines gave similar results, as shown in Table 2. One pair of these cell lines was tested for the ability to form metastases in lungs following intravenous injection into syngeneic mice. Analysis of transformation in vitro Following selection and expansion of the ras transformed cell lines, ras, cathepsin L, and OPN mRNA levels were analysed and normalized to β-actin mRNA levels. Cathepsin L expression has been shown in some cases to be, like OPN, induced by activated ras (Chambers et al, 1992b), and so the level of this mRNA was used as a means of assessing ras activity in the individual cell The ability of transformed cells to form colonies in soft agar is the parameter that most closely correlates with tumor forming ability in vivo (Freedman and Shin, 1974). Accordingly, the ability of several of the wildtype and OPN-deficient cell lines to form tumors after injection subcutaneously into mice was assessed. Four sets of cell lines (wildtype and OPN-deficient) were selected British Journal of Cancer (2000) 83(2), 156–163 © 2000 Cancer Research Campaign 160 Y Wu et al 160 Y Wu et al 160 Table 2 Growth characteristics and tumour formation of different cell lines with high efficiency of growth in soft agar. The indicated cell lines were injected subcutaneously into mice. At the same passage, the growth rate of the cells on plastic and in soft agar was measured. Table 2 Growth characteristics and tumour formation of different cell lines with high efficiency of growth in soft agar. The indicated cell lines were injected subcutaneously into mice. At the same passage, the growth rate of the cells on plastic and in soft agar was measured. Growth on Growth in Tumour size Tumour size Cell line Genotype plastic × 105a agarb (syngeneic)c n (nude)c mm3 mm3 n 279–3–7 +/+ 4.5 ± 0.1 772 ± 64 3848,2636 2 2576 ± 667* 3 279–3–12 +/+ 3.1 ± 0.4 793 ± 110 2354,3203 2 2851 ± 758* 3 275–3–2 +/+ 6.5 ± 0.5 753 ± 70 1206 ± 237* 3 2946,5535 2 275–1–4 +/+ 1.6 ± 0.2 628 ± 35 1151 ± 778* 3 nd 247–3–8 –/– 2.2 ± 0.0 955 ± 203 137 ± 106 3 287 ± 156* 3 247–3–12 –/– 1.9 ± 0.3 1342 ± 239 335,264 2 673 ± 489* 3 277–3–4 –/– 4.6 ± 0.0 810 ± 122 46 ± 64* 3 372 ± 253 4 277–1–11 –/– 2.8 ± 0.4 1502 ± 343 44 ± 32* 3 nd a Growth curves were constructed for each cell line as described in Methods. Analysis of transformation in vitro The designation and genotype of each cell line is shown above the lanes. Cell line 275-1-4 expresses very low levels of ras, and may be derived from spontaneously transformed cells. 1000 750 500 250 0 Colony number +/+ –/– – Tet + Tet Genotype or treatment Figure 6 ras protein level in wildtype and OPN-deficient cells used for tumour formation. Cell lysates were prepared from each cell line shown in Table 2, and equal amounts of protein separated on 12% SDS Page gels. Ras protein was detected with antibody F235. The designation and genotype of each cell line is shown above the lanes. Cell line 275-1-4 expresses very low levels of ras, and may be derived from spontaneously transformed cells. Genotype or treatment 4000 3000 2000 1000 0 0 5 10 15 20 25 30 35 Days after injection Tumour volume (mm3) Figure 7 Growth of tumours in nude mice after injection of wildtype cell line 279-3-7 (solid symbols) or OPN-deficient cell line 247-3-8 (open symbols). 5 × 105 cells in PBS were injected subcutaneously into the dorsal area of 3–4 mice. Tumour size was measured every other day, and tumour volume calculated as described in Methods. Each line represents results from a single mouse. Results for a representative pair of cell lines are shown – other cell lines gave similar results, as shown in Table 2. Figure 5 Colony-forming ability in soft agar of wildtype and OPN-deficient ras-transformed cell lines, and in cells with a tetracycline-responsive OPN construct, grown with and without tetracycline. +/+ = average colony formation per 104 cells of 19 wildtype cell lines; –/– = average colony formation per 104 cells of 15 OPN-deficient cells; –Tet = colony formation of 105 OPNTet cells in the absence of Tet (expressing OPN); +Tet = colony formation of 105 OPNTet cells in the presence of Tet (reduced OPN expression). Shown is the mean +/– SEM, P < 0.05. Figure 5 Colony-forming ability in soft agar of wildtype and OPN-deficient ras-transformed cell lines, and in cells with a tetracycline-responsive OPN construct, grown with and without tetracycline. Analysis of transformation in vitro Total cell number per well at 3 days, when the cells were still in exponential growth, is reported. Numbers represent the average of three determinations ± SD. b104 cells were plated in 0.3% agar, and the total number of colonies present at 14 days determined. Numbers represent the average of three determinations ± SD. c5 × 105 cells were injected subcutaneously into mice, and tumour size determined after 20 () or 21 days. The average total tumour size ± SD for three or four mice is shown, n represents the number of mice used. Where n = 2, results from individual animals are shown. a Growth curves were constructed for each cell line as described in Methods. Total cell number per well at 3 days, when the cells were still in exponential growth, is reported. Numbers represent the average of three determinations ± SD. b104 cells were plated in 0.3% agar, and the total number of colonies present at 14 days determined. Numbers represent the average of three determinations ± SD. c5 × 105 cells were injected subcutaneously into mice, and tumour size determined after 20 () or 21 days. The average total tumour size ± SD for three or four mice is shown, n represents the number of mice used. Where n = 2, results from individual animals are shown. 279-3-7, +/+ 277-3-4, –/– 279-3-12, +/+ 247-3-12, –/– 275-1-4, +/+ 277-1-11, –/– 275-3-2, +/+ 247-3-8, –/– ras Figure 6 ras protein level in wildtype and OPN-deficient cells used for tumour formation. Cell lysates were prepared from each cell line shown in Table 2, and equal amounts of protein separated on 12% SDS Page gels. Ras protein was detected with antibody F235. The designation and genotype of each cell line is shown above the lanes. Cell line 275-1-4 expresses very low levels of ras, and may be derived from spontaneously transformed cells. 279-3-7, +/+ 277-3-4, –/– 279-3-12, +/+ 247-3-12, –/– 275-1-4, +/+ 277-1-11, –/– 275-3-2, +/+ 247-3-8, –/– ras Figure 6 ras protein level in wildtype and OPN-deficient cells used for tumour formation. Cell lysates were prepared from each cell line shown in Table 2, and equal amounts of protein separated on 12% SDS Page gels. Ras protein was detected with antibody F235. The designation and genotype of each cell line is shown above the lanes. Analysis of transformation in vitro Cell line 275-1-4 expresses very low levels of ras, and may be derived from spontaneously transformed cells. 1000 750 500 250 0 Colony number +/+ –/– – Tet + Tet Genotype or treatment Figure 5 Colony-forming ability in soft agar of wildtype and OPN-deficient ras-transformed cell lines, and in cells with a tetracycline-responsive OPN construct, grown with and without tetracycline. +/+ = average colony formation per 104 cells of 19 wildtype cell lines; –/– = average colony formation per 104 cells of 15 OPN-deficient cells; –Tet = colony formation of 105 OPNTet cells in the absence of Tet (expressing OPN); +Tet = colony formation of 105 OPNTet cells in the presence of Tet (reduced OPN expression). Shown is the mean +/– SEM, P < 0.05. 1000 750 500 250 0 Colony number +/+ –/– – Tet + Tet Genotype or treatment Figure 5 Colony-forming ability in soft agar of wildtype and OPN-deficient ras-transformed cell lines, and in cells with a tetracycline-responsive OPN construct, grown with and without tetracycline. +/+ = average colony formation per 104 cells of 19 wildtype cell lines; –/– = average colony formation per 104 cells of 15 OPN-deficient cells; –Tet = colony formation of 105 OPNTet cells in the absence of Tet (expressing OPN); +Tet = colony formation of 105 OPNTet cells in the presence of Tet (reduced OPN expression). Shown is the mean +/– SEM, P < 0.05. 279-3-7, +/+ 277-3-4, –/– 279-3-12, +/+ 247-3-12, –/– 275-1-4, +/+ 277-1-11, –/– 275-3-2, +/+ 247-3-8, –/– ras Figure 6 ras protein level in wildtype and OPN-deficient cells used for tumour formation. Cell lysates were prepared from each cell line shown in Table 2, and equal amounts of protein separated on 12% SDS Page gels. Ras protein was detected with antibody F235. The designation and genotype of each cell line is shown above the lanes. Cell line 275-1-4 expresses very low levels of ras, and may be derived from spontaneously transformed cells. 4000 3000 2000 1000 Tumour volume (mm3) that could form colonies efficiently in soft agar and that had similar ras mRNA and protein levels (Figure 6, Table 2). These cells were collected, resuspended in PBS and injected subcutaneously into nude or syngeneic mice. The mice used for these injections were wildtype as there are at present no syngeneic OPN-deficient hosts for these 129 × C57Bl/6 F2 cells. Analysis of transformation in vitro Every second day after the tumors became palpable, the tumor size was measured with calipers In every case the wildtype cells 1000 750 500 250 0 Colony number +/+ –/– – Tet + Tet Genotype or treatment Figure 5 Colony-forming ability in soft agar of wildtype and OPN-deficient ras-transformed cell lines, and in cells with a tetracycline-responsive OPN construct, grown with and without tetracycline. +/+ = average colony formation per 104 cells of 19 wildtype cell lines; –/– = average colony formation per 104 cells of 15 OPN-deficient cells; –Tet = colony formation of 105 OPNTet cells in the absence of Tet (expressing OPN); +Tet = colony formation of 105 OPNTet cells in the presence of Tet (reduced OPN expression). Shown is the mean +/– SEM, P < 0.05. 279-3-7, +/+ 277-3-4, –/– 279-3-12, +/+ 247-3-12, –/– 275-1-4, +/+ 277-1-11, –/– 275-3-2, +/+ 247-3-8, –/– ras Figure 6 ras protein level in wildtype and OPN-deficient cells used for tumour formation. Cell lysates were prepared from each cell line shown in Table 2, and equal amounts of protein separated on 12% SDS Page gels. Ras protein was detected with antibody F235. The designation and genotype of each cell line is shown above the lanes. Cell line 275-1-4 expresses very low levels of ras, and may be derived from spontaneously transformed cells. 4000 3000 2000 1000 0 0 5 10 15 20 25 30 35 Days after injection Tumour volume (mm3) Figure 7 Growth of tumours in nude mice after injection of wildtype cell line 279-3-7 (solid symbols) or OPN-deficient cell line 247-3-8 (open symbols). 5 × 105 cells in PBS were injected subcutaneously into the dorsal area of 3–4 mice. Tumour size was measured every other day, and tumour volume calculated as described in Methods. Each line represents results from a single mouse. Results for a representative pair of cell lines are shown – other cell lines gave similar results, as shown in Table 2. 1000 750 500 250 0 Colony number +/+ –/– – Tet + Tet 279-3-7, +/+ 277-3-4, –/– 279-3-12, +/+ 247-3-12, –/– 275-1-4, +/+ 277-1-11, –/– 275-3-2, +/+ 247-3-8, –/– ras Figure 6 ras protein level in wildtype and OPN-deficient cells used for tumour formation. Cell lysates were prepared from each cell line shown in Table 2, and equal amounts of protein separated on 12% SDS Page gels. Ras protein was detected with antibody F235. DISCUSSION The development of mice deficient for osteopontin expression (Rittling et al, 1998; Liaw et al, 1998) has provided a unique system in which to compare directly the transformed properties of otherwise very similar cells in the presence and absence of osteo- pontin expression. The work presented here is important in that it provides an uncomplicated analysis of the role of OPN in multiple aspects of cellular transformation. Our results confirm and extend the long-held idea that OPN is important in the transformation process, in that the transformed phenotype was attenuated in the absence of OPN. Thus we observed lower rates on average of colony formation in soft agar, and dramatically reduced rates of primary tumor and metastasis growth in syngeneic and nude mice. The only aspect of transformation for which we did not detect a reduction in the OPN-deficient cells was in the process of focus formation: both the wildtype and osteopontin-deficient cells formed foci efficiently after ras transformation. This phenotype, focus formation, most likely represents a loss of contact inhibition of growth resulting from the expression of oncogenic ras, and we conclude that OPN, and by deduction also the integrins to which OPN binds in these cells, do not play a major role in this process. We also did not detect an effect of osteopontin on the process of spontaneous immortalization. This process requires an alteration in the cells, probably a mutation (Rittling, 1996) that enables the cells to overcome the growth restraints of senescence. We have considered several hypotheses to explain the mecha- nism of OPN’s ability to enhance tumor formation. It is unlikely that OPN is acting in an autocrine manner to stimulate growth of the tumor cells themselves since these cells already show efficient anchorage-independent growth. Alternatively, high level OPN expression could protect cells from apoptosis in the first few days following tumor cell injection. A role for the OPN-binding inte- grins α5β1 and αvβ3 in preventing apoptosis (or anoikis (Frisch and Ruoslahti, 1997)) of transformed cells has previously been demon- strated (Montgomery et al, 1994; Zhang et al, 1995). Denhardt and Chambers (1994) have proposed that OPN, by virtue of its ability to inhibit iNOS activity, protects tumor cells from attack by cyto- toxic cell in vivo, and this may be a possible mechanism of action of OPN in stimulating tumor growth. DISCUSSION The possible conflicting role of macrophages in OPN-dependent tumorigenesis has been discussed recently (Crawford et al, 1998). Finally, OPN has been shown to stimulate migration of endothelial cells (Liaw et al, 1995), particularly in association with VEGF (Senger et al, 1996). Possibly, expression of OPN in the injected cells is required for optimal angiogenesis. This idea is supported by the observations that ligation of the αvβ3 integrin protects endothelial cells against apoptosis (Brooks et al, 1994a; Strömblad et al, 1996) and stimu- lates angiogenesis (Brooks et al, 1994b), and that surface-bound OPN can protect endothelial cells from apoptosis (Scatena et al, 1998). Work is in progress to distinguish these possibilities. Our results indicate that the effects of OPN expression on anchorage-independent growth are complex. While antisense experiments suggested that OPN was necessary for the formation of colonies in soft agar (Su et al, 1995; Gardner et al, 1994), we clearly show here that OPN-deficient cell lines can form colonies efficiently in soft agar, indicating that OPN is not absolutely required for this process in ras-transformed cells. However, our data show that OPN can enhance anchorage-independent growth. Thus, OPN is not strictly required for anchorage-independent growth, but can enhance such growth in some cell lines. Anchorage-independent growth in ras-transformed cells probably results from stimulation by activated ras of signaling pathways that mediate both growth factor and integrin-initiated signals (reviewed in Schwartz, 1997). For example, MAPK activation by growth factors in normal cells requires adhesion through integrins (Renshaw et al, 1997), while oncogenic ras stimulates MAPK independently of both growth factor and integrin ligation to allow anchorage-independent growth. The binding of soluble OPN to integrins such as the αvβ3 may stimulate the signal throughput of these pathways, even in the presence of activated ras, thereby enhancing anchorage-independent growth, much as transformed cells that are growth factor independent show higher rates of growth in the presence of growth factors. Experiments with transformed cells are hampered by the well- known heterogeneity of such cells, making it difficult to make direct comparisons between different transformed cell lines. Our analysis of a large number of cell lines has allowed us to develop a consensus of the effects of OPN in vitro, despite considerable vari- ability among individual cell lines. The results with tumor forma- tion, however, were less variable, in that every cell line tested showed a slower rate of tumor growth. DISCUSSION Thus, it may be that the effects of OPN are more pronounced in the whole animal than in culture, possibly reflecting an important role for this protein in mediating cellular interactions. ACKNOWLEDGEMENTS We wish to thank Aaron Kowalski for help with various aspects of this work, and Dr David Axelrod for critically reviewing the manuscript. Supported by NIH R01 # CA72740 to SRR and NIH grants DC01295 and AR44434 to DTD. Submitted in partial fulfillment of the MSc degree by YW. The most pronounced effect of a lack of OPN that we observed was in the ability of the OPN-deficient transformed cells to form tumors. For these experiments, a subset of ras-transformed cell lines was selected that had similar ras levels and formed colonies efficiently in soft agar. While the OPN-deficient clones were able to form tumors in syngeneic or nude mice, they did so at a substan- tially slower rate than the wildtype cells. Thus, OPN clearly can enhance tumor formation in vivo. It is important to note here that the recipients for these tumor cell injections were wildtype mice, so OPN could have been produced by the stromal cells in the vicinity of the injection of the OPN-deficient cells. Thus host- derived OPN may have contributed to the ultimate ability of the Osteopontin is required for maximal transformation 161 OPN-deficient cells to form tumors. Indeed, we have found (Wu, Feng and Rittling, manuscript in preparation) that tumors arising from the OPN-deficient cells contain measurable amounts of OPN, which may have been supplied by infiltrating macrophages (Brown et al, 1994; Casson et al, 1997) or other sources. © 2000 Cancer Research Campaign Analysis of transformation in vitro The OPN-deficient cells were found to form fewer metastases, (5.5 ± 1.8 metastases per lung section for wt vs 1.2 ± 1.0 for OPN –/– cells; n = 3, P < 0.05), and those metastases were much smaller than those formed by WT cells (the mean cross-sectional area of the largest metastatic tumor examined in each section was 0.79 ± 0.42 mm2 for wt and 0.162 ± 0.16 mm2 for OPN –/– (n = 9; P < 0.01)). British Journal of Cancer (2000) 83(2), 156–163 © 2000 Cancer Research Campaign Osteopontin is required for maximal transformation Brooks PC, Clark RAF and Cheresh DA (1994a) Requirement of vascular integrin αvβ3 for angiogenesis. 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https://openalex.org/W2481659380	https://www.repository.cam.ac.uk/bitstream/1810/260441/4/3563.full.pdf	English	null	Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease	Biochemical journal	2,016	cc-by	16,775	Research Article Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson’s disease Felipe Roberti Teixeira1,2,3,, Suzanne J. Randle2,, Shachi P. Patel2,, Tycho E.T. Mevissen4, Grasilda Zenkeviciute2, Tie Koide3, David Komander4 and Heike Laman2 1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.; 3Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; and 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. Correspondence: Heike Laman (hl316@cam.ac.uk) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson’s disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within speciﬁc functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deﬁciencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early- onset PD. To date, relatively few substrates of the ligase have been identiﬁed. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in prote- asome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF recep- tor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To dis- cover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identiﬁed and from these we validated glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mito- chondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo. Ubiquitin chain restriction analyses revealed that Fbxo7 modiﬁed Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localiza- tion. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our ﬁndings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD. Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson’s disease Felipe Roberti Teixeira1,2,3,, Suzanne J. Randle2,, Shachi P. Patel2,, Tycho E.T. Mevissen4, Grasilda Zenkeviciute2, Tie Koide3, David Komander4 and Heike Laman2 Felipe Roberti Teixeira1,2,3,, Suzanne J. Randle2,, Shachi P. Patel2,, Tycho E.T. Mevissen4, Grasilda Zenkeviciute2, Tie Koide3, David Komander4 and Heike Laman2 1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.; 3Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; and 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. 1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.; 3Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; and 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. 1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.; 3Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; and 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. Correspondence: Heike Laman (hl316@cam.ac.uk) Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson’s disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within speciﬁc functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deﬁciencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early- onset PD. To date, relatively few substrates of the ligase have been identiﬁed. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in prote- asome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF recep- tor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To dis- cover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson’s disease A total of 338 new targets were identiﬁed and from these we validated glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mito- chondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo. Ubiquitin chain restriction analyses revealed that Fbxo7 modiﬁed Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localiza- tion. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our ﬁndings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD. Introduction F-box proteins assemble with Skp1, Cullin 1 and Rbx1 to form SCF-type E3 ubiquitin ligases [1–3]. These enzymes act in a cascade with a ubiquitin-activating enzyme (E1) and a ubiquitin-carrier enzyme (E2) to mediate the ubiquitination of their substrates, the consequences of which range from promoting proteasomal degradation to changing the localization or activity of the modiﬁed protein. Individual substrates are thought to be targeted for ubiquitination by E3 ligases by factors such as post-translational modiﬁcations (PTMs), such as phosphorylation, glycosylation, or the phase of the cell cycle and cellular localization of the substrate and/or the ligase [4]. The biological importance of F-box proteins stems from their involvement in the regulation of many core processes such as cell Equal first authors. Accepted Manuscript online: 8 August 2016 Version of Record published: 11 October 2016 Received: 27 April 2016 Revised: 2 August 2016 Accepted: 8 August 2016 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 3563 Research Article Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson’s disease Felipe Roberti Teixeira1,2,3,, Suzanne J. Randle2,, Shachi P. Patel2,, Tycho E.T. Mevissen4, Grasilda Zenkeviciute2, Tie Koide3, David Komander4 and Heike Laman2 1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.; 3Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; and 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. C d H ik L (hl316@ k) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Research Article Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson’s disease Felipe Roberti Teixeira1,2,3,, Suzanne J. Randle2,, Shachi P. Patel2,, Tycho E.T. Mevissen4, Grasilda Zenkeviciute2, Tie Koide3, David Komander4 and Heike Laman2 1Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil; 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.; 3Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; and 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. C d H ik L (hl316@ k) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Introduction In contrast, in pro-B cells and pro-erythroblasts, Fbxo7 negatively regulates cell proliferation and differentiation [11]. Within other cell types, altered Fbxo7 activity may be linked to pathological changes. For example, four studies reported on many SNPs in FBXO7 that corre- lated with changes in human red blood cell traits such as mean cell volume and mean cell hemoglobin, which are associated with adverse health outcomes such as anemia, cardiovascular diseases, and cancer [13–16]. [ ] Recessive mutations in FBXO7, also designated PARK15, have been identiﬁed in patients with phenotypes of idiopathic Parkinson’s disease (PD) and an early-onset form of PD [17–20]. Pathological point mutations are located within speciﬁc domains which hint at Fbxo7 function(s) that might be compromised. In addition to its F-box domain, which enables incorporation into an SCF complex via Skp1 binding, Fbxo7 contains an Ubl (ubiquitin-like) domain (aa 1–79), which interacts with Parkin and PINK1; a 40 amino acid linker (aa 129– 169), which binds to Cdk6; a dimerization domain (aa 182–325), which enables homo-dimerization and hetero-dimerization with PI31; and a PRR (proline-rich region) (aa 423–522), which mediates protein–protein interactions with hepatoma up-regulated protein (HURP) and Cdk6 (reviewed in ref. [9]). One PD-associated mutation, T22M, lies within the N-terminal Ubl domain and compromises its ability to interact with Parkin and to promote mitophagy [21]. A second missense mutation R378G abuts the F-box domain and compro- mises its ability to interact with Skp1 [22]. Finally, a point mutation R498X within the PRR truncates the ﬁnal 24 aa [17]. The loss of interaction with Skp1 and a substrate-interacting domain resulting from the R378G and R498X mutations suggests that SCFFbxo7-mediated ubiquitination is compromised in PD patients. However, the identity of critical targets and the functional effect of their ubiquitination are not known. Fb To date, there are only a handful of ubiquitinated proteins reported for SCFFbxo7. These include HURP/ DLG7 (hepatoma up-regulated protein), a regulator of mitotic spindle assembly, which is up-regulated in hepa- tocellular carcinoma, colon cancer, breast cancer, and transitional cell carcinoma [23,24]. SCFFbxo7 also pro- motes ubiquitination of c-IAP1 (inhibitor of apoptosis) and TRAF2 resulting in decreased NF-κB signaling activity [25,26]. Most recently, NRAGE (neurotrophin receptor-interacting MAGE) was reported to be ubiquiti- nated by SCFFbxo7, which positively regulated bone morphogenic protein (BMP) signaling [27]. Interestingly, Fbxo7 increased formation of a BMP receptor–NRAGE–TAK1–TAB1 complex and up-regulated NF-κB activ- ity. Introduction Equal first authors. Accepted Manuscript online: 8 August 2016 Version of Record published: 11 October 2016 Received: 27 April 2016 Revised: 2 August 2016 Accepted: 8 August 2016 F-box proteins assemble with Skp1, Cullin 1 and Rbx1 to form SCF-type E3 ubiquitin ligases [1–3]. These enzymes act in a cascade with a ubiquitin-activating enzyme (E1) and a ubiquitin-carrier enzyme (E2) to mediate the ubiquitination of their substrates, the consequences of which range from promoting proteasomal degradation to changing the localization or activity of the modiﬁed protein. Individual substrates are thought to be targeted for ubiquitination by E3 ligases by factors such as post-translational modiﬁcations (PTMs), such as phosphorylation, glycosylation, or the phase of the cell cycle and cellular localization of the substrate and/or the ligase [4]. The biological importance of F-box proteins stems from their involvement in the regulation of many core processes such as cell Accepted Manuscript online: 8 August 2016 Version of Record published: 11 October 2016 3563 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Comm ss article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 cycle (Fbxl1, Fbxw5, Fbxw7, and Fbxw1), differentiation and development (Fbxw1, Fbxl14, Fbxo11, Fbxo32, and Fbxw7), cell death (Fbxw1 and Fbxw7), intracellular signaling (Fbxo25), and others (reviewed in ref. [5–8]). cycle (Fbxl1, Fbxw5, Fbxw7, and Fbxw1), differentiation and development (Fbxw1, Fbxl14, Fbxo11, Fbxo32, and Fbxw7), cell death (Fbxw1 and Fbxw7), intracellular signaling (Fbxo25), and others (reviewed in ref. [5–8]). ) ( ) g g ( ) ( [ ]) Fbxo7 is a multi-functional F-box protein, with both SCF-dependent and -independent functions that are capable of impacting on many core cellular processes, and is associated with human diseases (reviewed in ref. [9]). For example, the effects of Fbxo7 on the cell cycle are independent of its ubiquitin ligase activity and are mediated instead through its interactions with cell cycle proteins, Cdk6 and p27 [10,11]. Remarkably, Fbxo7’s functional effects on cell proliferation and transformation, for example, are dependent on cell type. For example, Fbxo7 overexpression transforms immortalized ﬁbroblasts and imparts tumorigenic properties to p53 null hematopoietic stem and progenitor cells [10,12]. Introduction The contrasting effects of Fbxo7 expression on NF-κB signaling may reﬂect cell-type-speciﬁc effects of Fbxo7 on this pathway, or alternatively, reﬂect the inﬂuence of other signaling inputs, like cellular stresses. There is a potential relationship between NF-κB signaling, long-term neuroinﬂammation and PD, as suggested, for example, by the symptoms demonstrated by the c-Rel knockout mouse [28]; however, evidence for the role of NF-κB signaling in PD remains controversial (as reviewed in ref. [29]). g g There are currently no studies that deﬁne ubiquitination substrates for SCFFbxo7 at a proteome-wide scale, which would greatly assist in clarifying its function(s) in its multiple clinically relevant settings. Given the con- trasting biological effects of Fbxo7 expression in different cellular environments, we sought to bypass considera- tions of cell-type speciﬁcity, cell cycle phase, or subcellular localization, which might limit the interaction of Fbxo7 with its substrates. We undertook an in vitro high-throughput experimental approach utilizing a human protein microarray that displays ∼9500 individual proteins on a slide, to identify mammalian substrates for ubi- quitination by SCFFbxo7. This powerful approach has been used to identify substrates for ubiquitin ligases, such as yeast Rsp5 [30] and human NEDD4/NEDD4L [31], SCFFbxo25 [32], and SMURF1 [33]. Our in vitro ubiqui- tination screen identiﬁed 338 unique, high-conﬁdence Fbxo7 substrates distributed across different cellular compartments, and which are involved in many biological processes. To validate this screen, we assessed indi- vidually the in vitro and in vivo ubiquitination of two candidate proteins, Gsk3β (glycogen synthase kinase 3β) and Tomm20 (translocase of outer mitochondrial membrane 20). In addition, the type of ubiquitin chain lin- kages introduced by Fbxo7 onto these substrates was investigated using ubiquitin chain restriction analysis, [34] and the functional effects of their ubiquitination were tested. 3564 an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Experimental materials and methods Puriﬁcation of SCF complexes The SCF components such as human inﬂuenza hemagglutinin (HA)-Skp1, Cul1, Myc-Rbx1, and FLAG-Fbxo7 or FLAG-Fbxo7(ΔF-box) were transfected in HEK293T cells by using polyethylenimine. After 48 h of transfec- tion, the cells were harvested and resuspended in lysis buffer (LB) (25 mM Tris–HCl, pH 7.5, 225 mM KCl and 1% NP-40) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). The lysates were incubated with agarose anti-FLAG M2 beads (Sigma-Aldrich, St. Louis, MO) for 6 h at 4°C with rocking. Beads were washed with LB and the SCF com- plexes eluted with FLAG elution buffer (300 mg/ml of peptide FLAG in 10 mM HEPES pH 7.9, 225 mM KCl, 1.5 mM MgCl2, and 0.1% NP-40) for 1 h at 4°C with rocking. The eluates were stored in 15% glycerol at −20°C until use. To evaluate the puriﬁcation of SCF complexes, immunoblotting was performed and probed using anti-Fbxo7 (ABN1038, Merck Millipore, Watford, UK), anti-HA (Abcam, Cambridge, UK), anti-Gsk3β (Santa Cruz Biotechnologies, CA, USA), anti-Tomm20 (Abcam, Cambridge, UK), or anti-myc (Cell Signaling Technologies, MA, USA). The concentration of the complexes was determined against known concentrations of BSA by Coomassie blue staining of the gel. The densitometry of the bands was determined by ImageJ. In vitro ubiquitination assays The plasmids encoding human Gsk3β-HA and Tomm20 were purchased from Addgene (14 753 and 40 291, respectively). Human cIAP-1-myc was kindly provided by Dr Yasuko Matsuzawa (Sanford-Burnhan Medical Research Institute, La Jolla, CA, USA). Tomm20 was cloned into pcDNA3 in fusion with HA at the C-terminus. cIAP was truncated (183–570) and cloned in fusion with HA at the C-terminus. The substrates cIAP-1(183–570)-HA, Gsk3β-HA, and Tomm20-HA were produced by in vitro transcription/translation (IVT), and the crude programmed reticulocyte lysates were added to in vitro ubiquitination reactions. For the ubiquiti- nation reactions, puriﬁed SCF complexes were used at the indicated concentrations in combination with ubiqui- tin mix [ubiquitination buffer, E1(100 nM), E2(500 nM), biotin-ubiquitin (20 mM), Mg-ATP (2 mM; Boston Biochem)], and the puriﬁed substrates and incubated for 90 min at 30°C. Proteins were resolved by SDS–PAGE and immunoblotting was performed using anti-Gsk3β, anti-Tomm20, or anti-HA antibodies. To determine which E2(s) enabled SCFFbxo7 ligase activity, an in vitro E2 screening with 10 different E2 enzymes, each at 500 nM was performed. Auto-ubiquitination was observed with UBE2D1 (UbcH5a), UBE2D2 (UbcH5b), and UBE2D3 (UbcH5c; Supplementary Figure S1), and UBE2D1 (UbcH5a) was used in further experiments. Reactions were resolved by SDS–PAGE and detected by probing with streptavidin–HRP (Thermo Pierce, MA, USA). Analysis of ligases Samples were resolved ∼1 cm into a pre-cast SDS–polyacrylamide gel, and the entire lane was excised and cut into four equal slices. Proteins were reduced and alkylated, then digested in-gel using trypsin. The resulting peptides were analyzed by LC–MS/MS using an Orbitrap XL (Thermo) coupled to a nanoAcquity UPLC (Waters). Data were acquired in a DDA fashion with MS/MS in the LTQ triggered at 1000 counts. Raw ﬁles were converted into mzML using MSconvert (ProteoWizard) and submitted to MASCOT 2.3.0 to search a human Uniprot database (20 264 entries, downloaded on 09 June 14). Carbamidomethyl cysteine was set as a ﬁxed modiﬁcation with oxidized methionine and deamidation of asparagine and glutamine as potential variable modiﬁcations. Peptide and protein validation were performed using Scaffold 4.3.2. Peptides required a minimum of 95% probability and proteins required a minimum of 90% probability and two peptides in order to be counted. Data acquisition and analysis Software used for Protoarray® image acquisition was GenePix Pro 4.1 (Molecular Devices). The experimental design comprised two biological replicates [25 or 50 nM ligase of wild type (WT) or mutant F-box protein, ΔF] with two intraslide technical replicates. Each slide has 3004 negative control spots (Supplementary Table S2) and 576 positive control spots (Supplementary Table S3). The intensity value of each array feature was considered as the average raw intensity of all pixels in the delimited spot region minus the median intensity of pixels immediately surrounding the spot region (local background). Background-subtracted intensities were subjected to normalization to make them directly com- parable among different Protoarray® slides (replicates and conditions). Assuming that the manufacturer-produced positive controls should present the same theoretical intensities among replicates, their background-corrected values were obtained and a single centering (normalization) value per slide was deﬁned as the average of all known positive control spots. An interslide, study-wide overall positive control value was obtained by averaging all the intraslide centering/normalization factors. Finally, all array features had their background-subtracted intensities corrected by this overall factor to end up with comparable normalized inten- sities (IWT,25 nM, IWT,50 nM, IΔF,25 nM, and IΔF,50 nM). To determine spots with statistically signiﬁcant signals, we used all the negative control spots to estimate non-parametrically the null density distribution (NC) for each slide using a Gaussian kernel density estimator [35]. The intensity value that encompasses the vast majority of null probability mass was chosen as cutoff (c) for each slide: P(NC > c) = 0.005. This yielded four cutoff values: cWT,25 nM; cWT,50 nM; cΔF,25 nM, and cΔF,50 nM. Selected spots presented intensities IWT > cWT and IΔF < cΔF for each concentration and log fold change [FC = log2(IWT/IΔF)] greater than 5-fold: log2(IWT/IΔF) > log2(5). To increase stringency, only proteins which met these criteria simultaneously on both concentrations were deﬁned as possible substrates. Mean FC values of possible substrates were calculated and used to rank the proteins (Supplementary Table S4). Selected spots presented intensities IWT > cWT and IΔF < cΔF for each concentration and log fold change [FC = log2(IWT/IΔF)] greater than 5-fold: log2(IWT/IΔF) > log2(5). To increase stringency, only proteins which met these criteria simultaneously on both concentrations were deﬁned as possible substrates. Mean FC values of possible substrates were calculated and used to rank the proteins (Supplementary Table S4). In vivo ubiquitination assays and co-immunoprecipitation assays In vivo ubiquitination assays and co-immunoprecipitation assays HEK293T cells were transfected with empty vector, FLAG-Fbxo7 or FLAG-Fbxo7-ΔF-box, or truncated FLAG-Fbxo7 alleles where indicated, in combination with Gsk3β-HA or Tomm20-HA, with or without ubiquitin-6xHis-myc. Where indicated, cells were treated with 10 mM of MG132 prior to lysis with LB for 30 min on ice. Cells were centrifuged and supernatants were subjected to immunoprecipitation (IP) with agarose-anti-HA or agarose-anti-FLAG. The proteins were eluted by Laemmli buffer and the eluates were resolved by SDS–PAGE. The ubiquitinated proteins were visualized using antibodies to HA, the substrates, or anti-myc antibody. In vitro ubiquitination assays of protein arrays q y p y Protoarray® v5.0 was obtained from Life Technologies (catalog number PAH0525101). Supplementary Table S1 contains an Excel ﬁle of Protoarray® v5.0 protein content. Protocols were followed according to the manufac- turer’s instructions (Protoarray® v5.0, Invitrogen, MA, USA). Slides were incubated in Protoarray® Synthetic Block for 1 h at 4°C with shaking at 50 rpm. During this time, reactions were prepared in a volume of 120 ml as follows: 25 or 50 nM of the puriﬁed SCFFbxo7 or Fbxo7(ΔF-box) in combination with ubiquitin mix [E1 (100 nM), UbcH5a (500 nM), Mg-ATP (2 mM), and biotin-ubiquitin 0.1 mg/ml in ubiquitination buffer; Boston Biochem]. The slides were washed with assay buffer (AB; 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM 3565 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 MgSO4, 0.1% Tween 20, 1% BSA, and 1 mM DTT) and 110 ml of the reaction was added to the slide and over- laid with a coverslip followed by incubation for 1.5 h at 30°C in a humidiﬁed chamber. Slides were washed in 0.5% SDS and AB and then incubated with 1 mg/ml of streptavidin–AlexaFluor 647 for 45 min at 4°C with shaking. The arrays were washed with AB, once with distilled water, and ﬁnally dried by centrifugation at 1000 × g for 2 min, before being scanned on a GenePix Personal 4100A (Axon–Molecular Devices). MgSO4, 0.1% Tween 20, 1% BSA, and 1 mM DTT) and 110 ml of the reaction was added to the slide and over- laid with a coverslip followed by incubation for 1.5 h at 30°C in a humidiﬁed chamber. Slides were washed in 0.5% SDS and AB and then incubated with 1 mg/ml of streptavidin–AlexaFluor 647 for 45 min at 4°C with shaking. The arrays were washed with AB, once with distilled water, and ﬁnally dried by centrifugation at 1000 × g for 2 min, before being scanned on a GenePix Personal 4100A (Axon–Molecular Devices). Data acquisition and analysis The selected list of proteins was subjected to Gene Enrichment analysis using DAVID Bioinformatics Resources v6.7 [36] using Protoarray® v5.0 content (Supplementary Table S1) or the human proteome as the background. DAVID’s P-values up to 0.05 were considered signiﬁcant (Supplementary Table S4). Immunoﬂuorescence U2OS cells were plated onto glass coverslips, transfected with FLAG-Fbxo7, and 24 h later ﬁxed in 4% parafor- maldehyde in PBS for 15 min. Cells were permeabilized in 0.2% Triton X-100 in PBS, blocked for 1 h in 10% donkey serum, and incubated overnight with anti-FLAG and anti-Gsk3β antibodies. Cells were repeatedly washed in PBS, incubated with anti-rabbit AlexaFluor 488 and anti-mouse AlexaFluor 647 for 1 h, washed again, and then counterstained with Hoechst 33342 to visualize nuclei. Cells were visualized using a Zeiss ApoTome microscope. In vitro binding assays g y HEK293T cells were transfected with empty vector or various FLAG-Fbxo7 truncation mutants. Cells were lysed in NETN lysis buffer [NB; 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.2% NP-40 con- taining a protease inhibitor cocktail and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4)], and clari- ﬁed lysates were subjected to IP with anti-FLAG antibodies immobilized on agarose beads. Gsk3β-HA protein was IP from transfected HEK293T cells by anti-HA antibodies immobilized on agarose beads and eluted using HA peptide. Gsk3β-HA protein was added to immobilized FLAG-Fbxo7 proteins and rotated for 3 h at 4°C. Beads were washed twice in NB and twice in RIPA buffer, then eluted by Laemmli buffer and the eluates resolved by SDS–PAGE. 3566 pen access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Gsk3β activity reporter The Gsk3β reporter pCS2-GFP-Gsk3-MAPK and mutant version pCS2-GFP-Gsk3mut-MAPK described in ref. [37] were purchased from Addgene (29 689 and 29 690, respectively). U2OS cells were transfected with empty vector, FLAG-Fbxo7, or FLAG-Fbxo7(ΔF-box) and 24 h later transfected with pCS2-GFP-Gsk3-MAPK or pCS2-GFP-Gsk3mut-MAPK, in quadruplicate. After a further 24 h, cells were trypsinized and analyzed by ﬂow cytometry using a Cytek analyzer. The GFP mean ﬂuorescence intensity was determined and levels were expressed as a percentage of the mutant GFP-Gsk3mut-MAPK-expressing cells. Fbxo7 protein knockdown Cells were infected with miR30-based retroviruses encoding independent human FBXO7-speciﬁc shRNAs and selected using puromycin (2 μg/ml), as described previously [11]. Transient knockdown (KD) of Fbxo7 was performed by transfecting dsRNA targeting Fbxo7 into cells using Effectene (Invitrogen) as described previ- ously [10]. Luciferase assays U20S cells, seeded at 5000 cells per well of a 96-well plate, were transfected in quintuplicate with 1.5 ng pCMV-Renilla luciferase control plasmid, 75 ng minimal promotor ﬁreﬂy luciferase (pTA-luc) or LEF/TCF responsive ﬁreﬂy luciferase reporters (TOP-FLASH), and 75 ng empty, FLAG-Fbxo7 or FLAG-Fbxo7(ΔF-box) vectors. Where stated, cells were treated after 24 h with 50 mM LiCl or NaCl and 24 h later, luciferase levels were assayed using the Dual Glo luciferase assay system (Promega, Southampton, UK). Ubiquitin chain restriction analysis q y Ubiquitin chain restriction (UbiCRest) analyses were performed as described recently [34]. In vivo expressed substrates were IP from HEK293T cells transfected with FLAG-Fbxo7 and the tagged-HA substrates using agarose-anti-HA. Cell lysates were obtained as mentioned above and the in vivo polyubiquitinated substrates were eluted by HA peptide at 300 mg/ml in FLAG elution buffer and stored at −80°C. Deubiquitinating enzymes (DUBs) were diluted with 2× dilution buffer (50 mM Tris, pH 7.4, 300 mM NaCl, and 20 mM DTT) and added to the samples for 30 min at 37°C, and reactions were stopped by Laemmli buffer. Samples were run on SDS–PAGE, and the blots were probed for anti-polyubiquitin (Santa Cruz Biotechnologies, CA, USA). Statistical analysis Statistical differences in normalized protein levels in shRNA lines were compared with vector control levels using Student’s t-tests. For Gsk3β reporter assays, normalized data were analyzed by one-way ANOVA, with a post hoc Dunnett’s multiple comparisons test to determine statistical signiﬁcance compared with control cells. For luciferase assays, one-way ANOVA with a post hoc Tukey’s multiple comparisons test was used to determine differences between samples. substrates using a protein array Identiﬁcation of novel SCFFbxo7 substrates using a protein array We next optimized the SCF E3 ligase to perform a large-scale in vitro ubiquitination experiment using a protein microarray. The commercially available Protoarray® v5.0 (Invitrogen) contained over 9000 unique, full- length native proteins puriﬁed and spotted in duplicate under non-denaturing conditions onto a nitrocellulose slide. Ligase activity was ﬁrst titrated, and a 50 nM concentration, which demonstrated robust auto- ubiquitination activity, was chosen for use on the array (Supplementary Figure S1C). Ligase (25 nM) was used in a replicate experiment, where more overall signal was seen on the array with the WT than the mutant ligase (Figure 2A,B). Overviews of the experimental design (Figure 2A) and the data analysis (Supplementary Figure S2, Materials and Methods) are provided. To increase the stringency of our screen, only proteins which met deﬁned criteria for statistically signiﬁcant intensities at both 25 and 50 nM of ligase were categorized as possible substrates. This analysis yielded 338 unique proteins as substrates of SCFFbxo7 (Supplementary Table S5). ) Candidate substrates were next grouped in accordance with their cellular compartment and their biological pathway using the Gene Onthology and KEGG databases for data enrichment (Supplementary Table S5) [39,40]. Forty-six candidate substrates were localized to the nuclear lumen, 39 to the cytosol, 13 to the ribo- some, and 10 to the microtubule-organizing centre. This wide distribution pattern of candidate substrates is in agreement with the reported localization of Fbxo7 [10,22], and with the analyses of Fbxo7 ligases (Supplementary Table S4). Consistent with the presence of a mitochondrial-targeting sequence (MTS) at the N-terminus of Fbxo7, 29 candidates were localized to mitochondria, where Fbxo7 interacts with Parkin and PINK1 to mediate stress-induced mitophagy [21]. Such candidates represent potential Fbxo7 substrates that may be important for this process. In addition, although PINK1 is present on the Protoarray® (Supplementary Table S1), it was not ubiquitinated by SCFFbxo7, suggesting that their reported interaction does not result in PINK1 ubiquitination. An analysis of the substrates using Enrichment by Biological Process indicated that Fbxo7 ubiquitinates multiple proteins that have an impact on ErbB2 (10 substrates), Wnt (10 substrates), and MAPK (13 substrates) signalling, ribosomes (9 substrates), axonal guidance (10 substrates), and pathways in cancer (16 substrates). bxo7 complexes are active in vitro p SCF complexes were puriﬁed from mammalian HEK293T cells using mild conditions to maintain activity and structure. WT Fbxo7, but not a mutant with a deletion of the F-box domain (denoted ΔF-box, lacking amino acids 335–367), co-puriﬁed with other SCF components: Skp1, Cullin1, and Rbx1 (Figure 1A,B). To test for ligase activity, we performed in vitro ubiquitination assays using SCFFbxo7 or the mutant Fbxo7(ΔF-box) in con- junction with both E1 and E2 enzymes. We found UBE2D1 (UbcH5a), UBE2D2 (UbcH5b), UBE2D3 (UbcH5c), and UBE2E1 (UbcH6), all promoted robust levels of auto-ubiquitination in a screen of E2 enzymes 3567 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 (Supplementary Figure S1A,B). To test the ability of SCFFbxo7 to ubiquitinate one of its known substrates, we used cIAP-1 [25,26]. This protein contains a RING box domain that can bind independently to E2 enzymes, promoting auto-ubiquitination. As this would obscure the ubiquitination signal generated by SCFFbxo7, this RING domain was deleted, creating cIAP-1(183–570)-HA, for these assays. cIAP-1(183–570)-HA was synthe- sized by IVT using reticulocyte lysates, and the programmed lysates were added to in vitro ubiquitination reac- tions. A smear of higher molecular weight (MW) ubiquitinated species of cIAP-1, which intensiﬁed with increasing concentration of ligase, was seen in the presence of the WT SCFFbxo7, but not mutant Fbxo7 (ΔF-box; Figure 1C). These experiments established that the puriﬁed SCFFbxo7 ligase showed robust ligase activ- ity and could ubiquitinate itself and one of its known substrates. In addition to probing for known subunits of an SCF-type E3 ligase (Figure 1B), we also conducted an unbiased mass spectrometry (MS)-based screen on the puriﬁed WT ligase and the mutant Fbxo7(ΔF-box). Alongside expected SCF components, NEDD8, two proteasome subunits (PSMB3 and PSMB6), EIF4A2, FBL, HDLBP, and DNAJB6, appeared exclusively associated with SCFFbxo7 and not the vector control or the mutant F-box protein made from transfection with Fbxo7(ΔF-box; Supplementary Table S4). A longer list of 107 pro- teins that interacted with both the WT SCFFbxo7 ligases and the mutant Fbxo7 protein, included the prote- asome inhibitor, PI31, a previously identiﬁed dimerization partner of Fbxo7 [38]. The association of PI31 with the ligases was conﬁrmed by immunoblotting, where an Fbxo7(V253E) mutant that prevents binding to PI31 served as a negative control (Figure 1D). bxo7 complexes are active in vitro Analysis of the longer list using KEGG databases for data enrichment identiﬁed proteins from the ribosome, proteasome, and spliceosome as being signiﬁcantly represented (Supplementary Table S4). substrates using a protein array We reported that Fbxo7 regulated cyclin D/Cdk6/p27 complexes which transformed cells, via an ubiquitin-independent mechanism [10], and in agreement with this, cyclin D3, Cdk6, and p27 were present on the Protoarray® v5.0 (Supplementary Table S1), but not ubiquitinated. Our dataset further indi- cates that SCFFbxo7 also ubiquitinates proteins important in cancer progression. With regard to concordance with known SCFFbxo7 substrates, cIAP-1, HURP, and NRAGE were not present on the protein arrays, and although TRAF2 was, it did not make the ﬁnal list of substrates due to high levels of signal with the negative control Fbxo7(ΔF-box). We speculate that as TRAF2 is also an E3 ligase, it catalyzed auto-ubiquitination in the 3568 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Com an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 re 1. Fbxo7, but not a mutant version Fbxo7(ΔF-box), forms an active SCF complex. Puriﬁed SCF ligases resolved by SDS–PAGE and visualized with Coomassie brilliant blue stain (n = 3). (B) Immunoblot ponents of SCF holoenzyme that co-immunoprecipitate with FLAG-Fbxo7 or FLAG-Fbxo7-ΔF-box (n = 2). (C) Titratio igase activity of puriﬁed SCFFbxo7 complexes or mutant Fbxo7(ΔF-box) protein on cIAP-1(183–570)-HA. A concentrat ient (5, 10, 20, and 40 nM) of SCFFbxo7 or Fbxo7(ΔF-box) protein was used for in vitro ubiquitination assays in combi ubiquitin mix (ubiquitin buffer, E1, UBE2D1, and ATP) and puriﬁed cIAP-1(183–570). Membranes were probed with a bodies to visualize the ubiquitination proﬁle and anti-FLAG antibodies to evaluate the amount of E3 ligase (n = 2). mmunoblot for proteins that co-immunoprecipitate with FLAG-tagged SCF Fbxo7 ligases (WT, ΔF-box, or V253E). Figure 1. Fbxo7, but not a mutant version Fbxo7(ΔF-box), forms an active SCF complex. (A) Puriﬁed SCF ligases resolved by SDS–PAGE and visualized with Coomassie brilliant blue stain (n = 3). (B) Immunoblots for components of SCF holoenzyme that co-immunoprecipitate with FLAG-Fbxo7 or FLAG-Fbxo7-ΔF-box (n = 2). (C) Titration of the ligase activity of puriﬁed SCFFbxo7 complexes or mutant Fbxo7(ΔF-box) protein on cIAP-1(183–570)-HA. A concentration gradient (5, 10, 20, and 40 nM) of SCFFbxo7 or Fbxo7(ΔF-box) protein was used for in vitro ubiquitination assays in combination with ubiquitin mix (ubiquitin buffer, E1, UBE2D1, and ATP) and puriﬁed cIAP-1(183–570). substrates using a protein array Membranes were probed with anti-HA antibodies to visualize the ubiquitination proﬁle and anti-FLAG antibodies to evaluate the amount of E3 ligase (n = 2). (D) Immunoblot for proteins that co-immunoprecipitate with FLAG-tagged SCF Fbxo7 ligases (WT, ΔF-box, or V253E). presence of the ubiquitin mix used in this experiment, hence the high background observed in the negative control. Fb 7 These data demonstrate the robust ligase activity of the SCFFbxo7 ligase and its potential to ubiquitinate a large number of proteins. Importantly, there was no overlap between the SCFFbxo7 substrates with those identi- ﬁed in a previously reported SCFFbxo25 Protoarray® screen [41], arguing that this methodology identiﬁes speciﬁc candidate substrates for individual SCF-type E3 ligases. 3569 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Figure 2. Identiﬁcation of new Fbxo7 ubiquitinated substrates using protein arrays. (A) Schematic of the experimental design. Two concentrations of SCFFbxo7 or Fbxo7(ΔF-box) protein were used for the ubiquitination of Protoarrays®. Unique substrates were grouped according to the Gene Ontology Cell Compartment or Biological Process through the use of Cytoscape (plugin ClueGO + CluePedia; Supplementary Table S4). (B) Both concentrations of the SCFFbxo7, but not Fbxo7(ΔF-box) protein, promoted ubiquitination of Gsk3β and Tomm20. Quantiﬁcation of the ﬂuorescence intensity of individual spots. Gsk3β and Tomm20 were ubiquitinated in vitro and in vivo by SCFFbxo7 O h h h f ll l h d h ﬁd Figure 2. Identiﬁcation of new Fbxo7 ubiquitinated substrates using protein arrays. (A) Schematic of the experimental design. Two concentrations of SCFFbxo7 or Fbxo7(ΔF-box) protein were used for the ubiquitination of Protoarrays®. Unique substrates were grouped according to the Gene Ontology Cell Compartment or Biological Process through the use of Cytoscape (plugin ClueGO + CluePedia; Supplementary Table S4). (B) Both concentrations of the SCFFbxo7, but not Fbxo7(ΔF-box) protein, promoted ubiquitination of Gsk3β and Tomm20. Quantiﬁcation of the ﬂuorescence intensity of individual spots. Figure 2. Identiﬁcation of new Fbxo7 ubiquitinated substrates using protein arrays. (A) Schematic of the experimental design. Two concentrations of SCFFbxo7 or Fbxo7(ΔF-box) protein were used for the ubiquitination of Protoarrays®. Unique substrates were grouped according to the Gene Ontology Cell Compartment or Biological Process through the use of Cytoscape (plugin ClueGO + CluePedia; Supplementary Table S4). (B) Both concentrations of the SCFFbxo7, but not Fbxo7(ΔF-box) protein, promoted ubiquitination of Gsk3β and Tomm20. Quantiﬁcation of the ﬂuorescence intensity of individual spots. he N-terminus of Fbxo7 can mediate an interaction with Gsk3β The N-terminus of Fbxo7 can mediate an interaction with Gsk3β To determine the region within Fbxo7 required for interacting with these substrates in cells, co-immunoprecipitation assays were conducted. HEK293T cells were co-transfected with plasmids encoding C-terminally HA-tagged Gsk3β and various N-terminally FLAG-tagged Fbxo7 constructs: WT or Fbxo7 con- structs wherein the Ubl domain (1–88), a linker region (89–128), the PRR (399–522), or the last 24 aa at the C-terminus (R498X) was deleted either singly or in combination. The R498X mutation is a naturally occurring pathogenic mutation causing an early-onset PD. For Gsk3β, the loss of either the C-terminal 24 aa (R498X) or the PRR (1–398) did not substantially affect binding (Figure 4A). Loss of the N-terminal Ubl domain, as tested with the 89–522 construct, also did not affect Fbxo7 interaction with Gsk3β. However, loss of both the Ubl domain and the linker, as seen with a 129–522 construct, ablated their interaction. At the Fbxo7 C-terminus, loss of the PRR alone, 1–398, weakened binding, but the additional loss of the Ubl domain, as tested with the 89–398 construct, ablated Gsk3β binding altogether. These data suggest that the PRR, together with the Ubl/ linker sequences, contributes to the interaction of Fbxo7 with Gsk3β in cells, but is not sufﬁcient to mediate binding (Figure 4A). These experiments indicate multiple binding sites and/or a bipartite interaction for Gsk3β with Fbxo7. These interaction experiments are summarized in Figure 4B. To further validate the binding sites of Gsk3β on Fbxo7, we performed in vitro binding assays using FLAG-Fbxo7 deletion mutants immunopuriﬁed from cells and immobilized on agarose, in binding assays with puriﬁed Gsk3β (Figure 4C). In support of the in vivo co-immunoprecipitation studies, Fbxo7 constructs lacking the ﬁrst 129 amino acids, deleting the Ubl and linker of Fbxo7 were unable to interact with Gsk3β. Fbxo7 1–398 lacking only the PRR could still bind to Gsk3β, as could ΔF-box in this in vitro setting. To test this further, we created a ligase lacking the N-terminal 128 amino acids for its ability to ubiquitinate Gsk3β. We found that SCFFbxo7(129–522) was an E3 ubiquitin ligase with equivalent activity to SCFFbxo7 (Supplementary Figure S4A), which showed reduced activity against Gsk3β tested in in vitro ubiquitination assays (Figure 4D). These data argue for the N-terminus of Fbxo7 being the domain through which Gsk3β can be recruited for ubiquitination. Gsk3β and Tomm20 were ubiquitinated in vitro and in vivo by SCFFbxo7 Gsk3β and Tomm20 were ubiquitinated in vitro and in vivo by SCFFbxo7 One caveat inherent in using protein arrays is that the full-length proteins spotted on the arrays are puriﬁed from insect cells in fusion with both His and GST tags, which may affect ubiquitination. Also, these proteins would lack the posited PTMs and/or cofactors, which may be needed for ligase recruitment within cells. We therefore wished to retest our ﬁndings from the arrays using independent methodologies. To take this forward, we selected Gsk3β, ranked 38th, and Tomm20, ranked 44th (Supplementary Table S5), because of the existing literature arguing that they act within pathways that are potentially involved in the pathobiology of PD, and because inhibitors of these pathways are being pursued as potential therapeutics [21,42–47]. Their ubiquitina- tion signal on the arrays is shown in Figure 2B. We produced these potential substrates by in vitro transcrip- tion/translation in reticulocyte lysates using plasmids encoding C-terminally HA-tagged substrates, Gsk3β-HA or Tomm20-HA, and used these programmed lysates for in vitro ubiquitination assays (Figure 3A,B and Supplementary Figure S3A,C). We also used puriﬁed Gsk3β-HA or Tomm20-HA isolated by immunoprecipita- tion from cells and eluated with HA peptide as the substrates for in vitro ubiquitination assays (Supplementary Figure S3B,D). In the case of Gsk3β, a smear of higher MW bands was seen in crude reticulocyte lysates when no exogenous ligase was added to the reaction, suggesting that Gsk3β modiﬁcation occurred in reticulocyte 3570 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Common pen access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 lysates (Figure 3A and Supplementary Figure S3A). However, there was an increased intensity of higher MW bands in the presence of WT SCFFbxo7 ligase, which was dependent on the F-box domain (Figure 3A and Supplementary Figure S3A,B). For Tomm20, the addition of WT SCFFbxo7 ligase promoted discrete laddering, suggesting mono- or multi-mono-ubiquitination, and was also dependent on the F-box domain of Fbxo7 (Figure 3B, arrows). In addition, higher MW bands, dependent on Fbxo7 ligase activity, were also detectable using antibodies to Tomm20 (Supplementary Figure S3C,D, arrows). Gsk3β and Tomm20 were ubiquitinated in vitro and in vivo by SCFFbxo7 Taken together, these data support the idea that SCFFbxo7 catalyzed ubiquitination of these proteins. lysates (Figure 3A and Supplementary Figure S3A). However, there was an increased intensity of higher MW bands in the presence of WT SCFFbxo7 ligase, which was dependent on the F-box domain (Figure 3A and Supplementary Figure S3A,B). For Tomm20, the addition of WT SCFFbxo7 ligase promoted discrete laddering, suggesting mono- or multi-mono-ubiquitination, and was also dependent on the F-box domain of Fbxo7 (Figure 3B, arrows). In addition, higher MW bands, dependent on Fbxo7 ligase activity, were also detectable using antibodies to Tomm20 (Supplementary Figure S3C,D, arrows). Taken together, these data support the idea that SCFFbxo7 catalyzed ubiquitination of these proteins. b y q p To further test whether these candidate targets are substrates for SCFFbxo7, we carried out ubiquitination assays in HEK293T cells co-transfected with plasmids encoding C-terminally HA-tagged substrates including Gsk3β-HA or Tomm20-HA and N-terminally FLAG-tagged Fbxo7 or Fbxo7(ΔF-box). Substrates were IP via their HA tag, and immunoblotted with antibodies to the protein (Gsk3β or Tomm20; Figure 3C,D). In the samples co-transfected with WT Fbxo7, a smear of higher MW proteins was seen for Gsk3β (Figure 3C), while discrete laddering was observed for Tomm20 (Figure 3D). Similar results were obtained when experiments were performed with endogenous ubiquitin (Supplementary Figure S3E,F). In addition, in in vivo ubiquitina- tion assays performed with exogenous myc-tagged ubiquitin, Tomm20 showed discrete laddering, and both proteins showed robust smears of high-MW polyubiquitinated proteins over 100 kDa (Supplementary Figure S3G,H). These data support the idea that Fbxo7 promoted ubiquitination of both Gsk3β and Tomm20, and taken together, with the in vitro ubiquitination assays indicate that these two candidates identiﬁed on the protein arrays are ubiquitinated substrates of SCFFbxo7. he N-terminus of Fbxo7 can mediate an interaction with Gsk3β Similar experiments to map the interaction of Fbxo7 with Tomm20 were conducted in cells (Figure 4E) and by in vitro binding assays, although the latter were unsuccessful. In cells, the interaction of Tomm20 with Fbxo7 was substantially weakened when the N-terminus was deleted, see 89–522 and 129–522, suggesting that amino acids 1–128 also mediated the interaction of Fbxo7 and Tomm20. However, unlike Gsk3β, which is reported to localize in the cytosol, nucleus, and mitochondria, Tomm20 is part of a tranlocation complex and resident in the outer membrane of mitochondria. Therefore, an alternative explaination for this loss of binding could be the deletion of the MTS of Fbxo7, which lies at the start of isoform 1 (Figure 4B) and is essential for its recruitment to mitochondria [21]. To test this directly, an in vitro ubiquitination assay with SCFFbxo7(129–522) against Tomm20 was performed and showed that the ligase lacking the N-terminus of Fbxo7 was still able to mono-ubiquitinate Tomm20, and that there was only minor loss of ubiqutinated higher MW species 3571 open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC B © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Comm Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Figure 3. Fbxo7 promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. In vitro ubiquitination reactions contained puriﬁed SCFFbxo7 ligase or Fbxo7(ΔF-box) protein and ubiquitin mix (E1, UBE2D1 ubiquitin, and ATP) in combination with HA-tagged substrates Gsk3β (n = 2) (A) or Tomm20 (n = 2) (B). The proteins were resolved by SDS–PAGE, and immunoblots were performed with the indicated antibodies. (C and D) HEK293T cells were transfected with the indicated plasmids and the substrates were IP with anti-HA beads; Gsk3β (n = 3) (C) and Tomm20 (n = 3 (D). Proteins were separated by SDS–PAGE, and immunoblots were probed with antibodies to the substrates. Figure 3. Fbxo7 promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. In vitro ubiquitination reactions contained puriﬁed SCFFbxo7 ligase or Fbxo7(ΔF-box) protein and ubiquitin mix (E1, UBE2D1, ubiquitin, and ATP) in combination with HA-tagged substrates Gsk3β (n = 2) (A) or Tomm20 (n = 2) (B). The proteins were resolved by SDS–PAGE, and immunoblots were performed with the indicated antibodies. he N-terminus of Fbxo7 can mediate an interaction with Gsk3β (C and D) HEK293T cells were transfected with the indicated plasmids and the substrates were IP with anti-HA beads; Gsk3β (n = 3) (C) and Tomm20 (n = 3) (D). Proteins were separated by SDS–PAGE, and immunoblots were probed with antibodies to the substrates. Figure 3. Fbxo7 promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. In vitro ubiquitination reactions contained puriﬁed SCFFbxo7 ligase or Fbxo7(ΔF-box) protein and ubiquitin mix (E1, UBE2D1, ubiquitin, and ATP) in combination with HA-tagged substrates Gsk3β (n = 2) (A) or Tomm20 (n = 2) (B). The proteins were resolved by SDS–PAGE, and immunoblots were performed with the indicated antibodies. (C and D) HEK293T cells were transfected with the indicated plasmids and the substrates were IP with anti-HA beads; Gsk3β (n = 3) (C) and Tomm20 (n = 3 (D). Proteins were separated by SDS–PAGE, and immunoblots were probed with antibodies to the substrates. Figure 3. Fbxo7 promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. In vitro ubiquitination reactions contained puriﬁed SCFFbxo7 ligase or Fbxo7(ΔF-box) protein and ubiquitin mix (E1, UBE2D1, ubiquitin, and ATP) in combination with HA-tagged substrates Gsk3β (n = 2) (A) or Tomm20 (n = 2) (B). The proteins were resolved by SDS–PAGE, and immunoblots were performed with the indicated antibodies. (C and D) HEK293T cells were transfected with the indicated plasmids and the substrates were IP with anti-HA beads; Gsk3β (n = 3) (C) and Tomm20 (n = 3) (D). Proteins were separated by SDS–PAGE, and immunoblots were probed with antibodies to the substrates. promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. Figure 3. Fbxo7 promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. In vitro ubiquitination reactions contained puriﬁed SCFFbxo7 ligase or Fbxo7(ΔF-box) protein and ubiquitin mix (E1, UBE2D1, ubiquitin, and ATP) in combination with HA-tagged substrates Gsk3β (n = 2) (A) or Tomm20 (n = 2) (B). The proteins were resolved by SDS–PAGE, and immunoblots were performed with the indicated antibodies. (C and D) HEK293T cells were transfected with the indicated plasmids and the substrates were IP with anti-HA beads; Gsk3β (n = 3) (C) and Tomm20 (n = 3) (D). Proteins were separated by SDS–PAGE, and immunoblots were probed with antibodies to the substrates. e 3. Fbxo7 promotes ubiquitination of Gsk3β and Tomm20 in vitro and in cells. (Figure 4F). he N-terminus of Fbxo7 can mediate an interaction with Gsk3β This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons A Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 compare 89–522 binding with 89–398). Potential reasons for this include that the PRR inhibits Fbxo7 inter- action with Tomm20 or the smaller 89–398 construct can localize to the mitochondria. Combined, these data indicate that sequences between 89 and 398 of Fbxo7 can bind to Tomm20 in cells. These sequences include the F-box domain (335–372), and we also noted a diminished interaction of Tomm20 with an Fbxo7 construct lacking the F-box domain similar to Gsk3β (Figure 4A,E). However, this was likely to be due to a change in the localization of Fbxo7 as a result of a deletion of an NES embedded within the ﬁrst helix of the F-box domain [22]. We previously demonstrated that the NES of Fbxo7 is important for its cell cycle-dependent cytoplasmic/ nuclear shuttling, and if mutated, Fbxo7 becomes a predominantly nuclear protein. To test whether the ΔF-box mutant does not interact with Tomm20 because of its localization to the nucleus, we utilized an Fbxo7 quadru- ple point mutant, called H1, which mutates the F-box domain such that it does not bind to Skp1 but leaves a functional NES. The H1 mutant localizes similarly to WT, predominantly cytoplasmic with nuclear shuttling [22]. We directly compared the H1 mutant with the ΔF-box mutant for their interaction with Tomm20 using co-immunoprecipitation studies, and found the H1 mutant can bind to Tomm20, whereas the ΔF-box mutant does not (Figure 4G), supporting the nuclear localization of the NES/ΔF-box mutant is the likely cause for the lack of interaction with Tomm20. Both mutants do not bind to Skp1, which also indicates that Fbxo7 can bind to Tomm20 independent of it being part of an E3 ligase. SCFFbxo7 promotes non-degradative chain formation on Gsk3β and Tomm20 The type of polyubiquitin chain linkages catalyzed by an E3 ligase on a substrate induces different functional consequences [48]. To investigate the types of ubiquitin chains ligated by SCFFbxo7 onto Gsk3β, we carried out UbiCRest analyses by using DUBs as described in ref. [34]. The source of the ubiquitinated protein used in DUB assays was obtained by immunoprecipitating Gsk3β-HA from cells also transfected with Fbxo7 as in Figure 3A, where Fbxo7 expression enhanced a smear of ubiquitination. he N-terminus of Fbxo7 can mediate an interaction with Gsk3β Ubiquitinated Gsk3β was subjected to cleavage using a panel of DUBs, including as a positive control, USP21, a non-speciﬁc DUB which cleaves all types of ubiquitin chains (Figure 5A). Only increasing amounts of the K63-speciﬁc OTUD1 concentration diminished the intensity of the smear of polyubiquitinated Gsk3β (Figure 5A, lanes 6 and 7), indicating that Fbxo7 catalyzed predominantly K63-linked ubiquitin chains. To independently test for the presence of Fbxo7-enhanced K63 chains on Gsk3β, HEK293T cells were co-transfected with Gsk3β-HA and Fbxo7 or ΔF-box domain. Immunoprecipitates of Gsk3β-HA were probed for the presence of K63 chains, and these were detected when Fbxo7, but not ΔF-box, was overexpressed (Figure 5B). Fb To test the effect of SCFFbxo7 ubiquitination on Gsk3β and Tomm20, their total levels were assayed by immunoblotting of HEK293T cell lysates where constitutive knockdown (KD) of Fbxo7 expression was achieved by stable expression of two independent short hairpin miRNA constructs. Consistent with the identiﬁ- cation of mainly K63 polyubiquitination by SCFFbxo7 from UbiCRest analyses, reducing Fbxo7 expression did not alter Gsk3β levels (Figure 5C). These results were also conﬁrmed by similar experiments in SHSY-5Y KD cells (data not shown) and in experiments where cells were transiently transfected with dsRNA targeting Fbxo7 in U2OS cells (Supplementary Figure S4B). These ﬁndings suggest that ubiquitination may modulate its local- ization or function. Gsk3β is an upstream regulator of β-catenin, which has roles both in transcription through activation of TCF/LEF-binding sites and in adhesion through its binding to cadherins. We ﬁrst tested whether Fbxo7 affected transcriptional activation using a TCF/LEF luciferase reporter, which is responsive to β-catenin, in U2OS cells. We found that the overexpression of Fbxo7 increased transactivation from this reporter, and this was dependent on the F-box domain (Figure 5D). We also show that inhibition of Gsk3β upon LiCl treatment strongly activates the TCF/LEF reporter, and no further induction of reporter activity is seen with Fbxo7 expression, arguing for its effect being via Gsk3β inhibition (Supplementary Figure S4C). We also tested whether Gsk3β localization was changed as a result of Fbxo7 expression, but no signiﬁcant differences were observed by immunoﬂuorescence assays, neither when Fbxo7 was overexpressed nor when its levels were reduced (Supplementary Figure S4D,E). Gsk3β levels were also not signiﬁcantly altered by Fbxo7 overexpression (Supplementary Figure S4F). To determine if Fbxo7 affected Gsk3β activity, we utilized a GFP reporter with a degron that is sensitive to levels of Gsk3β activity [37]. he N-terminus of Fbxo7 can mediate an interaction with Gsk3β These data argue that other sites downstream of amino acid 128 in Fbxo7 bind to Tomm20. Deletion of the PRR at the C-terminus of the Fbxo7 (1–398) did not compromise binding to Tomm20, but suprisingly, deletion of the PRR restored Tomm20 binding to Fbxo7 lacking the MTS/Ubl domain (Figure 4, 3572 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Com Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 e 4. The N-terminus of Fbxo7 mediates its interaction with Gsk3β. EK293T cells were transfected with the indicated FLAG-Fbxo7 plasmids and Gsk3β-HA and the lysates were IP FLAG beads. Proteins were resolved by SDS–PAGE, and immunoblots were probed with the indicated antibodie ummary of the interaction mapping for Gsk3β and Tomm20 with various Fbxo7 constructs. (C) In vitro binding a immunopuriﬁed and eluted Gsk3β-HA protein added to various Fbxo7 proteins lacking N- and C-terminal dom obilized on anti-FLAG antibodies on agarose. (D) In vitro ubiquitination assay using an SCFFbxo7(129–522) ligase la minus against immunopuriﬁed and eluted Gsk3β-HA protein as the substrate. (E) HEK293T cells were transfecte ated FLAG-Fbxo7 plasmids and Tomm20-HA and the lysates were IP with anti-FLAG beads, as in (A). (F) In vitro Figure 4. The N-terminus of Fbxo7 mediates its interaction with Gsk3β. (A) HEK293T cells were transfected with the indicated FLAG-Fbxo7 plasmids and Gsk3β-HA and the lysates were IP with anti-FLAG beads. Proteins were resolved by SDS–PAGE, and immunoblots were probed with the indicated antibodies. (B) Summary of the interaction mapping for Gsk3β and Tomm20 with various Fbxo7 constructs. (C) In vitro binding assays using immunopuriﬁed and eluted Gsk3β-HA protein added to various Fbxo7 proteins lacking N- and C-terminal domains immobilized on anti-FLAG antibodies on agarose. (D) In vitro ubiquitination assay using an SCFFbxo7(129–522) ligase lacking the N-terminus against immunopuriﬁed and eluted Gsk3β-HA protein as the substrate. (E) HEK293T cells were transfected with the indicated FLAG-Fbxo7 plasmids and Tomm20-HA and the lysates were IP with anti-FLAG beads, as in (A). (F) In vitro ubiquitination assay using an SCFFbxo7(129–522) ligase lacking the N-terminus against immunopuriﬁed and eluted Tomm20-HA protein as the substrate. (G) In vivo co-immunoprecipitation assays conducted as in (E). 3573 16 The Author(s). he N-terminus of Fbxo7 can mediate an interaction with Gsk3β For comparison, a GFP reporter with alanine substitu- tions of the Gsk3β phosphoacceptors in the degron was used. The co-expression of Fbxo7, but not the ΔF-box mutant, with the GFP reporter signiﬁcantly increased the mean ﬂuorescence intensity of the cells compared with vector only expressing cells, indicating a decrease in Gsk3β kinase activity (Figure 5E). Taken together, these data argue for a repressive effect of SCFFbxo7 ubiquitination on Gsk3β activity. 3574 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Common an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Figure 5. Analysis of ubiquitin chain linkages catalyzed on SCFFbxo7 substrates Gsk3β and Tomm20. (A) UbiCRest analyses using DUBs for polyubiquitinated Gsk3β puriﬁed from cells (n = 2). The non-speciﬁc DUB (USP21) Figure 5. Analysis of ubiquitin chain linkages catalyzed on SCFFbxo7 substrates Gsk3β and Tomm20. (A) UbiCRest analyses using DUBs for polyubiquitinated Gsk3β puriﬁed from cells (n = 2). The non-speciﬁc DUB (USP21) cleaved all ubiquitin chains from polyubiquitinated Gsk3β, and K63-speciﬁc DUB, OTUD1, showed concentration-dependent activity against polyubiquitinated protein. (B) Immunoprecipitation of Gsk3β from cells and detecting the presence of K63 polyubiquitin chains using a K63-speciﬁc antibody (n = 2). Asterisk indicates heavy or light chains. (C) Effect of reducing Fbxo7 expression on expression of Gsk3β and Tomm20. HEK293T cell lines expressing two independent shRNAs for Fbxo7 (Fbxo7 sh1 and sh2) or vector control were used. Immunoblotting with the indicated antibodies was performed and protein levels were determined relative to GAPDH. The ratio of the densitometry value for each substrate relative to GAPDH is graphed (n = 3). (D) U2OS cells were co-transfected with an empty luciferase vector (pTA-luc) or a β-catenin-responsive TCF/LEF luciferase reporter, along with a Renilla luciferase reporter as a transfection control. Cells were also co-transfected with an empty vector (vec), Fbxo7 or Fbxo7(ΔF-box). After 48 h, cells were harvested and luciferase expression was assayed in quintuplicate from cell lysates using the Dual Glo kit according to the manufacturer’s instructions (Promega), and luciferase levels were normalized to Renilla levels and expressed relative to pTA-luc control levels (n = 3). he N-terminus of Fbxo7 can mediate an interaction with Gsk3β (E) U2OS cells were transfected with MAPK-Gsk3β-GFP or MAPK-Gsk3βmut-GFP reporters with empty vector, Fbxo7 or Fbxo7(ΔF-box), and GFP expression was determined by ﬂow cytometry. GFP mean ﬂuorescence intensity (MFI) was expressed as a percentage of MAPK-Gsk3βmut-GFP levels (n = 6). (F) U2OS cells were co-transfected with Tomm20-HA and the indicated plasmids bearing Fbxo7 WT or PD-associated mutant alleles, and immunoblots on total cell lysates were performed for the transfected proteins as indicated, and GAPDH used as a loading control. P-value is <0.001, P-value is <0.01. Figure 5. Analysis of ubiquitin chain linkages catalyzed on SCFFbxo7 substrates Gsk3β and Tomm20. Figure 5. Analysis of ubiquitin chain linkages catalyzed on SCFFbxo7 substrates G Figure 5. Analysis of ubiquitin chain linkages catalyzed on SCF substrates Gsk3β and Tomm20. (A) UbiCRest analyses using DUBs for polyubiquitinated Gsk3β puriﬁed from cells (n = 2). The non-speciﬁc DUB (USP21) cleaved all ubiquitin chains from polyubiquitinated Gsk3β, and K63-speciﬁc DUB, OTUD1, showed concentration-dependent activity against polyubiquitinated protein. (B) Immunoprecipitation of Gsk3β from cells and detecting the presence of K63 polyubiquitin chains using a K63-speciﬁc antibody (n = 2). Asterisk indicates heavy or light chains. (C) Effect of reducing Fbxo7 expression on expression of Gsk3β and Tomm20. HEK293T cell lines expressing two independent shRNAs for Fbxo7 (Fbxo7 sh1 and sh2) or vector control were used. Immunoblotting with the indicated antibodies was performed and protein levels were determined relative to GAPDH. The ratio of the densitometry value for each substrate relative to GAPDH is graphed (n = 3). (D) U2OS cells were co-transfected with an empty luciferase vector (pTA-luc) or a β-catenin-responsive TCF/LEF luciferase reporter, along with a Renilla luciferase reporter as a transfection control. Cells were also co-transfected with an empty vector (vec), Fbxo7 or Fbxo7(ΔF-box). After 48 h, cells were harvested and luciferase expression was assayed in quintuplicate from cell lysates using the Dual Glo kit according to the manufacturer’s instructions (Promega), and luciferase levels were normalized to Renilla levels and expressed relative to pTA-luc control levels (n = 3). (E) U2OS cells were transfected with MAPK-Gsk3β-GFP or MAPK-Gsk3βmut-GFP reporters with empty vector, Fbxo7 or Fbxo7(ΔF-box), and GFP expression was determined by ﬂow cytometry. GFP mean ﬂuorescence intensity (MFI) was expressed as a percentage of MAPK-Gsk3βmut-GFP levels (n = 6). he N-terminus of Fbxo7 can mediate an interaction with Gsk3β (F) U2OS cells were co-transfected with Tomm20-HA and the indicated plasmids bearing Fbxo7 WT or PD-associated mutant alleles, and immunoblots on total cell lysates were performed for the transfected proteins as indicated, and GAPDH used as a loading control. *P-value is <0.001, P-value is <0.01. (A) UbiCRest analyses using DUBs for polyubiquitinated Gsk3β puriﬁed from cells (n = 2). The non-speciﬁc DUB (USP21) cleaved all ubiquitin chains from polyubiquitinated Gsk3β, and K63-speciﬁc DUB, OTUD1, showed concentration-dependent activity against polyubiquitinated protein. (B) Immunoprecipitation of Gsk3β from cells and detecting the presence of K63 polyubiquitin chains using a K63-speciﬁc antibody (n = 2). Asterisk indicates heavy or light chains. (C) Effect of reducing Fbxo7 expression on expression of Gsk3β and Tomm20. HEK293T cell lines expressing two independent shRNAs for Fbxo7 (Fbxo7 sh1 and sh2) or vector control were used. Immunoblotting with the indicated antibodies was performed and protein levels were determined relative to GAPDH. The ratio of the densitometry value for each substrate relative to GAPDH is graphed (n = 3). (D) U2OS cells were co-transfected with an empty luciferase vector (pTA-luc) or a β-catenin-responsive TCF/LEF luciferase reporter, along with a Renilla luciferase reporter as a transfection control. Cells were also co-transfected with an empty vector (vec), Fbxo7 or Fbxo7(ΔF-box). After 48 h, cells were harvested and luciferase expression was assayed in quintuplicate from cell lysates using the Dual Glo kit according to the manufacturer’s instructions (Promega), and luciferase levels were normalized to Renilla levels and expressed relative to pTA-luc control levels (n = 3). (E) U2OS cells were transfected with MAPK-Gsk3β-GFP or MAPK-Gsk3βmut-GFP reporters with empty vector, Fbxo7 or Fbxo7(ΔF-box), and GFP expression was determined by ﬂow cytometry. GFP mean ﬂuorescence intensity (MFI) was expressed as a percentage of MAPK-Gsk3βmut-GFP levels (n = 6). (F) U2OS cells were co-transfected with Tomm20-HA and the indicated plasmids bearing Fbxo7 WT or PD-associated mutant alleles, and immunoblots on total cell lysates were performed for the transfected proteins as indicated, and GAPDH used as a loading control. *P-value is <0.001, P-value is <0.01. In the case of Tomm20, there was a signiﬁcant decrease in its endogenous levels when Fbxo7 was knocked down constitutively or by transient transfection of siRNA (Figure 5C and Supplementary Figure S4B), suggest- ing that Fbxo7 stabilizes Tomm20 levels. Conversely, a small and reproducible 50% increase was observed in 3575 © 2016 The Author(s). he N-terminus of Fbxo7 can mediate an interaction with Gsk3β This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Comm Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Figure 5. Continued Figure 5. Continued Figure 5. Continued endogenous Tomm20 levels when Fbxo7 was overexpressed, (Supplementary Figure S4F). These data indicate a direct correlation between endogenous Fbxo7 and Tomm20 expression levels. However, results in Figure 4G argue that stabilization of Tomm20 occurred independently of Fbxo7 being part of an SCF complex. As can be seen in Figure 4G, overexpression of WT Fbxo7 and the H1 mutant both caused increased Tomm20-HA levels (Input, IB: HA, lanes 3 and 5), even though H1 does not recruit Skp1. These data suggest that the stabilization of Tomm20 does not require ubiquitination by SCFFbxo7. q q y We also tested whether the mutants in Fbxo7 associated with early-onset PD affected the ubiquitination of Tomm20 by co-transfecting cells with the mutant Fbxo7 alleles (T22M, R378G, and R498X) and Tomm20-HA (Figure 5F). The presence the higher MW mono- and di-ubiquitinated Tomm20 were not altered by co-transfection of mutant alleles of Fbxo7 when compared with WT Fbxo7. Importantly, as the T22M Fbxo7 mutant cannot bind to Parkin, these data strongly argue that the ubiquitination detected was not due to Parkin-mediated ubiquitination, but rather mediated by Fbxo7. These data indicate that mutant Fbxo7 alleles were capable of enhancing Tomm20 ubiquitination, and suggest that defects in Tomm20 ubiquitination do not contribute to the aetiology of the Fbxo7 cases of early-onset PD. Discussion Our data place Fbxo7 in the complex regulatory landscape of Gsk3β function and activ- ity, and indicate that the consequences of Gsk3β ubiquitination by SCFFbxo7 warrant further investigation in the context of neurodegenerative PD model systems. g y Fbxo7 has a reported role in mitophagy through its direct association with two other PD associated proteins, PINK1/PARK6 and Parkin/PARK2 [21]. Upon depolarization, Tomm20 is one of the mitochondrial proteins that is ubiquitinated by Parkin [64,65]. Tomm20 is a core component of the mitochondrial translocase complex, and its overexpression alone can promote mitophagy [44]. We demonstrate here that under normal conditions, WT Fbxo7 and the mutant alleles of Fbxo7 stabilized its levels, which did not depend on SCF for- mation, and promoted Tomm20 mono-, multi-mono-, or di-ubiquitination. The overexpression of human Fbxo7, but not the PD-associated alleles, rescues the phenotypes of parkin loss in a Drosophila model of neuro- degeneration [21]. However, the WT and mutant alleles tested have a similar ability to ubiquitinate Tomm20, indicating that improper ubiquitination of Tomm20 by Fbxo7 is unlikely to be the pathological deﬁciency. These included a T22M mutant of Fbxo7, which cannot recruit Parkin, indicating Parkin was not catalyzing Tomm20 ubiquitination in these experiments. An enrichment analysis of the substrates by GO Cellular Compartment revealed a further 29 mitochondrial proteins, which may indicate a more general role of Fbxo7 in mitochondrial biology, and suggests that Fbxo7 may affect other activities through ubiquitination of sub- strates, like ATP5C1, CHCHD2, and MTIF3, mitochondrial translation initiation factor 3 (Supplementary Table S5). We note that the only reported polymorphism within MTIF3, rs7669, has been reported to show a signiﬁcant association with risk of PD [66], and CHCHD2 also has been reported to be associated with cases of autosomal dominant PD [67–69]. These substrates indicate a connected network among the genes mutated in familial PD data and are an area for future study. b y In summary, we identiﬁed 338 new targets of SCFFbxo7 using a high-throughput, cell-independent proteomic approach and validated Gsk3β and Tomm20 as new substrates, and argue against defective regulation of Tomm20 by Fbxo7 as an underlying mechanism in PD. On the basis of our ﬁndings, we predict that Fbxo7 will impact on multiple biological functions, and this potentially explains why this F-box protein is of clinical importance in pathologies affecting many different tissue and cell types. Discussion In a study of the Cullin interactome using MS and where the abundance of the cullin-associated proteins was calculated based on peptide recovery, Fbxo7 was the ﬁfth most abundant F-box protein identiﬁed (behind Skp2, Fbxl18, Fbxo21, and Fbxo22), suggesting that SCFFbxo7 is a stable, abundant E3 ligase in a modiﬁed 293 cell line [49]. In agreement with this prediction, we found that the puriﬁed SCFFbxo7 ligase from HEK293T cells was indeed abundant, stable and robustly active when used for in vitro assays. A total of 338 unique pro- teins, or about 3.6% of the proteins on the array, were deﬁned as putative substrates of SCFFbxo7 ubiquitination using a cell-independent methodology to obtain a global view of its activity. Approximately 123 (36%) of the Fbxo7 ubiquinome are listed as ubiquitinated proteins in either HEK293T and/or U2OS cells [50,51], with 17% of this subset affected by treatment with a proteasomal inhibitor MG132. In comparison, the number of sub- strates identiﬁed in the present study is about four times those for SCFFbxo25 and SMURF1 where 89 and 75 substrates, respectively, were identiﬁed using a similar experimental approach [33,41]. The substrates for SCFFbxo7 do not overlap with these other screens, arguing for the speciﬁcity of these ligases in comparable set- tings. We hypothesize that within speciﬁc cell types, SCFFbxo7 activity and the chain linkages assembled will be reﬁned and dictated by the expression levels of Fbxo7, the abundance and potential PTM of its substrates, and the E2 ligases with which it engages. g g g An enrichment analysis of KEGG pathways revealed that several proteins directly involved in the Wnt signal- ing pathway, including Csnk1E, Gsk3β, Prickle2, and Nkd2, were ubiquitinated by SCFFbxo7 on the Protoarray®. Gsk3β phosphorylates proteins like β-catenin, Snail, and Smad, which creates a degron for E3 ligases SCFβ-TRCP 3576 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attr pen access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 and SMURF1 that ubiquitinates them to promote their proteasomal degradation [52–55]. The deregulation of this pathway is associated with several types of cancer [56,57]. Discussion Our dataset opens new perspectives and avenues for investigation in determining the role of SCFFbxo7 ligase activity in the biology of human diseases, including PD and cancer. Discussion In addition to its function in the Wnt pathway, Gsk3β phosphorylates a large number of proteins located throughout the cell and is involved in many cellular processes such as cell proliferation, differentiation, microtubule dynamics, cell cycle, and apoptosis [58]. With such pleiotropic function, Gsk3β is linked with many different diseases including diabetes, cancer, Alzheimer’s disease, osteoporosis, and cardiac hypertrophy, and also with PD [59]. Investigations into the role of Gsk3β in PD have uncovered many associations including a high level of kinase activity within the striatum and also the ﬁnding that a phosphorylated form of Gsk3β is found surrounding Lewy bodies, which may stem from the fact that Gsk3β can directly phosphorylate α-synuclein [60–62]. Gsk3β is also an interesting therapeutic target, and several small-molecule inhibitors have already been described [63]. We ﬁnd that SCFFbxo7 can ubiquitinate Gsk3β and promote K63 linkages. Fbxo7 did not affect Gsk3β endogenous levels or localization, but instead repressed its activity leading to increased expression of a Gsk3β-sensitive GFP reporter and increased β-catenin transactivation in cells. Our data place Fbxo7 in the complex regulatory landscape of Gsk3β function and activ- ity, and indicate that the consequences of Gsk3β ubiquitination by SCFFbxo7 warrant further investigation in the context of neurodegenerative PD model systems. and SMURF1 that ubiquitinates them to promote their proteasomal degradation [52–55]. The deregulation of this pathway is associated with several types of cancer [56,57]. In addition to its function in the Wnt pathway, Gsk3β phosphorylates a large number of proteins located throughout the cell and is involved in many cellular processes such as cell proliferation, differentiation, microtubule dynamics, cell cycle, and apoptosis [58]. With such pleiotropic function, Gsk3β is linked with many different diseases including diabetes, cancer, Alzheimer’s disease, osteoporosis, and cardiac hypertrophy, and also with PD [59]. Investigations into the role of Gsk3β in PD have uncovered many associations including a high level of kinase activity within the striatum and also the ﬁnding that a phosphorylated form of Gsk3β is found surrounding Lewy bodies, which may stem from the fact that Gsk3β can directly phosphorylate α-synuclein [60–62]. Gsk3β is also an interesting therapeutic target, and several small-molecule inhibitors have already been described [63]. We ﬁnd that SCFFbxo7 can ubiquitinate Gsk3β and promote K63 linkages. Fbxo7 did not affect Gsk3β endogenous levels or localization, but instead repressed its activity leading to increased expression of a Gsk3β-sensitive GFP reporter and increased β-catenin transactivation in cells. Acknowledgements We thank Dr Marcelo D. Gomes from the Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil for enabling the research exchange of FRT to the University of Cambridge. We also thank Dr Yasuko Matsuzawa, Sanford-Burnhan Medical Research Institute, La Jolla, CA, USA for kindly providing the human cIAP-1 plasmid. We thank Robin Antrobus and Yagnesh Umrania for expert technical help with LC/MS-MS. Funding g F.R.T. was funded by a BEPE-FAPESP Fellowship [2010/16464-8, 2012/09241-8]. S.J.R. and H.L. are funded by the Biotechnology and Biological Science Research Council [BB/J007846/1]. D.K. is funded by the European Research Council [309756], Medical Research Council [U105192732] and the Lister Institute for Preventive Medicine. T.E.T.M. was funded by the Marie Curie ITN ‘UPStream’. Author Contribution ut o Co t but o F.R.T., S.J.R., S.P.P., T.E.T.M., G.Z., and T.K. conducted experiments and analyzed data. D.K. and H.L. designed experiments, analyzed data and wrote the manuscript. All authors read and edited the manuscript. Competing Interests The Authors declare that there are no competing interests associated with the manuscript. Abbreviations AB, assay buffer; BMP, bone morphogenic protein; BSA, bovine serum albumin; c-IAP1, inhibitor of apoptosis protein 1; DDA, data-dependent acquisition; DTT, dithiothreitol; DUBs, deubiquitinating enzymes; E1, ubiquitin-activating enzyme; E2, ubiquitin-carrier enzyme; FC, fold change; Gsk3β, glycogen synthase kinase 3β; HA, human inﬂuenza hemagglutinin; HRP, horseradish peroxidase; HURP, hepatoma up-regulated protein; IVT, in vitro transcription/translation; LB, lysis buffer; LEF/TCF, lymphoid enhancer factor/T-cell factor; LQT, linear trap quadrupole; MTS, mitochondrial-targeting sequence; MW, molecular weight; NB, NETN lysis buffer; NES, nuclear export signal; NRAGE, neurotrophin receptor-interacting MAGE; PD, Parkinson’s disease; PTMs, post-translational modiﬁcations; SCF, Skp1-Cul1-F box protein; SNP, single nucleotide polymorphism; Tomm20, translocase of outer mitochondrial membrane 20; TRAF2, TNF receptor-associated factor 2; UbiCRest, ubiquitin chain restriction; WT, wild type. 3577 access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 Biochemical Journal (2016) 473 3563–3580 DOI: 10.1042/BCJ20160387 References (2005) Transforming activity of Fbxo7 is mediated speciﬁcally through regulation of cyclin D/cdk6. 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https://openalex.org/W3085335471	https://elib.dlr.de/146162/2/Fairushin_ResultsinPhysics_2020_1-s2.0-S221137972031826X-main.pdf	English	null	Direct evaluation of the physical characteristics of Yukawa fluids based on a simple approximation for the radial distribution function	Results in physics	2,020	cc-by	5,209	A R T I C L E I N F O We propose a simple analytical approximation for the radial distribution function (RDF) in a single-component Yukawa fluid. The proposed RDF depends on the two input parameters – the non-ideality parameter and the structure (screening) parameter , which determine the thermodynamic state of Yukawa systems. We demon- strate that various physical properties can be directly calculated using the proposed RDF. In particular, the internal energy and pressure, the excess entropy in the pair approximation, and the dispersion relation of longitudinal acoustic-like collective excitations are calculated. These theoretical results are compared with the results from molecular dynamics simulations and good overall agreement is observed in the investigated regime of screening and coupling parameters. Keywords: Yukawa fluid Radial distribution function Two-step approximation Thermodynamic properties Dispersion law of the longitudinal collective mode the electron charge, the potential becomes the electron charge, the potential becomes the electron charge, the potential becomes The structure of an equilibrium liquid is characterized by the pre- sence of the so-called short-range order, which determines significantly various physical properties of the liquid state. The radial distribution function (RDF) g r( ) is a structural characteristic, which contains in- formation about relative positions of the particles (molecules) in the system. This function characterizes pair correlations in many-particle systems [1–3] and it appears in the expressions for the basic thermo- dynamic properties such as the internal energy, pressure, and the pair excess entropy. Last quantity is used to approximate the actual excess entropy (with a varying degree of accuracy depending on the state point) and that stems from the difficulty of computing the higher order terms of the Nettleton-Green expansion [4,5]. In addition to the func- tion g r( ), it is also necessary to know the interparticle potential u r( ) in order to calculate analytically these thermodynamic properties. The pairwise interaction potential of the form = u r Ze r r ( ) ( ) exp( / ) 4 , s 2 0 (2) (2) where s is the Debye screening length, associated with the presence of neutralizing medium and 0 is the vacuum permittivity. The time scale of charge density fluctuations in the system is determined by the plasma frequency where s is the Debye screening length, associated with the presence of neutralizing medium and 0 is the vacuum permittivity. I.I. Fairushina,b,⁎, S.A. Khrapakb,c, A.V. Mokshina,⁎ a Kazan Federal University, 420008 Kazan, Russia b Joint Institute for High Temperatures, Russian Academy of Science, 125412 Moscow, Russia c Institut für Materialphysik im Weltraum, Deutsches Zentrum für Luft- und Raumfahrt (DLR), 82234 Weßling, Germany https://doi.org/10.1016/j.rinp.2020.103359 Received 28 April 2020; Received in revised form 12 August 2020; Accepted 26 August 2020 ⁎ Corresponding authors. E-mail addresses: fairushin_ilnaz@mail.ru (I.I. Fairushin), anatolii.mokshin@mail.ru (A.V. Mokshin). Available online 09 Septem ber 2020 2211-3797/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY licens (http://creativecom m ons.org/licenses/by/4.0/). Contents lists available at ScienceDirect Contents lists available at ScienceDirect Direct evaluation of the physical characteristics of Yukawa fluids based on a simple approximation for the radial distribution function I.I. Fairushina,b,⁎, S.A. Khrapakb,c, A.V. Mokshina,⁎ Results in Physics 19 (2020) 103359 Results in Physics 19 (2020) 103359 A R T I C L E I N F O Given the expressions (4) and (5), the formula for the potential can be rewritten in the dimensionless form as follows = u x x x ( ) exp( ) , = u x x x ( ) exp( ) , (6) (6) where = k T 1/( ) B and = x r a / . Recently, it has been demonstrated [7–9] that the so-called one-step approximation for the RDF, of the form = g x x x ( ) ( ) (1st) eff (7) = g x x x ( ) ( ) (1st) eff (7) can be used within the framework of the quasilocalized charge ap- proximation (QLCA) [10,7,11–13] to obtain particularly simple analy- tical expressions for the dispersion of longitudinal and transverse col- lective excitations in Yukawa fluids. Here x( ) is the Heaviside step- function and xeff is the effective correlational hole radius, which can be determined by requiring that the internal energy or pressure are cor- rectly evaluated from the approximation (7). Note that the approx- imation (7) is at first glance better appropriate for a rarefied gas of hard spheres (and real gases at sufficiently high temperatures [14]). How- ever, it turns out to be a good approximation also for fluids with soft long-range interactions, when the cumulative contribution from long distances (where g x( ) 1) is dominant. Nevertheless, the simplest form (7) does not account for the most prominent signature of the liquid state, which is manifested in the characteristic maximum of the RDF. Fig. 1. Radial distribution function. The solid line corresponds to the two-step approximation (8), the dashed line corresponds to a typical RDF of a liquid-like state. = x b b b 1 ln ( ) ( ) , 1 3 1 2 3 (9) where = x b b b 1 ln ( ) ( ) , 1 3 1 2 3 (9) where = b 1.575, 1 = b 1.575, 1 = + b ( ) 0.931 0.422 0.696 , 2 2 = + b ( ) 1.238 0.28 0.644 . 3 2 = + b ( ) 0.931 0.422 0.696 , 2 2 = + b ( ) 1.238 0.28 0.644 . A R T I C L E I N F O The time scale of charge density fluctuations in the system is determined by the plasma frequency = Z e m , p 2 2 0 (3) (3) where is the density of particles in the system and m is the particle mass.i The equilibrium thermodynamics of Yukawa systems is specified by the two key dimensionless parameters: the non-ideality (or coupling) parameter and the structural (or screening) parameter [1–3]. The non-ideality parameter u r r r ( )~ 1 exp , (1) u r r r ( )~ 1 exp , (1) = Ze ak T ( ) 4 B 2 0 (4) is known as the Yukawa (screened Coulomb) potential [1–3,6]. Yukawa potential has been proven to be a suitable model to test various ap- proximations in the theory of condensed matter. Its repulsive character mimics interaction between charged particles immersed into a neu- tralizing medium (like e.g. plasma). For example, in the case of the particles of the same charge Ze, where Z is the charge number and e is (4) represents the ratio of the pair interaction energy (excluding the screening effects) evaluated at the mean interparticle distance to the average energy of the thermal motion of the particles. In expression (4), the quantity = a (3/4 )1/3 is the so-called Wigner-Seitz radius. The https://doi.org/10.1016/j.rinp.2020.103359 Received 28 April 2020; Received in revised form 12 August 2020; Accepted 26 August 2020 ⁎ Corresponding authors. E-mail addresses: fairushin_ilnaz@mail.ru (I.I. Fairushin), anatolii.mokshin@mail.ru (A.V. Mokshin). Available online 09 Septem ber 2020 2211-3797/ © 2020 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/). Results in Physics 19 (2020) 103359 I.I. Fairushin, et al. Fig. 1. Radial distribution function. The solid line corresponds to the two-step approximation (8), the dashed line corresponds to a typical RDF of a liquid-like state. structural parameter is determined as the ratio of a to the Debye screening length s: structural parameter is determined as the ratio of a to the Debye screening length s: = a . s (5) = a . s (5) (5) In the limit = 0, the potential (2) reduces to the bare Coulomb potential. In the opposite limit , it approximates the hard-sphere interaction potential. A R T I C L E I N F O 3 2 = + b ( ) 0.931 0.422 0.696 , 2 2 Expression (9) allows us to determine the value of x1 with good accuracy (the maximum deviation from the simulation results does not exceed 5.6%). To determine the outer radius of the model RDF max- imum, x2, we apply the charge neutrality condition: A rather accurate parameterization of the Yukawa RDF has been devised in Ref. [15]. The expression is based on combining a Coulomb RDF parameterization with an appropriate effective coupling parameter that maps Yukawa fluids into Coulomb fluids (such an effective cou- pling parameter has been identified in molecular dynamics (MD) si- mulations in Ref. [16]). In spite of considerable success of this attempt, the fundamental difference between Coulomb and Yukawa systems renders impossible a perfect match between their static structures through an effective coupling parameter [15]. = g x x dx 1 ( ) 1 3 . 0 2 (10) (10) This condition is strictly valid for Coulomb systems, but remains a very good approximation for weakly screened Yukawa systems. After simple algebra we get f In this paper we propose a much simpler approximation for RDF, which depends explicitly on and and exhibits a maximum. Using this approximation we then calculate the internal energy, pressure, the pair excess entropy, and the dispersion relation of the longitudinal collective mode. The calculation is done for nine ( , ) state points with = 20, 50, 100 and = 1, 1.5, 2. Theoretical results are compared with the results of MD simulations and reasonable agreement is docu- mented. = x x g g 1 1 . m m 2 3 1 3 (11) (11) The quantity gm can be determined from a relation suggested in Ref. [16]: [16]: = + + a a g a g ( ) ( ) ( ) , m m 1 2 3 2 (12) (12) Namely, we suggest the following two-step approximation for the function g x( ): where the parameters a a ( ), ( ) 1 2 and a ( ) 3 satisfy the quadratic poly- nomial where the parameters a a ( ), ( ) 1 2 and a ( ) 3 satisfy the quadratic poly- nomial nomial = + g x g x x x x x x ( ) ( ) ( ) ( ). I.I. Fairushin, et al. (8), we find i = P g G x g g G x 2 ( ) 1 ( ) , m m m ex (2st) 2 1 2 (17) where = + + G x x x x ( ) (( ) 3 3)exp( ) 2 where = P g G x g g G x 2 ( ) 1 ( ) , m m m ex (2st) 2 1 2 (17) where = + + G x x x x ( ) (( ) 3 3)exp( ). 2 = + + G x x x x ( ) (( ) 3 3)exp( ). 2 The microscopic expression for the reduced pair excess entropy Sex2 in the two-particle approximation does not explicitly contain the in- teraction potential u r( ) and for an isotropic system reads [22] As follows from Eqs. (15), (17), (19) and (21), the quantities U P S , , ex ex ex2 and the dispersion relation k ( ) L (QLCA) can be directly cal- culated for a given ( , ) state-point. In addition, these quantities can be evaluated on the basis of microscopic expressions (14), (16), (18) and (20) if the actual RDF is known. The actual RDFs have been cal- culated using MD simulations [25–29]. These simulations have been performed for a Yukawa system consisting of 64000 particles in a cubic cell with the periodic boundary conditions. The simulations have been done the NVT ensemble. The time step for integration of the equations of motion has been chosen as = = t t 0.01/ /(200 3 ) p step th , where tth is the time required for the particle to overcome the average interparticle distance a 2 , moving with the thermal velocity = v k T m / B th . = + S g x g x g x x dx 3 2 ( )ln ( ) 1 ( ) , ex2 0 2 (18) (18) where pair excess entropy is expressed in units NkB. From Eq. (8) we find = + S g x g g g g g 1 2( 1) [ ln ( ln 1 )]. m m m m m m ex2 (2st) 1 3 (19) (19) The interparticle interaction and structural properties determine also the collective particle dynamics of a system [23,3]. A R T I C L E I N F O m (2st) 1 2 2 (8) = + g x g x x x x x x ( ) ( ) ( ) ( ). m (2st) 1 2 2 (8) = + + = a c c c ( ) , 1, 2, 3, 1 ( ) 2 ( ) 3 ( ) 2 = + + = a c c c ( ) , 1, 2, 3, 1 ( ) 2 ( ) 3 ( ) 2 According to Eq. (8), the first maximum of g x( ) is modelled by the rectangular shape. Here, the distances x1 and x2 determine the left and right boundaries of the first maximum of the RDF and gm is the mag- nitude of this maximum. The sketch of this two-step RDF is shown in Fig. 1, where approximation (8) is compared with a real RDF, typical for a liquid-like state. We require that the magnitude of the first peak of the model RDF (8) coincides with that of the real RDF. Other peaks of the RDF corresponding to the second, third, etc. coordination shells are ignored in this two-step approximation. We chose to determine the distance x1 from the condition = g x( ) 0.5 1 for a real RDF (see Fig. 1). Then, following Ref. [16], we can express x1 in terms of and as follows: and the dimensionless coefficients c c , 1 ( ) 2 ( ) and c3 ( ) are constants: = = c c 22.4, 7.88 1 (1) 2 (1) and = = = c c c 9.68; 70.09, 20.28 3 (1) 1 (2) 2 (2) and = = = c c c 32.48; 52.6, 12.71 3 (2) 1 (3) 2 (3) and = c 23.73 3 (3) . From Eq. (12) we obtain we obtain = + + g a a a a a a ( ) ( ) 4 ( ) ( ) 4 ( ) 2 ( ) . m 2 2 2 1 3 3 3 (13) (13) Expression (13) allows us to determine the value of gm with good accuracy (the maximum deviation from the simulation results does not exceed 3.3%). Then, the quantity r2 for a particular ( , ) state-point can be found by solving the system of Eqs. (9), (11) and (13). 2 I.I. Fairushin, et al. I.I. Fairushin, et al. The propaga- tion of a collective mode is characterized by the so-called dispersion relation k ( ), where is the frequency of these excitations, and k is the wave number. The exact expression for the dispersion law k ( ) L of the longitudinal acoustic-like mode in liquids is not known [24]. Therefore, the dispersion k ( ) L is calculated, as a rule, within the framework of some approximations or theoretical models [23]. For soft interactions in the plasma-related context, QLCA has been proven to adequately describe the dispersion relation k ( ) L , especially in the long-wave- length regime. For Yukawa fluids the QLCA model yields [10,7,11,12]: The results of numerical calculations for the reduced excess internal energy Uex, reduced excess internal pressure Pex and reduced pair excess entropy Sex2, obtained using the approximation (8) along with those obtained using the actual RDFs from MD simulations are presented in Table 1. The relative deviations between theoretical and simulation results are also given in this table (in percent). It is observed that the proposed model provides very accurate estimates of Uex and Pex. The relative deviations from “exact” MD results are usually less than 1%. This is comparable to the accuracy of other recent approximations [18,30,20,19]. More accurate integral equation theory models are A R T I C L E I N F O Results in Physics 19 (2020) 103359 Results in Physics 19 (2020) 103359 I.I. Fairushin, et al. The RDF g x( ) enters microscopic equations for various physical properties. For example, the reduced excess internal energy Uex of the Yukawa fluid [17–21] is = + + k k k D k ( ) ( ) , L p (QLCA) 2 2 2 (20) The RDF g x( ) enters microscopic equations for various physical properties. For example, the reduced excess internal energy Uex of the Yukawa fluid [17–21] is = + + k k k D k ( ) ( ) , L p (QLCA) 2 2 2 (20) The RDF g x( ) enters microscopic equations for various physical properties. For example, the reduced excess internal energy Uex of the Yukawa fluid [17–21] is = + + k k k D k ( ) ( ) , L p (QLCA) 2 2 2 (20) (20) where = k ka, where = k ka, Yukawa fluid [17 21] is = U x g x x dx 3 2 exp ( ) . ex 0 (14) Using Eq. (8), we immediately obtain from Eq. (14) = U g F x g g F x 3 2 ( ) 1 ( ) , m m m ex (2st) 2 1 2 (15) where = + F x x x ( ) ( 1)exp( ). The reduced excess internal pressure Pex of Yukawa fluids [17–21] is where = k ka, = D k K k g x x x dx ( ) ( , ) 1 ( ) exp( ) , 0 and = + + + + K k x x kx kx kx kx kx kr x kx kx ( , ) 2 1 ( ) 3 sin( ) 3cos( ) ( ) 3sin( ) ( ) ( ) 3 sin( ) 1 . 2 2 3 2 = U x g x x dx 3 2 exp ( ) . ex 0 (14) where = k ka, = D k K k g x x x dx ( ) ( , ) 1 ( ) exp( ) , 0 = U x g x x dx 3 2 exp ( ) . I.I. Fairushin, et al. ex 0 (14) = D k K k g x x x dx ( ) ( , ) 1 ( ) exp( ) , 0 = D k K k g x x x dx ( ) ( , ) 1 ( ) exp( ) , 0 Using Eq. (8), we immediately obtain from Eq. (14) and = U g F x g g F x 3 2 ( ) 1 ( ) , m m m ex (2st) 2 1 2 (15) a K = U g F x g g F x 3 2 ( ) 1 ( ) , m m m ex (2st) 2 1 2 (15) where = + F x x x ( ) ( 1)exp( ). The reduced excess internal pressure Pex of Yukawa fluids [17–21] is and = + + + + K k x x kx kx kx kx kx kr x kx kx ( , ) 2 1 ( ) 3 sin( ) 3cos( ) ( ) 3sin( ) ( ) ( ) 3 sin( ) 1 . 2 2 3 2 = U g F x g g F x 3 2 ( ) 1 ( ) , m m m ex (2st) 2 1 2 (15) and K k ( , ) (15) (15) where = + F x x x ( ) ( 1)exp( ). = + F x x x ( ) ( 1)exp( ). The reduced excess internal pressure Pex of Yukawa fluids [17–21] is The reduced excess internal pressure Pex of Yukawa fluids [17–21] is Using the approximation (8), we obtain from Eq. (20) = + P x x g x xdx 2 ( 1)exp ( ) . ex 0 (16) = + k g E x g g E x ( ) ( ) 1 ( ) , L p m m m (QLCA 2st) 1 2 (21) where = + + + + E x kx kx kx kx x k kx kx k k x ( ) 1 3 2cos( ) ( ) 2sin( ) ( ) ( 1) ( cos( ) sin( )) ( ) exp( ). 2 3 2 2 2 (16) (21) Then, using Eq. (8), we find Then, using Eq. Table 1 Here, kmax is the position of the main maximum in the static structure factor S k ( ). The symbols represent MD results based on the analysis of the spectral density of longitudinal current fluctuations. Solid curves are the results obtained using Eq. (20) with the actual RDF taken from MD simulations. The dashed curves represent the results of Eq. (21) with the two-step approximation for RDF and the dash-dotted curves correspond to Eq. (20) with the one-step approximation (7) for RDF [7]. The symbols • represent positions of the maxima in the theoretical spectra of the dynamic structure factor from work [3]. vibrational dynamics in the wave number range corresponding to the generalized hydrodynamics: < < k k 0 ( /2) max . In particular, the theo- retical model yields the correct values of the sound velocity: = c k k lim ( )/ s k L 0 . For example, for the state with = 20and = 1 we find = c a /( ) 0.97 s p , and for the state = 100 and = 2 we obtain = c a /( ) 0.44 s p . Note that the dispersion relations found within the one- step approximation (7) also reproduce the MD simulation results with a very good accuracy at long wavelengths [7,8]. available, albeit rather more complicated, such as for instance the variational modified hypernetted chain approach [31], and the iso- morph-based empirically modified hypernetted chain approximation [32] and the empirical bridge function approach [33]. The appeal of the present approach is mostly in terms of its simplicity and physical transparency. For the reduced pair excess entropy the agreement is not so good, but increases when the coupling parameter increases. The entropy is more sensitive to the exact shape of the RDF. This is abso- lutely not surprising: From Eq. (18) we see that in contrast to the energy and pressure, the contribution to the reduced pair excess entropy from the region g x( ) 1 is vanishingly small. The main results of this work can be summarized as follows. The two-step approximation for the RDF proposed in this work yields thermodynamic quantities such as the reduced excess internal energy, the reduced excess internal pressure, the reduced pair excess entropy and also the dispersion relation k ( ) L of the longitudinal collective mode for the Yukawa fluids. Table 1 Reduced excess internal energy Uex, reduced excess internal pressure Pex and reduced pair excess entropy Sex2 of Yukawa fluids evaluated using the actual RDF from MD simulations. The same quantities (U P , ex (2st) ex (2st) and Sex2 (2st)) are calculated from Eqs. (15), (17) and (19). The relative deviations Uex , Pex and Sex2 between the theoretical and simulation values are given [in percents]. l MD simulations. The same quantities (U P , ex (2st) ex (2st) and Sex2 (2st)) are calculated from Eqs. (15), (17) and (19). The relative deviations Uex , Pex and Sex2 between the theoretical and simulation values are given [in percents]. Uex Uex(2st) Uex Pex Pex(2st) Pex Sex2 Sex2 (2st) Sex2 1 20 21.132 21.213 0.38 24.995 25.267 1.09 −0.63 −0.856 35.87 1 50 51.562 51.681 0.23 61.805 62.363 0.9 −1.136 −1.242 9.33 1 100 101.968 101.72 0.24 122.994 123.717 0.59 −1.839 −1.604 12.78 1.5 20 7.029 7.026 0.04 9.371 9.371 0.0 −0.532 −0.738 38.7 1.5 50 16.495 16.525 0.18 22.647 22.621 0.11 −0.937 −1.038 10.8 1.5 100 31.965 31.924 0.13 44.552 44.334 0.49 −1.478 −1.418 4.06 2 20 2.948 2.902 1.56 4.311 4.274 0.86 −0.434 −0.626 44.2 2 50 6.509 6.534 0.38 9.994 10.003 0.09 −0.75 −0.944 25.9 2 100 12.154 12.215 0.5 19.184 19.151 0.17 −1.164 −1.238 6.4 3 I.I. Fairushin, et al. Results in Physics 19 (2020) 103359 Fig. 2. Reduced frequency k ( )/ L p of the longitudinal collective mode of Yukawa fluids versus the reduced wave vector k k / max for nine ( , ) state-points. Here, kmax is the position of the main maximum in the static structure factor S k ( ). The symbols represent MD results based on the analysis of the spectral density of longitudinal current fluctuations. Solid curves are the results obtained using Eq. (20) with the actual RDF taken from MD simulations. The dashed curves represent the results of Eq. (21) with the two-step approximation for RDF and the dash-dotted curves correspond to Eq. (20) with the one-step approximation (7) for RDF [7]. The symbols • represent positions of the maxima in the theoretical spectra of the dynamic structure factor from work [3]. Fig. 2. Reduced frequency k ( )/ L p of the longitudinal collective mode of Yukawa fluids versus the reduced wave vector k k / max for nine ( , ) state-points. CRediT authorship contribution statement [6] Tareeva EE, Ryzhov VN. Theoret Math Phys 2016;189:1806. [7] Khrapak S, Klumov B, Couedel L, Thomas H. Phys Plasmas 2016;23:2. Khrapak S. AIP Adv 2017;7:125026. [8] Khrapak S. AIP Adv 2017;7:125026. I.I. Fairushin: Conceptualization, Data curation, Writing - original draft, Investigation. S.A. Khrapak: Conceptualization, Methodology, Investigation, Writing - review & editing. A.V. Mokshin: Conceptualization, Investigation, Writing - original draft, Project ad- ministration. [9] Khrapak S, Khrapak A. IEEE Trans Plasma Sci 2018;46:737 [10] Kalman GJ, Golden KI. Phys Rev A 1990;41:10. [10] Kalman GJ, Golden KI. Phys Rev A 1990;41:10. [11] Golden KI, Kalman GJ. Phys Plasmas 2000;7:1. [12] Donkó Z, Kalman GJ, Hartmann P. J Phys: Condens Matter 2008;20:413101. [ ] , , y ; [13] Khrapak S, Klumov B, Thomas H. Phys Plasmas 2017;24:023702. [13] Khrapak S, Klumov B, Thomas H. Phys Plasmas 2017;24:0237 [14] Stishov SM. JETP Lett 1980;31:249. [14] Stishov SM. JETP Lett 1980;31:249. [15] Desbiens N, Arnault P, Clerouin J. Phys Plasm 2016;23:092120 [15] Desbiens N, Arnault P, Clerouin J. Phys Plasm 2016;23:092120. Declaration of Competing Interest [16] Ott T, Bonitz M, Stanton L, Murillo MS. Phys Plasmas 2014;2 [17] Farouki RT, Hamaguchi S. J Chem Phys 1994;101:9885. [18] Tolias P, Ratynskaia S, de Angelis U. Phys Rev E 2014;90:05310 [19] Tolias P, Ratynskaia S, de Angelis U. Phys Plasmas 2015;22:083703. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper. [20] Khrapak SA, Kryuchkov NP, Yurchenko SO, Thomas HM. J Chem Phys 2015;142:194903. [21] Filippov AV, Reshetnyak VV, Starostin AN, Tkachenko IM, Fortov VE. JETP Lett 2019;110:659. [22] Baranyai A, Evans DJ. Phys Rev A 1989;40:3817. Table 1 In the investigated intermediate screening and moderate-to-strong coupling regimes, energy, pressure, and the long-wavelength dispersion relations are all in excellent agreement with MD simulation results. For these quantities the exact structure of the RDF is not essential and properly designed models can be quite useful. For the reduced pair excess entropy the agreement is not so good, illustrating much stronger sensitivity to the exact shape of the RDF. In addition, it should be noted that the two-step approximation for the RDF proposed in this work for a homogeneous Yukawa fluid can also be generalized for the cases of inhomogeneous fluids, which, for example, can form at the surface of solid walls [35], as well as for the cases of two or more component Yukawa fluids [36]. In these cases, additional information is needed on the position x1 of the left boundary of the first maximum in the two-step for the RDF approximation, de- termined from the condition = g x( ) 0.5 1 . The dispersion relations k ( )/ L p of the longitudinal acoustic-like mode are presented in Fig. 2 for the nine ( , ) state-points in- vestigated. Here, the theoretical results obtained using Eq. (21) are compared with the results obtained in the framework of the one-step approximation (7) and MD simulations. In MD simulations, the dis- persion relations have been evaluated from the location of the maxima in the spectral density of the longitudinal current (for details see Ref. [34]).In addition, we estimated the positions of the maxima in the theoretical spectra of the dynamic structure factor from work [3], which obtained using the results of the sum-rule approach. As can be seen from Fig. 2, theoretical results reproduce properly the dispersion law for the case of low temperature states with = 100 and 50. Some discrepancy between the theoretical and MD simulation results is ob- served only for the case of = 20 for the wave number < < k k k ( /2) max max. The main reason for disagreement here is the ne- glect of the kinetic contribution to the dispersion relation (which is reasonable at strong coupling, but is not so appropriate at weaker coupling). It is noteworthy that for all considered ( , ) states, the theoretical model (21) reproduces very well the features of collective 4 I.I. Fairushin, et al. Results in Physics 19 (2020) 103359 Acknowledgement [23] Mokshin AV. Theoret Math Phys 2015;183:449. [24] Hansen JP, McDonald IR. Theory of Simple Liqu [24] Hansen JP, McDonald IR. Theory of Simple Liquids. London: Aca [24] Hansen JP, McDonald IR. Theory of Simple Liquids. London: Academic Press; 2006. [25] Choi Y, Dharuman G, Murillo MS. Phys Rev E 2019;100:013206. [25] Choi Y, Dharuman G, Murillo MS. Phys Rev E 2019;100:013206. The authors are grateful to Khusnutdinoff R.M. and Galimzyanov B.N. for help with molecular dynamics simulations. We thank V. Yaroshenko for reading the manuscript. This work is supported by the Russian Science Foundation (project 19-12-00022). [26] Galimzyanov BN, Ladyanov VI, Mokshin AV. J Cryst Growth 201 [27] Khusnutdinoff RM, Mokshin AV. J Cryst Growth 2019;524:125182. f [28] Mokshin AV, Galimzyanov BN, Yarullin DT. JETP Lett 2019;110:511.f [29] Khusnutdinoff RM, Mokshin AV. JETP Lett 2019;110:557. f [30] Khrapak S, Thomas H. 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https://openalex.org/W2952043270	https://www.nature.com/articles/s41419-018-1096-6.pdf	English	null	Integrative analysis of transcriptomic and clinical data uncovers the tumor suppressive activity of MITF in prostate cancer	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	10,096	© The Author(s) 2018 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Abstract The dysregulation of gene expression is an enabling hallmark of cancer. Computational analysis of transcriptomics data from human cancer specimens, complemented with exhaustive clinical annotation, provides an opportunity to identify core regulators of the tumorigenic process. Here we exploit well-annotated clinical datasets of prostate cancer for the discovery of transcriptional regulators relevant to prostate cancer. Following this rationale, we identify Microphthalmia-associated transcription factor (MITF) as a prostate tumor suppressor among a subset of transcription factors. Importantly, we further interrogate transcriptomics and clinical data to reﬁne MITF perturbation-based empirical assays and unveil Crystallin Alpha B (CRYAB) as an unprecedented direct target of the transcription factor that is, at least in part, responsible for its tumor-suppressive activity in prostate cancer. This evidence was supported by the enhanced prognostic potential of a signature based on the concomitant alteration of MITF and CRYAB in prostate cancer patients. In sum, our study provides proof-of-concept evidence of the potential of the bioinformatics screen of publicly available cancer patient databases as discovery platforms, and demonstrates that the MITF-CRYAB axis controls prostate cancer biology. A R T I C L E A R T I C L E O p e n A c c e s s Integrative analysis of transcriptomics and clinical data uncovers the tumor- suppressive activity of MITF in prostate cancer Lorea Valcarcel-Jimenez1, Alice Macchia1, Natalia Martín-Martín1,2, Ana Rosa Cortazar1,2, Ariane Schaub-Clerigué1, Mikel Pujana-Vaquerizo1, Sonia Fernández-Ruiz1, Isabel Lacasa-Viscasillas3, Aida Santos-Martin3, Ana Loizaga-Iriarte3, Miguel Unda-Urzaiz3, Ivana Hermanova1, Ianire Astobiza1, Mariona Graupera4, Julia Starkova5, James Sutherland 1, Rosa Barrio 1, Ana M. Aransay 1, Arkaitz Carracedo 1,2,6 and Verónica Torrano1,2 Correspondence: Verónica. Torrano (vtorrano@cicbiogune.es) 1CIC bioGUNE, Bizkaia Technology Park, 801ª bld, 48160 Derio, Bizkaia, Spain 2CIBERONC, Madrid, Spain Full list of author information is available at the end of the article. These authors contributed equally: Lorea Valcarcel-Jimenez, Alice Macchia Edited by B. Rotblat Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 DOI 10.1038/s41419-018-1096-6 Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 DOI 10.1038/s41419-018-1096-6 Cell Death & Disease Cell Death & Disease Ofﬁcial journal of the Cell Death Differentiation Association Introduction maintaining the transcriptional homeostasis in normal cells and places gene expression deregulation at the core of cancer research interests. Balanced integration of intracellular circuits operates within a normal cell to sustain physiological homeostasis. Alterations in some, if not all, of these circuits converge in changes on gene expression, which will eventually enable the acquisition and sustenance of the hallmarks of cancer cells1. This event emphasizes the importance of In the last decades, transcriptomics data derived from cancer specimens have become an important resource for the classiﬁcation, stratiﬁcation, and molecular driver iden- tiﬁcation in tumors. We and others have demonstrated that deregulation of gene expression is a key node for cancer pathogenesis and progression2–6. Prostate cancer (PCa) research exempliﬁes the effort in deciphering the genomics and transcriptomics landscape of tumors, and extremely valuable data have been generated7–13. In spite of the public © The Author(s) 2 Ofﬁcial journal of the Cell Death Differentiation Association Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 2 of 12 In the present study, by combining an exhaustive interrogation of seven publically available PCa databases with reﬁned empirical assays, we have identiﬁed MITF as a prostate tumor suppressor. In addition, we have unveiled CRYAB as a novel direct target of the tran- scription factor that is, at least in part, responsible for its tumor-suppressive activity in PCa. Importantly, the tumor-suppressive role for this novel MITF-CRYAB axis is supported by the enhanced prognostic potential of a signature based on the concomitant alteration of both genes in PCa patients. availability of these relevant data, they are still under- exploited by the scientiﬁc community to understand PCa biology. In this regard, the computational tools and dataset selection strategies to carry out these studies are a bottle- neck for the cancer research ﬁeld. availability of these relevant data, they are still under- exploited by the scientiﬁc community to understand PCa biology. In this regard, the computational tools and dataset selection strategies to carry out these studies are a bottle- neck for the cancer research ﬁeld. By combining integrated-bioinformatics screening of clinically relevant PCa datasets with in vivo and in vitro molecular biology assays, we have recently described the metastasis suppressor activity of peroxisome proliferator- activated receptor γ (PPARγ) coactivator alpha (PGC1α)14,15. Bioinformatics screening identiﬁes MITF as a transcription factor altered in prostate cancer p We have recently demonstrated that the reduced expression of the transcriptional coactivator PGC1α is a causal event for metastatic PCa14. We sought to identify transcriptional regulators related to the alteration in PGC1α expression. We designed a bioinformatics strategy based on the analysis of 16 genes directly linked to the regulation of PGC1A gene17,22,34–38, in order to identify transcription factors that could be relevant to PCa biol- ogy. For the candidate screen we applied selection criteria based on the consistency of, ﬁrst, the correlation with PGC1A expression and second, the expression of each individual candidate in seven publicly available PCa datasets7,9–13 (Fig. 1a). We selected those candidates whose expression in primary tumors correlated with PGC1A (R ≥0.2 and p value ≤0.05 in more than 50% of the datasets) (Supplementary Figure 1A) and was altered when compared to normal specimens. For genes exhi- biting various transcript variants, the correlation analysis was initially performed using the average signal (Supple- mentary Figure 1A) and, when available (only Taylor dataset11), the correlation was conﬁrmed in all the indi- vidual isoforms (Supplementary Table 1). The transcrip- tion factor MITF was the sole candidate that complied with the established criteria. We observed a consistent correlation between PGC1A and MITF in four out of the seven datasets analyzed (Fig. 1b and Supplementary Fig- ure 1A). In addition, not only the mean expression but also the expression of the individual MITF isoforms were reduced in primary tumor specimens when compared with the normal prostate tissue samples (Fig. 1c and Supplementary Figure 1B). Taken together, our data reveal MITF as a PGC1A-associated transcription factor that is consistently downregulated in PCa. MITF is a basic helix-loop-helix leucine zipper (bHLHZIP) transcription factor that regulates the expression of lineage commitment programs that are essential for propagation of the melanocyte lineage19. The existence of different MITF transcript variants is the result of both alternative splicing and promoter activa- tion that results in the cell-type-speciﬁc expression of the different MITF isoforms (A, CX, MC, C, E, H, D, B, M, J)20. The melanoma-speciﬁc isoform M-MITF is the best studied isoform and, despite some controversy, its expression is generally deregulated in melanoma. Although MITF alone cannot act as a classical oncogene, it has been called a “lineage survival oncogene” for mel- anoma19,21. Bioinformatics screening identiﬁes MITF as a transcription factor altered in prostate cancer Importantly, the presence or absence of the M-MITF-PGC1α regulatory axis has stratiﬁcation potential in melanoma and informs on the efﬁcacy of BRAF inhibitor treatments18,22. Although the expression of MITF has been detected in other types of tumors different from melanoma23,24, its active role in the pro- gression of these diseases, including PCa, remains unexplored. Crystallin Alpha-B (CRYAB) is a ubiquitous small heat- shock protein that is expressed in response to a wide range of physiological and nonphysiological conditions preventing aggregations of denatured proteins. In a wide variety of tumor types CRYAB has been found to be overexpressed and associated with disease progression25–29 and poor prognosis30,31. However, in PCa and nasopharyngeal cancers, CRYAB expression is decreased32,33, pointing at possible tumor-suppressive activity of CRYAB in these cancer scenarios. Introduction This transcriptional coactivator is a major regulator of mitochondrial biogenesis and function, and has an inherent capacity to integrate environmental sig- nals and cellular energetic demands. This ability empowers PGC1α to be a driver in shaping responses to metabolic stress during different physiologic and tumorigenic processes16. As might be expected due to its fundamental role in normal and cancer scenarios, the regulation of PGC1α expression, from the genomic to the protein level, is complex and dynamic17. At the level of mRNA expression, one of the well-deﬁned direct reg- ulators of PGC1α is the Microphthalmia-associated transcription factor (MITF)18. Ofﬁcial journal of the Cell Death Differentiation Association MITF exhibits tumor-suppressive activity in PCa c MITF expression in normal prostate and primary tumor (PT) specimens in different datasets9–11. Correlation (b) and expression (c) data from Taylor dataset corresponds to the mean signal of all isoforms of the transcripts. In (b) and (c), each dot corresponds to an individual specimen. Sample sizes: Grasso et al. (Normal, n = 12; PT, n = 45); Lapointe et al. (Normal, n = 9; PT, n = 13); Taylor et al. (Normal, n = 29; PT, n = 131). Error bars represent s.e.m. Statistic test: Spearman correlation R (b) and Mann−Whitney test (c). p, p- value Fig. 1 MITF expression correlates with PGC1A expression and is downregulated in PCa. a Schematic representation of candidate screening to mediate PGC1A downregulation in PCa. Candidate selection was performed by applying two different selection criteria based on the consistency within the datasets used (>50%): the expression of the candidate must be consistently (i) correlated with the PGC1A’s and (ii) altered in the disease. b Correlation analysis between PGC1A and MITF expression in primary tumor (PT) specimens of different PCa datasets (refs. 9–11 and TCGA provisional). Sample sizes: Grasso n = 45; Lapointe n = 13; Taylor n = 131 and TCGA provisional n = 495. c MITF expression in normal prostate and primary tumor (PT) specimens in different datasets9–11. Correlation (b) and expression (c) data from Taylor dataset corresponds to the mean signal of all isoforms of the transcripts. In (b) and (c), each dot corresponds to an individual specimen. Sample sizes: Grasso et al. (Normal, n = 12; PT, n = 45); Lapointe et al. (Normal, n = 9; PT, n = 13); Taylor et al. (Normal, n = 29; PT, n = 131). Error bars represent s.e.m. Statistic test: Spearman correlation R (b) and Mann−Whitney test (c). p, p- value mRNA isoforms of MITF in normal, PCa primary tumors, and PCa cell lines (Supplementary Fig. 2A–C). MITFA was the isoform predominantly expressed in the three scenarios analyzed, and we pursued the studies further with this isoform. Next, we aimed to analyze the biological consequences of ectopic expression of MITFA in PC3 PCa cells. We transduced PC3 cells with a lentiviral vector containing a doxycycline-inducible cassette for the expression of MITFA resulting in the generation of the PC3 TRIPZ-MITFA cell line. The induction of MITFA expression (Fig. 2a, b) as well as the regulation of known target genes, including PGC1A14,15 (Supplementary Fig. MITF exhibits tumor-suppressive activity in PCa The expression proﬁle of MITF in PCa, together with its direct correlation with PGC1A, was suggestive of a tumor- suppressive activity of the transcription factor. We ﬁrst examined the differential expression of the distinct Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 3 of 12 Fig. 1 MITF expression correlates with PGC1A expression and is downregulated in PCa. a Schematic representation of candidate screening to mediate PGC1A downregulation in PCa. Candidate selection was performed by applying two different selection criteria based on the consistency within the datasets used (>50%): the expression of the candidate must be consistently (i) correlated with the PGC1A’s and (ii) altered in the disease. b Correlation analysis between PGC1A and MITF expression in primary tumor (PT) specimens of different PCa datasets (refs. 9–11 and TCGA provisional). Sample sizes: Grasso n = 45; Lapointe n = 13; Taylor n = 131 and TCGA provisional n = 495. c MITF expression in normal prostate and primary tumor (PT) specimens in different datasets9–11. Correlation (b) and expression (c) data from Taylor dataset corresponds to the mean signal of all isoforms of the transcripts. In (b) and (c), each dot corresponds to an individual specimen. Sample sizes: Grasso et al. (Normal, n = 12; PT, n = 45); Lapointe et al. (Normal, n = 9; PT, n = 13); Taylor et al. (Normal, n = 29; PT, n = 131). Error bars represent s.e.m. Statistic test: Spearman correlation R (b) and Mann−Whitney test (c). p, p- value on correlates with PGC1A expression and is downregulated in PCa. a Schematic representation of candidate screening to Fig. 1 MITF expression correlates with PGC1A expression and is downregulated in PCa. a Schematic represe Fig. 1 MITF expression correlates with PGC1A expression and is downregulated in PCa. a Schematic representation of candidate screening to mediate PGC1A downregulation in PCa. Candidate selection was performed by applying two different selection criteria based on the consistency within the datasets used (>50%): the expression of the candidate must be consistently (i) correlated with the PGC1A’s and (ii) altered in the disease. b Correlation analysis between PGC1A and MITF expression in primary tumor (PT) specimens of different PCa datasets (refs. 9–11 and TCGA provisional). Sample sizes: Grasso n = 45; Lapointe n = 13; Taylor n = 131 and TCGA provisional n = 495. Ofﬁcial journal of the Cell Death Differentiation Association MITF exhibits tumor-suppressive activity in PCa Statistic test: One-sample t test (a, d, and e) and Student’s t test (c, f p < 0.05, p < 0.01, *p < 0.001 angiogenesis (Supplementary Fig. 2K). Altogether these results demonstrate that MITFA isoform exhibits tumor- suppressive activity in PCa. d), with no effect of doxycycline treatment by itself14. In line with its known function as an inhibitor of cell cycle progression39, the increased expression of MITFA in PC3 cells resulted in a decrease in BrdU incorporation, a surrogate readout of proliferation (Fig. 2e). MITF exhibits tumor-suppressive activity in PCa 2 D–E) was conﬁrmed. We next evaluated the biological outcome of MITFA ectopic expression in PC3 cells and observed that its upregulation signiﬁcantly reduced two- dimensional and anchorage-independent growth (Fig. 2c, Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 4 of 12 Fig. 2 MITF exhibits tumor-suppressive activity in PC3 PCa cell line. a, b Analysis and quantiﬁcation of MITF expression by qRTPCR (a, n = 8)) and western blot (b, representative experiment out of three independent ones) in PC3 TRIPZ-MITFA cells after treatment with 0.5 μg mL−1 doxycycline (Dox). c Relative cell number quantiﬁcation by crystal violet in doxycycline-treated and nontreated PC3 TRIPZ-MITFA cells. Data are normalized to day 0. Asterisks indicate statistics of ﬁve independent experiments. One representative experiment out of ﬁve is shown. Error bars represent standard deviation. d, e Effect of MITF induction on anchorage-independent growth (d, soft agar; n = 4 independent experiments) and BrdU incorporation (e, n = 3 independent experiments). f Impact of MITF induction in tumor growth rate of PC3 TRIPZ-MITFA cells (n = 7 animals per group; 14 injections/tumors). No dox: MITFA noninduced conditions; Dox: MITFA-induced conditions. Error bars represent s.e.m. (a, d, and e) or minimum and maximum values (f). Statistic test: One-sample t test (a, d, and e) and Student’s t test (c, f p < 0.05, p < 0.01, p < 0.001 Fig. 2 MITF exhibits tumor-suppressive activity in PC3 PCa cell line. a, b Analysis and quantiﬁcation of MITF expression by qRTPCR (a, n = 8)) and western blot (b, representative experiment out of three independent ones) in PC3 TRIPZ-MITFA cells after treatment with 0.5 μg mL−1 doxycycline (Dox). c Relative cell number quantiﬁcation by crystal violet in doxycycline-treated and nontreated PC3 TRIPZ-MITFA cells. Data are normalized to day 0. Asterisks indicate statistics of ﬁve independent experiments. One representative experiment out of ﬁve is shown. Error bars represent standard deviation. d, e Effect of MITF induction on anchorage-independent growth (d, soft agar; n = 4 independent experiments) and BrdU incorporation (e, n = 3 independent experiments). f Impact of MITF induction in tumor growth rate of PC3 TRIPZ-MITFA cells (n = 7 animals per group; 14 injections/tumors). No dox: MITFA noninduced conditions; Dox: MITFA-induced conditions. Error bars represent s.e.m. (a, d, and e) or minimum and maximum values (f). Ofﬁcial journal of the Cell Death Differentiation Association Candidate screening of genes mediating the tumor- suppressive activity of MITF We next performed correlation analysis between MITF and each of the six dif- ferentially expressed genes obtained from the microarray (Fig. 3a and Supplementary Figure 3A). The correlation analysis in PCa primary tumor specimens showed that a single gene, Crystallin Alpha B (CRYAB), had a consistent correlation (in more than 50% of datasets) with MITF, both the mean of isoforms (Fig. 3b and Supplementary Fig- ure 3A) and the individual isoform A (Supplementary Table 4). The MITF−CRYAB correlation was conﬁrmed using an independent cohort of PCa patients from a local hospital (Basurto cohort, Supplementary Figure 3A). Moreover, the expression of CRYAB either at the level of mRNA (from public datasets and Basurto cohort) and protein (from Basurto cohort) was consistently down- regulated through the progression of the disease (Fig. 3c, d and Supplementary Figure 3B–D), supporting the associa- tion of MITF and CRYAB expression in PCa. We next asked whether the functional association between MITF and CRYAB could be employed to identify PCa patients with high disease aggressiveness. We thus ascertained the stratiﬁcation potential of the MITF- CRYAB axis in PCa by means of consistency and robustness. We download the mRNA expression raw data together with the clinical data (recurrence or not recur- rence) from Taylor11, Glinsky8, and TCGA7 datasets. The individual or average expression signal of CRYAB and MITF genes was calculated for each patient in each dataset. Patients were separated by quartiles according to the individual or average signal of CRYAB and MITF genes and then Kaplan−Meyer survival curves were plotted comparing patients with low expression (Quartile 1—(Q1)) of the individual genes or the gene combination (CRYAB and MITF) vs the rest of the cohort (Q2 + Q3 + Q4). Strikingly, the signature formed by the average signal of MITF and CRYAB outperformed the prognostic potential of each individual gene, strongly suggesting that the pathway described herein is strongly associated to PCa aggressiveness (Fig. 4f and Supplementary Figure 5). The regulation of CRYAB expression by MITFA was further validated in vitro by western blot and quantitative real-time PCR (qRTPCR) in doxycycline-treated PC3 TRIPZ-MITFA cell lines and in vivo by qRTPCR in the xenograft samples (Supplementary Figure 3E–G). MITF is a transcription factor that regulates gene expression through the DNA binding to E-boxes (Myc-binding sites)19. Candidate screening of genes mediating the tumor- suppressive activity of MITF Sample sizes: Taylor, n = 131; Grasso, n = 49; Lapointe, n = 13; TCGA provisional data, n = 495; and Glinsky, n = 78. c CRYAB expression in normal prostate and primary tumor (PT) specimens in different PCa datasets9–13. Sample sizes: Taylor (N, n = 29; PT, n = 130); Grasso (N, n = 12; PT, n = 49); Varambally (N, n = 6; PT, n = 7); Lapointe et al. (N, n = 9; PT, n = 13), and Tomlins (N, n = 22; PT, n = 32). Data from Taylor dataset correspond to the mean signal of all isoforms of the transcripts. In (b) and (c), each dot corresponds to an individual specimen. d Western blot analysis of CRYAB expression in benign prostatic hyperplasia (BPH) and PCa specimens from Basurto University Hospital cohort (BPH n = 7 patient specimens; PCa n = 14 patient specimens). e Chromatin immunoprecipitation (ChIP) of exogenous MITF on CRYAB promoter in PC3 TRIPZ-MITFA cells after induction with 0.5 µg mL−1 doxycycline for 3 days (n = 4–5). Binding to ANGPT4 was used as a negative control. Final data were normalized to IgG (negative-immunoprecipitation control) and to No dox condition. No dox: MITFA noninduced conditions; Dox: MITFA-induced conditions. Statistic tests: Spearman correlation (b); Mann−Whitney test (c); one-sample t test (e); Error bars represent s.e.m. p < 0.05, *p < 0.01 this aim, we constitutively silenced the expression of CRYAB by RNAi using two independent short hairpin RNA (sh#1 and sh#2) in PC3 TRIPZ-MITFA cells. After validating that RNAi was achieved (Fig. 4a and Supple- mentary Figure 4A–C) the tumor-suppressive activity of MITFA was monitored in control and CRYAB-silenced conditions (PC3 TRIPZ-MITFA scr, sh#1 or sh#2 cell lines). CRYAB silencing blunted the antiproliferative effects of MITFA in vitro in two-dimensional and anchorage-independent growth when compared with scramble shRNA (Fig. 4b, c). Moreover, the reduction in BrdU induced by MITFA was prevented when CRYAB was silenced (Fig. 4d). Importantly, the requirement of CRYAB for the tumor-suppressive activity of MITFA was corroborated in vivo (Fig. 4e and Supplementary Fig- ure 4D–F). The in vitro and in vivo data demonstrate that the induction of CRYAB is a major effector involved in the tumor-suppressive activity of the transcription factor MITF in PCa. the induction of the transcription factor (Supplementary Table 2; yellow bold highlighted). Candidate screening of genes mediating the tumor- suppressive activity of MITF In order to conﬁrm the direct regulation of CRYAB expression by MITFA, we screened the promoter of the chaperon and performed chromatin immunopreci- pitation assays in two Myc-binding sites (UCSC-Genome browser; Supplementary Figure 3H). As predicted, upon doxycycline treatment we detected differential binding of MITFA in both regions of CRYAB promoter (Fig. 3e). Taken together, these data presented CRYAB as a direct target of MITFA and the best candidate to mediate its tumor-suppressive activity in PCa. Candidate screening of genes mediating the tumor- suppressive activity of MITF In order to ascertain whether the regulation of endo- genous PGC1A (Supplementary Fig. 2E) was required for the antiproliferative effect of MITFA in PC3 cells, we aim at silencing PGC1A by using constitutive (pLKO) expression of short hairpins against it (Supplementary Fig. 2F). Transduction with the shRNA prevented the upregulation of PGC1A upon MITFA induction (Sup- plementary Fig. 2G) but the antiproliferative effect of the transcription factor remained unaffected (Supplementary Fig. 2H). These data suggested that the reduced pro- liferation induced by MITFA was not dependent on the regulation of endogenous PGC1A in PC3 cells. In order to decipher the molecular mechanism driving the tumor-suppressive role of MITFA, we performed gene expression proﬁling of both doxycycline-treated and control PC3 TRIPZ-MITFA cells and identiﬁed 101 probes that showed statistically differential signal between both conditions (Supplementary Table 2; GEO Series accession number GSE114345). We ﬁrst performed a gene enrichment analysis using the functional enrichment tool contained in CANCERTOOL40 with those genes that displayed upregulated expression (76 genes) upon MITFA overexpression (Fig. 3a and Supplementary Table 3), as the number of downregulated genes (25) was not sufﬁcient to obtain any gene enrichment. Next, we aimed at identifying potential MITFA effectors of relevance in human PCa. To this end, we established a threshold of 1.5-fold change over MITFA noninduced cells, which resulted in eight probes (corresponding to six annotated genes) upregulated upon Importantly, the overall reduction in cell proliferation induced by MITFA was conﬁrmed in vivo. Using sub- cutaneous xenografts assays we observed that MITFA overexpression in PC3 cells (Supplementary Fig. 2I) led to a marked reduction in the tumor volume (Supplementary Fig. 2J) and growth rate (Fig. 2f), with no changes in Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 5 of 12 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 6 of 12 (see ﬁgure on previous page) Fig. 3 CRYAB is the candidate to mediate its tumor-suppressive activity in PCa. a Workﬂow of the candidate screening. b Correlation analysis between MITF and CRYAB expression in primary tumor (PT) specimens of different PCa datasets. CRYAB mediates the tumor-suppressive activity of MITF in PCa We next studied the functional relevance of CRYAB for the tumor-suppressive activity of MITFA in PCa. Towards Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 7 of 12 Fig. 4 CRYAB mediates the tumor-suppressor activity of MITF. a Analysis of MITFA and CRYAB protein expression in doxycycline-treated PC3 TRIPZ-MITFA cells transduced with shScramble (scr) or two independent shCRYAB (sh#1 and#2) (one representative experiment with technical duplicates is shown; similar results were obtained in three independent experiments). b Relative cell number quantiﬁcation by crystal violet in doxycycline-treated and nontreated PC3 TRIPZ-MITFA cells, in the presence (scr) or absence (sh#1, 2) of CRYAB (n = 5 independent experiments). Data are normali ed to da 0 and represented as cell n mber at da 6 relati e to No Do condition (depicted b a dotted line) c d Effect of CRYAB Fig. 4 CRYAB mediates the tumor-suppressor activity of MITF. a Analysis of MITFA and CRYAB protein expression in doxycycline-treated PC3 TRIPZ-MITFA cells transduced with shScramble (scr) or two independent shCRYAB (sh#1 and#2) (one representative experiment with technical duplicates is shown; similar results were obtained in three independent experiments). b Relative cell number quantiﬁcation by crystal violet in doxycycline-treated and nontreated PC3 TRIPZ-MITFA cells, in the presence (scr) or absence (sh#1, 2) of CRYAB (n = 5 independent experiments). Data are normalized to day 0 and represented as cell number at day 6 relative to No Dox condition (depicted by a dotted line). c, d Effect of CRYAB silencing on anchorage-independent growth (c, soft agar; n = 4 independent experiments) and BrdU incorporation (d, n = 3 independent experiments) in PC3 TRIPZ-MITFA cells after treatment with 0.5 μg mL−1 doxycycline. e Impact of CRYAB silencing on tumor growth rate of MITF- induced cells (n = 10 animals per group-scr or sh#1; 2 injections per mice (scr No dox, n = 10 tumors; sh#1 No dox, n = 8 tumors; scr Dox, n = 6 tumors; sh#1 Dox, n = 11 tumors). f Association of the mean signal of MITF and CRYAB with disease-free survival (DFS) in three PCa datasets (Q1: ﬁrst quartile distribution; rest: second, third, and fourth quartile distribution. Sample sizes: Taylor, primary tumors n = 131; TCGA provisional data primary tumors n = 490; Glinsky, primary tumors n = 78. No dox: MITFA noninduced conditions; Dox: MITFA-induced conditions. HR: hazard ratio. Ofﬁcial journal of the Cell Death Differentiation Association CRYAB mediates the tumor-suppressive activity of MITF in PCa Statistic Fig. 4 CRYAB mediates the tumor-suppressor activity of MITF. a Analysis of MITFA and CRYAB protein expression in doxycycline-treated PC3 TRIPZ-MITFA cells transduced with shScramble (scr) or two independent shCRYAB (sh#1 and#2) (one representative experiment with technical duplicates is shown; similar results were obtained in three independent experiments). b Relative cell number quantiﬁcation by crystal violet in doxycycline-treated and nontreated PC3 TRIPZ-MITFA cells, in the presence (scr) or absence (sh#1, 2) of CRYAB (n = 5 independent experiments). Data are normalized to day 0 and represented as cell number at day 6 relative to No Dox condition (depicted by a dotted line). c, d Effect of CRYAB silencing on anchorage-independent growth (c, soft agar; n = 4 independent experiments) and BrdU incorporation (d, n = 3 independent experiments) in PC3 TRIPZ-MITFA cells after treatment with 0.5 μg mL−1 doxycycline. e Impact of CRYAB silencing on tumor growth rate of MITF- induced cells (n = 10 animals per group-scr or sh#1; 2 injections per mice (scr No dox, n = 10 tumors; sh#1 No dox, n = 8 tumors; scr Dox, n = 6 tumors; sh#1 Dox, n = 11 tumors). f Association of the mean signal of MITF and CRYAB with disease-free survival (DFS) in three PCa datasets (Q1: ﬁrst quartile distribution; rest: second, third, and fourth quartile distribution. Sample sizes: Taylor, primary tumors n = 131; TCGA provisional data primary tumors n = 490; Glinsky, primary tumors n = 78. No dox: MITFA noninduced conditions; Dox: MITFA-induced conditions. HR: hazard ratio. Statistic tests: One-sample t test (b, c and d—No dox vs Dox conditions); Unpaired Student’s t test (t) (b, c and d—Dox-treated scr vs Dox-treated sh#1/2); Log-rank (Mantel–Cox) test (f). Error bars represent s.e.m. /$p < 0.05, **/$$p < 0.01. Asterisks indicate statistic between No dox and Dox conditions and dollar symbol between Dox-treated scr and Dox-treated sh#1 or 2 Ofﬁcial journal of the Cell Death Differentiation Association Page 8 of 12 Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Our data provide solid evidence of an unprecedented MITF-CRYAB transcriptional axis that exerts tumor- suppressive activity in PCa and could positively contribute to disease prognosis. and chronic myeloid leukemia23,52. Moreover, the novelty of our study relies on the observation and deﬁnition of the tumor-suppressive activity of MITF in PCa. In this con- text, MITF upregulation was associated with a reduction in cell proliferation and DNA replication. CRYAB mediates the tumor-suppressive activity of MITF in PCa As occurs in melanoma, the modulation of MITF expression in PCa cells induces the expression of the cell cycle inhibitor p21 but no changes in the cell cycle inhibitor p16 were observed (data not shown). Thus, our results in PCa are in line with the canonical function of MITF in cell cycle progression and proliferation in melanoma39,50,51. Ofﬁcial journal of the Cell Death Differentiation Association Discussion Technological advances in the molecular understanding of cancer have led to a paradigmatic change in the way that we combat the disease41. We are now able to deconstruct a tumor at a molecular level using genomics, transcriptomics, proteomics, and metabolomics42. This, in turn, enables us to foresee, identify, and demonstrate the potential of patient stratiﬁcation3,14,43. Speciﬁcally, the transcriptomics characterization of tumors is an invalu- able strategy to identify clinically relevant genes that play key roles in the progression of cancer, especially for those types with poorer prognosis14. Thus, the comprehensive and integrative analysis of gene expression changes and clinical parameters in cancer has become a mainstream in cancer research44–46. Mining cancer-associated tran- scriptome datasets is an emerging approach used by top cancer research groups, but better tools are needed to increase its power and user-friendliness. In order to face this challenge, new interfaces to exploit OMICs data, such as cBioportal and CANCERTOOL45,47,40, are being designed to help scientists interrogate, integrate, and visualize large amount of information contained on multiple credible and qualiﬁed cancer datasets. It is important to highlight that the tissue-speciﬁc dif- ferences in MITF expression among different cancer types suggest that in order to fully comprehend MITF’s role in cancer, its expression and function has to be analyzed in the context of each particular cell and tissue type. p yp CRYAB is a member of the small heat-shock protein family that functions as stress-induced molecular cha- perone. It inhibits the aggregation of denatured proteins, promotes cell survival, and inhibits apoptosis in the context of cancer53–55. Paradoxically, CRYAB is highly expressed in some cancer types but decreased in others and in both scenarios an association with cancer pro- gression and prognosis has been reported25,26,28–32,56–60. In spite of the amount of information regarding the changes in CRYAB expression in cancer, the transcrip- tional regulation of this chaperone has been poorly explored56. In this study, we described a novel direct transcriptional regulation of CRYAB by MITF. Although there is no direct nor mechanistic evidence of the MITF- CRYAB transcriptional axis in other cancer types, in melanoma both MITF and CRYAB expression are upre- gulated by BRAF/MEK-inhibitor treatments57,60, sug- gesting that this regulation can go beyond both PCa scenario. Indeed, we observed that the correlation between MITF and CRYAB is also present in colorectal cancer, but not in breast nor lung cancer (data available in CANCERTOOL40). Molecular assays Western blot was performed as previously described14. Antibodies used: HA-Tag (Cell Signalling #3724; dilution 1:10,000); MITF (Thermo Fisher Scientiﬁc MA5-14146; dilution 1:1000); β-Actin (Cell Signalling #3700; dilution 1:2000); GAPDH (clone 14C10; Cell Signalling #2218; dilution 1:1000); CRYAB (Cell Signalling #45844s; dilu- tion 1:1000). Our study endorses the potential of transcriptional deregulation analysis, as either a cause or a consequence of cancer, and its impact to support the discovery of novel cancer-related genes and long-term development of novel cancer treatment strategies. RNA was extracted using NucleoSpin® RNA isolation kit from Macherey-Nagel (ref: 740955.240C). For patients and animal tissues a Trizol-based implementation of the NucleoSpin® RNA isolation kit protocol was used as reported14. One microgram of total RNA was used for cDNA synthesis using MaximaTM H Minus cDNA Synthesis Master Mix (Invitrogen M1682). Quantitative Real-Time PCR (qRTPCR) was performed as previously described14. Universal Probe Library (Roche) primers and probes employed are detailed in Supplementary Table 5. GAPDH (Hs02758991_g1) housekeeping assay from Applied Biosystems was used for data normalization. Discussion However, our data showing enhanced prog- nostic potential of the combined signature provides a new Here we show that MITF is downregulated in PCa when compared with normal specimens, in contrast to the elevated expression reported in hepatocellular carcinoma Ofﬁcial journal of the Cell Death Differentiation Association Page 9 of 12 Page 9 of 12 Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 9 of 12 and exciting perspective of the functional interaction of these genes in PCa. Materials and methods Cell culture and reagents Human prostate carcinoma cell lines (PC3) were pur- chased from Leibniz-Institut DSMZ—Deutsche Samm- lung von Mikroorganismen und Zellkulturen GmbH, who provided authentication certiﬁcate. The cell line used in this study was not found in the database of commonly misidentiﬁed cell lines maintained by ICLAC and NCBI Biosample. Cells were transduced with a modiﬁed TRIPZ (Dharmacon) doxycycline-inducible lentiviral construct in which the RFP and miR30 region was substituted by HA- Flag-MITF. Lentiviral shRNA constructs targeting PGC1A (#1-TRCN0000001165 and #2-TRCN000000 1166) and CRYAB (#1-TRCN0000010822 and #2- TRCN0000010823) were purchased from Sigma and a scramble shRNA (hairpin sequence: CCGGCAACAAGA TGAAGAGCACCAACTCGAGTTGGTGCTCTTCATC TTGTTG) was used as control. For PGC1A and CRYAB shRNAs, Puromycin resistance cassette was replaced by Hygromycin cassette from pLKO.1 Hygro (Addgene Ref. 24150) using BamHI and KpnI sites. Cell lines were routinely monitored for mycoplasma contamination and quarantined while treated if positive. Doxycycline hyclate (Dox) and Puromycin were purchased from Sigma, and Hygromycin from Invitrogen. For transcriptomics analysis in PC3 TRIPZ-HA-Flag- MITFA cells, Illumina whole genome -HumanHT- 12_V4.0 (DirHyb, nt) method was used as reported14. A hypergeometric test was used to detect enriched dataset categories. Chromatin immunoprecipitation Chromatin Immunoprecipitation (ChIP) was performed using the SimpleChIP® Enzymatic Chromatin IP Kit (Cat: 9003, Cell Signalling Technology, Inc). Four million PC3 cells were grown in 150 mm dishes either with or without 0.5 µg mL−1 doxycycline during 3 days. Cells from three 150 mm dishes were cross-linked with 35% formaldehyde for 10 min at room temperature. Glycine was added to dishes during 5 min at room temperature. Cells were then Cellular assays Cell number quantiﬁcation with crystal violet was per- formed as referenced14. For starvation experiments 100,000 cells per well were seeded in a six-well plate. Cells were initially plated in 10% FBS media for 24 h and then the media was changed to FBS free media and left overnight. Soft agar assays were performed as previously descri- bed14, seeding 5000 cells per well in six-well plates. Xenotransplant assays For BrDu incorporation, cells were seeded on glass cover slips in 12-well plates and after 4 days, cells were incubated with 3 µg mL−1 BrDu (Sigma B5002). Cells were ﬁxed with 4% para-formaldehyde, permeabilized with 1% Triton X-100 and incubated with a monoclonal anti-BrDu (MoBU-1) antibody (Invitrogen B35128) at a 1:100 dilution. Images were obtained with an AxioImager D1 microscope (Zeiss). At least three different areas per cover slip were quantiﬁed. All mouse experiments were carried out following the ethical guidelines established by the Biosafety and Welfare Committee at CIC bioGUNE. The procedures employed were carried out following the recommendations from AAALAC. Xenograft experiments were performed as previously described14, injecting 106 cells per condition in two ﬂanks per mouse (Nu/Nu immunodeﬁcient males; 6–12 weeks of age). PC3 TRIPZ-HA-MITFA cells alone or under CRYAB silencing were injected in each ﬂank of nude mice and 24 h post-injections mice were fed with chow or doxycycline diet (Research diets, D12100402). Discussion CRYAB is a member of the small heat-shock protein family that functions as stress-induced molecular cha- perone. It inhibits the aggregation of denatured proteins, promotes cell survival, and inhibits apoptosis in the context of cancer53–55. Paradoxically, CRYAB is highly expressed in some cancer types but decreased in others and in both scenarios an association with cancer pro- gression and prognosis has been reported25,26,28–32,56–60. In the present study, we exploited publicly available and well-annotated (transcriptomics and clinical data) PCa databases together with experimental assays to describe a novel tumor-suppressive activity of the transcription fac- tor MITF in PCa, which is executed, at least in part, through the direct regulation of CRYAB expression. The identiﬁcation of MITF emanates from the screening of reported upstream regulators of PGC1A. It is worth noting that transcriptome-wide correlative study with the gene of interest could represent a complementary approach to predict candidate upstream regulators and downstream effectors. In our study, the MITF-CRYAB transcriptional axis is reduced and exerts tumor-suppressive activity in PCa. This is in agreement with the reduced expression of CRYAB observed in PCa patients and its previous con- sideration as a protective gene against PCa32. Yet, the exact molecular mechanism underlying the tumor- suppressive activity of CRYAB remains to be elucidated. The functional implication of MITF in cancer has been best deﬁned in melanoma, in which the expression of the transcription factor is heterogeneous. Although some controversy exists regarding its oncogenic role in mela- noma, MITF has been deﬁned as a “lineage survival oncogene” with no data pointing out at a tumor- suppressive function19,21,39,48–51. Even though the expression of MITF has been detected in other cancer types23,24,52, no data supporting a functional role of MITF deregulation have been reported yet in a cancer scenario different from melanoma. Importantly, in the present manuscript, the extensive interrogation of PCa transcriptomes and associated clin- ical data has led us to propose the transcriptional axis MITF-CRYAB as a potential prognostic biomarker in PCa. The individual expression of CRYAB and MITF has been previously associated with poor prognosis in various tumor types26,29–31,58,59 and to therapy response in mel- anoma61–63. Whole-genome gene expression characterization No statistical method was used to predetermine the sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Unless otherwise stated, data analyzed by parametric tests are represented by the mean ± s.e.m. of pooled experiments and median ± interquartile range for experiments analyzed by non- parametric tests. n values represent the number of inde- pendent experiments performed, the number of individual mice or patient specimens. For each independent in vitro experiment, at least two technical replicates were used and a minimum number of three experiments were done to ensure adequate statistical power. For data mining analysis Student’s t test for two component comparisons. In the in vitro experiments, normal distribution was conﬁrmed or assumed (for n < 5) and Student’s t test was applied for two component comparisons. In the statistical analyses involving fold changes, one-sample t test with a hypothetical value of 1 was performed. The conﬁdence level used for all the statistical analyses was of 95% (alpha value = 0.05). Two-tail statistical analysis was applied for experimental design without predicted result, and one-tail for validation or hypothesis-driven experiments. Whole-genome expression characterization was con- ducted using Human HT12 v4 BeadChips (Illumina Inc.). In brief, cRNA synthesis was obtained with TargetAmp™ Nano-g™Biotin-aRNA Labeling Kit for the Illumina® System, Epicentre (Cat. Num. TAN07924) and sub- sequent ampliﬁcation, labeling and hybridization were performed according to Whole-Genome Gene Expression Direct Hybridization Illumina Inc.’s protocol. Raw data were extracted with GenomeStudio analysis software (Illumina Inc.) in the form of GenomeStudio’s Final Report (sample probe proﬁle). Correlation analysis Spearman correlation test was applied to analyze the relationship between paired genes. Gene enrichment The recently published tool, CANCERTOOL40, harbors 11 independent enrichment databases, including the basic Gene Ontology analysis (GO, biological process (GOBP), molecular function (GOMF) and cell compartment (GOCC)), pathways and pathophysiological processes (KEGG, Biocarta, Reactome, Biocarta, Onco, DOSE, HIPC, Connectivity Map), and the upstream regulatory cue prediction tool (TFT, MIR). The prevalence of such functions within the gene list was analyzed, and statistical signiﬁcance of the associations sieved according to the Benjamini–Hochberg correction (adjusted p value). Patient samples DNA quantiﬁcation was carried out using a Viia7 Real-Time PCR System (Applied Biosystems) with SybrGreen reagents and primers that amplify the predicted MITFA binding region to CRYAB (region 1; For: ttgtttcctcgtagggcttg, Rev: tttca- gagccaggagagagc- region 2; For: tctggaatggtgatgtcagg, Rev: attgggtgtggacagaaagc) and ANGPTL4 (For: gttgacccggctca- caat, Rev: ggaacagctcctggcaatc) as a negative binding control. of interest in three datasets. The recurrence of the disease was selected as the event of interest. Kaplan−Meier esti- mator was used to perform the test as it takes into account right-censoring, which occurs if a patient with- draws from a study. Gene expression array data analysis First, raw expression data were background-corrected, log2-transformed, and quantile-normalized using the lumi R package64, available through the Bioconductor reposi- tory65,66. Probes with a “detection p value” lower than 0.01 in at least one sample were considered expressed. For the Bioinformatics analysis and statistics Database normalization All the datasets used for the data mining analysis were downloaded from GEO and TCGA. Referenced acces- sions: TCGA https://cancergenome.nih.gov/, Grasso et al., GEO: GSE35988 (9); Lapointe et al., GEO: GSE3933 (10); Taylor et al., GEO: GSE21032 (11); Tomlins et al., GEO: GSE6099 (12); Varambally et al., GEO: GSE3325 (13); and Glinsky et al (8). GEO-downloaded data were subjected to background correction, log2 transformation and quartile normalization. In the case of using a preprocessed dataset, this normalization was reviewed and corrected if required. TCGA data were downloaded as upper quartile normal- ized RSEM count, which was been log2 transformed. Patient samples All samples were obtained from the Basque Biobank for research (BIOEF, Basurto University Hospital) upon informed consent and with evaluation and approval from the corresponding ethics committee (CEIC code OHEUN11-12 and OHEUN14-14). Ofﬁcial journal of the Cell Death Differentiation Association Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Page 10 of 12 Page 10 of 12 washed twice with ice-cold PBS, and scraped into PBS + PMSF. Pelleted cells were lysed and nuclei were harvested following the manufacturer’s instructions. Nuclear lysates were digested with micrococcal nuclease for 20 min at 37 °C and then sonicated in 500 μL aliquots on ice for 6 pulses of 20 s using a Branson sonicator. Cells were held on ice for at least 1 min between sonications. Lysates were clariﬁed at 11,000 × g for 10 min at 4 °C, and chromatin was stored at −80 °C. HA-Tag polyclonal antibody (Cat: C29F4, Cell Signalling Technology) and IgG antibody (Cat: 2729, Cell Signalling Technology, Inc), were incubated overnight (4 °C) with rotation and protein G magnetic beads were incubated 2 h (4 °C). Washes and elution of chromatin were performed following the manufacturer’s instructions. DNA quantiﬁcation was carried out using a Viia7 Real-Time PCR System (Applied Biosystems) with SybrGreen reagents and primers that amplify the predicted MITFA binding region to CRYAB (region 1; For: ttgtttcctcgtagggcttg, Rev: tttca- gagccaggagagagc- region 2; For: tctggaatggtgatgtcagg, Rev: attgggtgtggacagaaagc) and ANGPTL4 (For: gttgacccggctca- caat, Rev: ggaacagctcctggcaatc) as a negative binding control. washed twice with ice-cold PBS, and scraped into PBS + PMSF. Pelleted cells were lysed and nuclei were harvested following the manufacturer’s instructions. Nuclear lysates were digested with micrococcal nuclease for 20 min at 37 °C and then sonicated in 500 μL aliquots on ice for 6 pulses of 20 s using a Branson sonicator. Cells were held on ice for at least 1 min between sonications. Lysates were clariﬁed at 11,000 × g for 10 min at 4 °C, and chromatin was stored at −80 °C. HA-Tag polyclonal antibody (Cat: C29F4, Cell Signalling Technology) and IgG antibody (Cat: 2729, Cell Signalling Technology, Inc), were incubated overnight (4 °C) with rotation and protein G magnetic beads were incubated 2 h (4 °C). Washes and elution of chromatin were performed following the manufacturer’s instructions. Acknowledgements Th k f V T Grasso, C. S. et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239–243 (2012). 10. Lapointe, J. et al. Genomic proﬁling reveals alternative genetic pathways of prostate tumorigenesis. Cancer Res. 67, 8504–8510 (2007). 10. Lapointe, J. et al. Genomic proﬁling reveals alternative genetic pathways of prostate tumorigenesis. Cancer Res. 67, 8504–8510 (2007). 11. Taylor, B. S. et al. Integrative genomic proﬁling of human prostate cancer. Cancer Cell. 18, 11–22 (2010). 11. Taylor, B. S. et al. Integrative genomic proﬁling of human prostate cancer. Cancer Cell. 18, 11–22 (2010). 12. Tomlins, S. A. et al. Integrative molecular concept modeling of prostate cancer progression. Nat. Genet. 39, 41–51 (2007). 12. Tomlins, S. A. et al. Integrative molecular concept modeling of prostate cancer progression. Nat. Genet. 39, 41–51 (2007). 13. Varambally, S. et al. Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression. Cancer Cell 8, 393–406 (2005). Author details 1 b 1CIC bioGUNE, Bizkaia Technology Park, 801ª bld, 48160 Derio, Bizkaia, Spain. 2CIBERONC, Madrid, Spain. 3Department of Urology, Basurto University Hospital, 48013 Bilbao, Spain. 4Vascular Signalling Laboratory, Institut d ´Investigació Biomèdica de Bellvitge (IDIBELL), Gran Via de l’Hospitalet 199-203, Barcelona, Spain. 5CLIP-Childhood Leukaemia Investigation. Dept. of Pediatric Hematology and Oncology. Second Faculty of Medicine, Charles University, Prague, Czech Republic. 6Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), P.O. Box 64448080 Bilbao, Spain 14. Torrano, V. et al. The metabolic co-regulator PGC1alpha suppresses prostate cancer metastasis. Nat. Cell Biol. 18, 645–656 (2016). 15. Valcarcel-Jimenez, L., Torrano, V. & Carracedo, A. New insights on prostate cancer progression. Cell Cycle 16, 13–14 (2017). 16. Valcarcel-Jimenez, L., Gaude, E., Torrano, V., Frezza, C. & Carracedo, A. Mito- chondrial metabolism: yin and yang for tumor progression. Trends Endocrinol. Metab. 28, 748–757 (2017). 17. Hock, M. B. & Kralli, A. Transcriptional control of mitochondrial biogenesis and function. Annu. Rev. Physiol. 71, 177–203 (2009). References 1. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011). 1. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011). 2. Martin-Martin, N., Carracedo, A. & Torrano, V. Metabolism and transcription in cancer: Merging Two Classic Tales. Front. Cell Dev. Biol. 5, 119 (2017). 2. Martin-Martin, N., Carracedo, A. & Torrano, V. Metabolism and transcription in cancer: Merging Two Classic Tales. Front. Cell Dev. Biol. 5, 119 (2017). Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-1096-6). Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-1096-6). Received: 10 August 2018 Revised: 19 September 2018 Accepted: 25 September 2018 Quartile analysis in disease-free survival Patients biopsies from primary tumors were organized into four quartiles according to the expression of the gene Ofﬁcial journal of the Cell Death Differentiation Association Page 11 of 12 Page 11 of 12 Valcarcel-Jimenez et al. Cell Death and Disease (2018) 9:1041 Publisher's note detection of differentially expressed genes, a linear model was ﬁtted to the probe data and empirical Bayes moder- ated t statistics were calculated using the limma67 package from Bioconductor. Adjusted p values were estimated with Benjamini−Hochberg false discovery rate method (https://www.jstor.org/stable/2346101)68. Only genes with differential fold-change (FC) > 1.5 or <−1.5 and an adjusted p value < 0.05 were considered as differentially expressed. The transcriptomics data generated in this publication have been deposited in NCBI’s Gene Expres- sion Omnibus and are accessible through GEO Series accession number GSE114345. detection of differentially expressed genes, a linear model was ﬁtted to the probe data and empirical Bayes moder- ated t statistics were calculated using the limma67 package from Bioconductor. Adjusted p values were estimated with Benjamini−Hochberg false discovery rate method (https://www.jstor.org/stable/2346101)68. Only genes with differential fold-change (FC) > 1.5 or <−1.5 and an adjusted p value < 0.05 were considered as differentially expressed. The transcriptomics data generated in this publication have been deposited in NCBI’s Gene Expres- sion Omnibus and are accessible through GEO Series accession number GSE114345. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Authors’ contributions 18. Haq, R. et al. Oncogenic BRAF regulates oxidative metabolism via PGC1alpha and MITF. Cancer Cell 23, 302–315 (2013). L.V.-J. and A.M. performed the majority of in vitro and in vivo experiments, unless speciﬁed otherwise. N.M.-M. contributed to the in vivo experiments, experimental design and discussion. A.R.C. carried out the bioinformatics and biostatistical analysis. A.S.-C., M.P.-V., I.H. and I.A. contributed to the in vitro analysis and provided technical support. I.L.-V., A.S.-M., A.L.-I. and M.U.-U. provided BPH and PCa samples for gene expression analysis from Basurto University Hospital. M.G. carried out microvessel staining and quantiﬁcations. J. S. contributed as supervisor of IH. J.S. and R.B. performed or coordinated (RB) the cloning of MITFA in lentiviral vectors. A.M.A. contributed to experimental design and discussion. A.C. contributed to experimental design, data analysis and discussion. V.T. supervised the project, experimental design, data generation, analysis and discussion and wrote the manuscript. and MITF. Cancer Cell 23, 302–315 (2013). 19. Wellbrock, C. & Arozarena, I. Microphthalmia-associated transcription factor in melanoma development and MAP-kinase pathway targeted therapy. Pigment. Cell. Melanoma Res. 28, 390–406 (2015). 20. Tachibana, M. MITF: a stream ﬂowing for pigment cells. Pigment Cell Res. 13, 230–240 (2000). 21. Garraway, L. A. et al. Integrative genomic analyses identify MITF as a lineage survival oncogene ampliﬁed in malignant melanoma. Nature 436, 117–122 (2005). 21. Garraway, L. A. et al. Integrative genomic analyses identify MITF as a lineage survival oncogene ampliﬁed in malignant melanoma. 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Torrano is funded by Fundación Vasca de Innovación e Investigación Sanitarias, BIOEF (BIO15/CA/052), the AECC J.P. Bizkaia and the Basque Department of Health (2016111109). The work of A. Carracedo is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the Department of Education (IKERTALDE IT1106-16, also participated by A. Gomez-Muñoz), the BBVA Foundation, the MINECO (SAF2016-79381-R (FEDER/EU); Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020- MSCA-ITN-308 2016 721532), the AECC IDEAS16 (IDEAS175CARR) and the European Research Council (Starting Grant 336343, PoC 754627). CIBERONC was co-funded with FEDER funds. The work of M. Graupera is supported by the MINECO (SAF2014-59950-P). The work of A. Aransay is supported by the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek Programs), the Innovation Technology Department of Bizkaia County, CIBERehd Network and Spanish MINECO the Severo Ochoa Excellence Accreditation (SEV-2016-0644). R. Barrio acknowledges Spanish MINECO (BFU2014-52282-P, Consolider BFU2014-57703-REDC), the Departments of Education and Industry of the Basque Government (PI2012/42) and the Bizkaia County. J. Starková acknowledges the Ministry of Health of Czech Republic AZV NV15-28848A. We are thankful to the Basque Biobank for Research (BIOEF) for the custody and management of human prostate specimens used in this study. 3. Martin-Martin, N. et al. Stratiﬁcation and therapeutic potential of PML in metastatic breast cancer. Nat. Commun. 7, 12595 (2016). metastatic breast cancer. Nat. Commun. 7, 12595 (2016). 4. Martin-Martin, N. et al. PPARdelta elicits ligand-independent repression of trefoil factor family to limit prostate cancer growth. 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https://openalex.org/W4242667980	https://peerj.com/articles/3934v0.2/submission	English	null	Peer Review #1 of "Tolerance: the forgotten child of plant resistance (v0.1)"	null	2,017	cc-by	11,761	Manuscript to be reviewed 12 Abstract 13 Plant resistance against insect herbivory has greatly focused on antibiosis, whereby the plant has 14 a deleterious effect on the herbivore, and antixenosis, whereby the plant is able to direct the 15 herbivore away from it. Although these two types of resistance may reduce injury and yield loss, 16 they produce selection pressures on insect herbivores that lead to herbivore resistance to the plant 17 resistance type. Tolerance, on the other hand, is a more sustainable pest management strategy 18 because it involves only a plant response and therefore does not cause evolution of resistance in 19 target pest populations. Despite its attractive attributes, tolerance has been poorly studied and 20 understood. In this critical, interpretive review, we discuss tolerance to insect herbivory and the 21 biological and socioeconomic factors that have limited its use in plant resistance and integrated 22 pest management. First, tolerance is difficult to identify and the mechanisms conferring it are 23 mostly unknown. Second, the genetics of tolerance are mostly unknown. Third, several obstacles 24 hinder the establishment of high-throughput phenotyping methods for large-scale screening of 25 tolerance. Fourth, tolerance has received little attention from entomologists because, for most, 26 primary interest, research training, and funding opportunities are in mechanisms which affect 27 pest biology, not plant biology. Fifth, the efforts of plant resistance are directed at controlling 28 pest populations rather than managing plant stress. We conclude this paper by discussing future 29 research and development activities. 13 Plant resistance against insect herbivory has greatly focused on antibiosis, whereby the plant has 14 a deleterious effect on the herbivore, and antixenosis, whereby the plant is able to direct the 15 herbivore away from it. Although these two types of resistance may reduce injury and yield loss, 16 they produce selection pressures on insect herbivores that lead to herbivore resistance to the plant 17 resistance type. Tolerance, on the other hand, is a more sustainable pest management strategy 18 because it involves only a plant response and therefore does not cause evolution of resistance in 19 target pest populations. Despite its attractive attributes, tolerance has been poorly studied and 20 understood. In this critical, interpretive review, we discuss tolerance to insect herbivory and the 21 biological and socioeconomic factors that have limited its use in plant resistance and integrated 22 pest management. Tolerance: the forgotten child of plant resistance 1 Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana, United States 2 Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana, United States 3 School of Natural Resources, University of Nebraska - Lincoln, Lincoln, Nebraska, United States Corresponding Author: Robert K Peterson Email address: bpeterson@montana.edu Plant resistance against insect herbivory has greatly focused on antibiosis, whereby the plant has a deleterious effect on the herbivore, and antixenosis, whereby the plant is able to direct the herbivore away from it. Although these two types of resistance may reduce injury and yield loss, they can produce selection pressures on insect herbivores that lead to resistance. Tolerance, on the other hand, is a more sustainable pest management strategy because it involves only a plant response and therefore does not cause evolution of resistance in target pest populations. Despite its attractive attributes, tolerance has been poorly studied and understood. In this critical, interpretive review, we discuss tolerance to insect herbivory and the biological and socioeconomic factors that have limited its use in plant resistance and integrated pest management. First, tolerance is difficult to identify, and the mechanisms conferring it are poorly understood. Second, the genetics of tolerance are mostly unknown. Third, several obstacles hinder the establishment of high-throughput phenotyping methods for large-scale screening of tolerance. Fourth, tolerance has received little attention from entomologists because, for most, their primary interest, research training, and funding opportunities are in mechanisms which affect pest biology, not plant biology. Fifth, the efforts of plant resistance are directed at controlling pest populations rather than managing plant stress. We conclude this paper by discussing future research and development activities. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) 1 Tolerance: The Forgotten Child of Plant Resistance 2 Robert K. D. Peterson1, Andrea C. Varella2, Leon G. Higley3 3 4 1. Department of Land Resources and Environmental Sciences, Montana State Univ., Bozeman, 5 MT 59717 6 2. Department of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT 59717 7 3. School of Natural Resources, University of Nebraska-Lincoln, Lincoln, NE 68583 8 9 Corresponding author: Robert K. D. Peterson, Department of Land Resources and 10 Environmental Sciences, Montana State University, Bozeman, Montana, 59717-3120, 406-994- 11 7927; bpeterson@montana.edu. Manuscript to be reviewe Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) 12 Abstract First, tolerance is difficult to identify and the mechanisms conferring it are 23 mostly unknown. Second, the genetics of tolerance are mostly unknown. Third, several obstacles 24 hinder the establishment of high-throughput phenotyping methods for large-scale screening of 25 tolerance. Fourth, tolerance has received little attention from entomologists because, for most, 26 primary interest, research training, and funding opportunities are in mechanisms which affect 27 pest biology, not plant biology. Fifth, the efforts of plant resistance are directed at controlling 28 pest populations rather than managing plant stress. We conclude this paper by discussing future 29 research and development activities. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) 32 INTRODUCTION Fourth, tolerance has received little attention 47 from entomologists because, for most, their primary interest, research training, and funding 48 opportunities are in mechanisms which affect pest biology, not plant biology. Fifth, the efforts of 49 plant resistance are still directed at controlling pest populations rather than managing plant stress. 50 In this paper, we discuss tolerance and the factors that have limited its use in plant resistance and 51 IPM. 53 Primary and secondary literature relevant to the topic of this paper was assessed using 54 Web of Science (Clarivate Analytics) and Google Scholar. Key words such as “plant tolerance,” 53 Primary and secondary literature relevant to the topic of this paper was assessed using 54 Web of Science (Clarivate Analytics) and Google Scholar. Key words such as “plant tolerance,” 32 INTRODUCTION 32 INTRODUCTION 33 Is tolerance the forgotten child of plant resistance? Its attributes are so appealing, yet it 34 has received the least attention of the three types of plant resistance. As an insect pest 35 management tactic, tolerance may be the consummate strategy (Pedigo & Higley 1992). This is 36 because a central tenet of integrated pest management (IPM) is that we tolerate some amount of 37 pest injury. By making plants more tolerant of injury, we are achieving this important goal. 38 Another goal is to use tactics that impose little selection pressure that will lead to pest resistance 39 to those tactics. Contrary to antixenosis and antibiosis, tolerance does not affect insect biology or 40 behavior (Smith 2005); therefore, pests cannot become resistant to tolerant plants. Clearly, the 41 conceptual advantages of tolerance in plant resistance cannot be discounted. 42 We believe there are several reasons why tolerance has not been developed as 43 successfully as antibiosis and antixenosis. First, tolerance is difficult to identify and the 44 mechanisms conferring it are poorly understood. Second, the genetics of tolerance are mostly 45 unknown. Third, several obstacles still hinder the establishment of high-throughput phenotyping 46 methods for large-scale screening of tolerance. Fourth, tolerance has received little attention 47 from entomologists because, for most, their primary interest, research training, and funding 48 opportunities are in mechanisms which affect pest biology, not plant biology. Fifth, the efforts of 49 plant resistance are still directed at controlling pest populations rather than managing plant stress. 50 In this paper, we discuss tolerance and the factors that have limited its use in plant resistance and 51 IPM. 52 SURVEY METHODOLOGY 33 Is tolerance the forgotten child of plant resistance? Its attributes are so appealing, yet it 34 has received the least attention of the three types of plant resistance. As an insect pest 35 management tactic, tolerance may be the consummate strategy (Pedigo & Higley 1992). This is 36 because a central tenet of integrated pest management (IPM) is that we tolerate some amount of 37 pest injury. By making plants more tolerant of injury, we are achieving this important goal. 38 Another goal is to use tactics that impose little selection pressure that will lead to pest resistance 39 to those tactics. 32 INTRODUCTION Contrary to antixenosis and antibiosis, tolerance does not affect insect biology or 40 behavior (Smith 2005); therefore, pests cannot become resistant to tolerant plants. Clearly, the 41 conceptual advantages of tolerance in plant resistance cannot be discounted. 33 Is tolerance the forgotten child of plant resistance? Its attributes are so appealing, yet it 34 has received the least attention of the three types of plant resistance. As an insect pest 35 management tactic, tolerance may be the consummate strategy (Pedigo & Higley 1992). This is 36 because a central tenet of integrated pest management (IPM) is that we tolerate some amount of 37 pest injury. By making plants more tolerant of injury, we are achieving this important goal. 38 Another goal is to use tactics that impose little selection pressure that will lead to pest resistance 39 to those tactics. Contrary to antixenosis and antibiosis, tolerance does not affect insect biology or 40 behavior (Smith 2005); therefore, pests cannot become resistant to tolerant plants. Clearly, the 41 conceptual advantages of tolerance in plant resistance cannot be discounted. 42 We believe there are several reasons why tolerance has not been developed as 43 successfully as antibiosis and antixenosis. First, tolerance is difficult to identify and the 44 mechanisms conferring it are poorly understood. Second, the genetics of tolerance are mostly 45 unknown. Third, several obstacles still hinder the establishment of high-throughput phenotyping 46 methods for large-scale screening of tolerance. Fourth, tolerance has received little attention 47 from entomologists because, for most, their primary interest, research training, and funding 48 opportunities are in mechanisms which affect pest biology, not plant biology. Fifth, the efforts of 49 plant resistance are still directed at controlling pest populations rather than managing plant stress. 50 In this paper, we discuss tolerance and the factors that have limited its use in plant resistance and 51 IPM. 42 We believe there are several reasons why tolerance has not been developed as 43 successfully as antibiosis and antixenosis. First, tolerance is difficult to identify and the 44 mechanisms conferring it are poorly understood. Second, the genetics of tolerance are mostly 45 unknown. Third, several obstacles still hinder the establishment of high-throughput phenotyping 46 methods for large-scale screening of tolerance. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed Tolerance is a type of resistance that causes 65 the plant to compensate for pest injury to a degree exceeding non-tolerant plants (Kogan & 66 Ortman 1978; Painter 1951; Smith 2005). In an evolutionary context, tolerance is defined as the 67 slope of the line describing the association between fitness and level of damage for a set of 68 genetically related plants (Strauss & Agrawal 1999). In agronomic situations, tolerant crop 69 varieties are able to withstand injury and produce acceptable yields (Flinn et al. 2001; Qiu et al. 70 2011; Webster 1990; Webster et al. 1991). From an ecological perspective, tolerant plants can 71 maintain fitness in response to pest injury (Núñez-Farfán et al. 2007; Rosenthal & Kotanen 72 1994). 62 Antibiosis is a type of resistance that contains at least one plant characteristic that affects 63 pest biology in a deleterious manner. Antixenosis is a type of resistance that contains at least one 64 plant characteristic that directs a pest away from it. Tolerance is a type of resistance that causes 65 the plant to compensate for pest injury to a degree exceeding non-tolerant plants (Kogan & 66 Ortman 1978; Painter 1951; Smith 2005). In an evolutionary context, tolerance is defined as the 67 slope of the line describing the association between fitness and level of damage for a set of 68 genetically related plants (Strauss & Agrawal 1999). In agronomic situations, tolerant crop 69 varieties are able to withstand injury and produce acceptable yields (Flinn et al. 2001; Qiu et al. 70 2011; Webster 1990; Webster et al. 1991). From an ecological perspective, tolerant plants can 71 maintain fitness in response to pest injury (Núñez-Farfán et al. 2007; Rosenthal & Kotanen 72 1994). 73 Both antibiosis and antixenosis involve a plant response and a pest response. However, in 74 the case of tolerance only a plant response is involved. Therefore, there is a nonreciprocal 75 process associated with tolerance (Smith 2005). This non-reciprocity has important ramifications 76 when considering the use of tolerant cultivars in IPM programs. 73 Both antibiosis and antixenosis involve a plant response and a pest response. However, in 74 the case of tolerance only a plant response is involved. Therefore, there is a nonreciprocal 75 process associated with tolerance (Smith 2005). This non-reciprocity has important ramifications 76 when considering the use of tolerant cultivars in IPM programs. Manuscript to be reviewed 55 “host plant resistance,” “plant resistance,” “insect resistance,” “plant breeding,” “pest 56 resistance,” “antibiosis,” and “antixenosis” were searched between 1 January and 31 May, 2017. 57 DEFINITIONS AND CONCEPTS 58 Before discussing the five factors above in detail, we first need to define tolerance. In this 59 instance, precisely defining terms is important because there continues to be considerable 60 overlap in plant resistance definitions. At the outset, we recognize tolerance as distinctly 61 different from the two other resistance types: antibiosis and antixenosis. 62 Antibiosis is a type of resistance that contains at least one plant characteristic that affects 63 pest biology in a deleterious manner. Antixenosis is a type of resistance that contains at least one 64 plant characteristic that directs a pest away from it. Tolerance is a type of resistance that causes 65 the plant to compensate for pest injury to a degree exceeding non-tolerant plants (Kogan & 66 Ortman 1978; Painter 1951; Smith 2005). In an evolutionary context, tolerance is defined as the 67 slope of the line describing the association between fitness and level of damage for a set of 68 genetically related plants (Strauss & Agrawal 1999). In agronomic situations, tolerant crop 69 varieties are able to withstand injury and produce acceptable yields (Flinn et al. 2001; Qiu et al. 70 2011; Webster 1990; Webster et al. 1991). From an ecological perspective, tolerant plants can 71 maintain fitness in response to pest injury (Núñez-Farfán et al. 2007; Rosenthal & Kotanen 72 1994). 73 Both antibiosis and antixenosis involve a plant response and a pest response. However, in 55 “host plant resistance,” “plant resistance,” “insect resistance,” “plant breeding,” “pest 56 resistance,” “antibiosis,” and “antixenosis” were searched between 1 January and 31 May, 2017. 58 Before discussing the five factors above in detail, we first need to define tolerance. In this 59 instance, precisely defining terms is important because there continues to be considerable 60 overlap in plant resistance definitions. At the outset, we recognize tolerance as distinctly 61 different from the two other resistance types: antibiosis and antixenosis. 62 Antibiosis is a type of resistance that contains at least one plant characteristic that affects 63 pest biology in a deleterious manner. Antixenosis is a type of resistance that contains at least one 64 plant characteristic that directs a pest away from it. Manuscript to be reviewed 77 Like antibiosis and antixenosis, tolerance is a type of resistance. Tolerance (as well as 78 antibiosis and antixenosis) is not a mechanism of resistance (Smith 1997). There are numerous 79 mechanisms conferring tolerance (Koch et al. 2016; Strauss & Agrawal 1999; Tiffin 2000), just 80 as there are numerous mechanisms for antibiosis and antixenosis (Du et al. 2009; War et al. 81 2012). Therefore, different and distinct mechanisms that enhance pest mortality collectively 82 belong to the antibiosis resistance type. 77 Like antibiosis and antixenosis, tolerance is a type of resistance. Tolerance (as well as 78 antibiosis and antixenosis) is not a mechanism of resistance (Smith 1997). There are numerous 79 mechanisms conferring tolerance (Koch et al. 2016; Strauss & Agrawal 1999; Tiffin 2000), just 80 as there are numerous mechanisms for antibiosis and antixenosis (Du et al. 2009; War et al. 81 2012). Therefore, different and distinct mechanisms that enhance pest mortality collectively 82 belong to the antibiosis resistance type. 83 What do we mean by stating that tolerant hosts can compensate for injury better than 84 non-tolerant hosts? Plant response to biotic injury depends on four factors: the intensity of injury, 85 the time of injury, the type of injury, the plant part injured, and interactions with environmental 86 factors (Peterson & Higley 2001). The intensity of injury is very important when considering the 87 potential impact of the stressor on host yield or fitness. The relationship was described in the 88 form of a damage curve by Tammes (1961), and has since been supported by substantial 89 empirical evidence (Shelton et al. 1990). 83 What do we mean by stating that tolerant hosts can compensate for injury better than 84 non-tolerant hosts? Plant response to biotic injury depends on four factors: the intensity of injury, 85 the time of injury, the type of injury, the plant part injured, and interactions with environmental 86 factors (Peterson & Higley 2001). The intensity of injury is very important when considering the 87 potential impact of the stressor on host yield or fitness. The relationship was described in the 88 form of a damage curve by Tammes (1961), and has since been supported by substantial 89 empirical evidence (Shelton et al. 1990). 90 Pedigo et al. (1986) defined portions of the damage curve more than two decades after its 91 inception (Fig. 1). Manuscript to be reviewed 73 Both antibiosis and antixenosis involve a plant response and a pest response. However, in 74 the case of tolerance only a plant response is involved. Therefore, there is a nonreciprocal 75 process associated with tolerance (Smith 2005). This non-reciprocity has important ramifications 76 when considering the use of tolerant cultivars in IPM programs. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed Manuscript to be reviewed The damage curve can be used to present some of the basic aspects of 92 tolerance. Although the initial portion of the damage curve is termed the tolerant region, there 93 are actually four portions that can theoretically be expressed differentially by tolerant plants 94 when compared with nontolerant plants. The damage curve can be altered by extending the 95 initial zero slope of the damage curve; i.e., no damage per unit injury is expressed at higher 96 levels of injury for tolerant plants than for nontolerant plants (Fig. 2a). Tolerant plants also may 97 be able to affect the compensation area of the damage curve in two ways. First, because this area 98 is curvilinear (with a negative decreasing slope), tolerant plants may express less damage per 99 unit injury (Fig. 2b). Second, the slope is not altered, but the curvilinear portion is extended into 90 Pedigo et al. (1986) defined portions of the damage curve more than two decades after its 91 inception (Fig. 1). The damage curve can be used to present some of the basic aspects of 92 tolerance. Although the initial portion of the damage curve is termed the tolerant region, there 93 are actually four portions that can theoretically be expressed differentially by tolerant plants 94 when compared with nontolerant plants. The damage curve can be altered by extending the 95 initial zero slope of the damage curve; i.e., no damage per unit injury is expressed at higher 96 levels of injury for tolerant plants than for nontolerant plants (Fig. 2a). Tolerant plants also may 97 be able to affect the compensation area of the damage curve in two ways. First, because this area 98 is curvilinear (with a negative decreasing slope), tolerant plants may express less damage per 99 unit injury (Fig. 2b). Second, the slope is not altered, but the curvilinear portion is extended into PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 100 higher levels of injury (Fig. 2c). The linear portion can also be affected by tolerant plants in two 101 ways. First, the constant, negative slope (constant damage per unit injury) may have a less 102 negative slope for tolerant plants (Fig. 2d). Second, the linear portion may be shorter. Therefore, 103 desensitization and inherent impunity would occur at a higher yield (Fig. 2e). The last portion, 104 overcompensation (increasing yield per unit injury), can be expressed by both tolerant plants and 105 nontolerant plants; however, tolerant plants may express a higher yield increase per unit injury 106 (Fig. 2f). 107 As we have suggested, the damage curve theoretically can be altered by plants expressing 108 tolerance. The challenge remains to empirically identify empirically the portion or portions of the 109 damage curve where tolerance is expressed by plants. In addition, simply because portions are 110 identified in which tolerance is expressed does not mean those portions would be practical 111 targets for plant breeding. The tolerance, overcompensation, and compensation portions (Fig. 112 2a,b,f) most likely would be the most practical, producer accepted, and economic targets for 113 enhancing tolerance. Enhancing tolerance in the linearity, desensitization, and inherent impunity 114 portions (Fig. 2c,d,e) most likely would not be acceptable to producers because economic yield 115 loss would already be occurring in these portions, except perhaps for lower injury areas of the 116 linearity portion. 117 Tolerance can also be expressed in the context of economic injury level (EIL) parameters. 118 The relationship between damage per unit injury and the EIL typically takes the form of Fig. 3. 119 Because a tolerant plant ultimately expresses less damage per unit injury, the EIL will be greater 120 for most levels of injury. This relationship can also be expressed when considering pest 100 higher levels of injury (Fig. 2c). The linear portion can also be affected by tolerant plants in two 101 ways. First, the constant, negative slope (constant damage per unit injury) may have a less 102 negative slope for tolerant plants (Fig. 2d). Second, the linear portion may be shorter. Therefore, 103 desensitization and inherent impunity would occur at a higher yield (Fig. 2e). The last portion, 104 overcompensation (increasing yield per unit injury), can be expressed by both tolerant plants and 105 nontolerant plants; however, tolerant plants may express a higher yield increase per unit injury 106 (Fig. 2f). Manuscript to be reviewed 117 Tolerance can also be expressed in the context of economic injury level (EIL) parameters. 118 The relationship between damage per unit injury and the EIL typically takes the form of Fig. 3. 119 Because a tolerant plant ultimately expresses less damage per unit injury, the EIL will be greater 120 for most levels of injury. This relationship can also be expressed when considering pest 121 population levels over time and the EIL (Fig. 3). 122 CONSTRAINTS ON THE DEVELOPMENT AND USE OF TOLERANCE PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 123 Identifying tolerance and characterizing tolerance mechanisms is difficult 124 A major factor contributing to the predominance of the use of antibiosis and antixenosis 125 in plant resistance is sheer amenability. Antibiosis mechanisms often have been relatively easy to 126 identify and breed for, mainly because they are, in many cases, determined by a single gene or 127 major quantitative trait locus (QTL) and because their effects on herbivorous arthropods are 128 readily apparent. We realize that the precise biochemical mechanisms for antibiosis in many 129 systems are not known. For example, larval survival of the wheat stem sawfly, Cephus cinctus, is 130 reduced by QTL on wheat chromosomes 2A, 3A, and 5B (Varella et al. 2015). Although specific 131 mechanisms causing larval mortality have yet to be determined, this constraint has not hindered 132 the identification of antibiosis and the ability to breed for wheat resistance to this pest. 133 Although antixenosis mechanisms are not as readily identifiable as antibiosis 134 mechanisms, they still are more apparent than tolerance mechanisms. This is because antixenotic 135 mechanisms usually involve plant morphological features that can be visually identified and 136 because insect responses can be typically observed and measured. For example, the frego bract 137 character in cotton and glandular trichomes in alfalfa (both of which discourage larval feeding 138 and oviposition) are very apparent and efficacious (Jenkins & Parrott 1971; Ranger & Hower 139 2001). Even less visually apparent mechanisms such as surface waxes, tissue thickness, and 140 chemical deterrents can be readily identified and assayed (Chamarthi et al. 2011; Jindal & 141 Dhaliwal 2011; Weaver et al. 2009). 142 In contrast to antixenosis and antibiosis relatively little is known about tolerance 23 Identifying tolerance and characterizing tolerance mechanisms is difficul 124 A major factor contributing to the predominance of the use of antibiosis and antixenosis 125 in plant resistance is sheer amenability. Antibiosis mechanisms often have been relatively easy to 126 identify and breed for, mainly because they are, in many cases, determined by a single gene or 127 major quantitative trait locus (QTL) and because their effects on herbivorous arthropods are 128 readily apparent. We realize that the precise biochemical mechanisms for antibiosis in many 129 systems are not known. For example, larval survival of the wheat stem sawfly, Cephus cinctus, is 130 reduced by QTL on wheat chromosomes 2A, 3A, and 5B (Varella et al. 2015). Although specific 131 mechanisms causing larval mortality have yet to be determined, this constraint has not hindered 132 the identification of antibiosis and the ability to breed for wheat resistance to this pest. 142 In contrast to antixenosis and antibiosis, relatively little is known about tolerance. 142 In contrast to antixenosis and antibiosis, relatively little is known about tolerance. 143 Tolerance to arthropod injury has been identified in alfalfa, barley, rice, sorghum, maize, wheat, 144 cotton, cowpea, okra, muskmelon, turnip, and tea (Velusamy & Heinrichs 1986), northern red 145 oak, Spanish cedar, Brassica rapa, tall fescue, and perennial ryegrass (Strauss & Agrawal 1999), 143 Tolerance to arthropod injury has been identified in alfalfa, barley, rice, sorghum, maize, wheat, 144 cotton, cowpea, okra, muskmelon, turnip, and tea (Velusamy & Heinrichs 1986), northern red 145 oak, Spanish cedar, Brassica rapa, tall fescue, and perennial ryegrass (Strauss & Agrawal 1999), PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 169 It is important to note that mechanisms that contribute to tolerance may vary with 170 herbivore specialization (e.g. specialists, generalists) (Agrawal & Fishbein 2006; Delaney et al. 171 2008; Delaney et al. 2009; Delaney & Higley 2006; Foyer et al. 2015), feeding guild (e.g. 172 chewing, sucking) (Zhou et al. 2015), the plant’s symbiotic relationships (e.g. several milkweed 173 species show increased tolerance to herbivory when associated with arbuscular mycorrhizal 174 fungi) (Tao et al. 2016) and environmental conditions (Wise & Abrahamson 2007). All of these 175 factors complicate the identification and characterization of tolerance mechanisms. Also, some 176 mechanisms are constitutively expressed while others are induced. Evaluation of germplasm 177 showing induced tolerance must be done in the presence of pest populations, which is often mor 178 challenging due to seasonal variation in pest infestation at any given location. 179 Many crop varieties expressing tolerance have been discovered fortuitously. 180 Development of resistant cultivars usually has been the result of general screening for any 181 expression of resistance. For example, the development of the alfalfa cultivar "Team," which is 182 tolerant to alfalfa weevil, Hypera postica, injury, was the result of large-scale screenings of 183 germplasm, in which more than two million seedlings were exposed to weevil infestation in an 184 attempt to identify any resistance. After 10 years of breeding, "Team" was released in 1970. The 185 cultivar is believed to express all three resistance types, but tolerance seems to be the dominant 186 resistance factor (Barnes et al. 1970). It should be noted that the goal of the researchers was not 187 to characterize mechanisms, but rather to produce a resistant variety. Large scale screenings 188 focusing exclusively on plant tolerance have also been successful (Dunn et al. 2011). 189 The genetics of tolerance are mostly unknown 190 The ability to predict phenotypic characteristics based on plant genotype is key to 169 It is important to note that mechanisms that contribute to tolerance may vary with 170 herbivore specialization (e.g. specialists, generalists) (Agrawal & Fishbein 2006; Delaney et al. 171 2008; Delaney et al. 2009; Delaney & Higley 2006; Foyer et al. 2015), feeding guild (e.g. 172 chewing, sucking) (Zhou et al. 2015), the plant’s symbiotic relationships (e.g. several milkweed 173 species show increased tolerance to herbivory when associated with arbuscular mycorrhizal 174 fungi) (Tao et al. 2016) and environmental conditions (Wise & Abrahamson 2007). Manuscript to be reviewed 146 lentils, sugarcane, soybean, potato, switchgrass, and cacao (Koch et al. 2016), cassava, tomato, 147 and strawberry (Byrne et al. 1982; Gilbert et al. 1966; Schuster et al. 1980). In some of these 148 commodities, tolerance is a very important resistance attribute. For example, the resistance of 149 sorghum to greenbug, Schizaphis graminum, is dependent on the survival of seedlings in 150 response to feeding injury. This is clearly a tolerance response because resistant cultivars have 151 no effect on greenbug biology or behavior (Schuster & Starks 1973). In barley, the identification 152 of Russian wheat aphid, Diuraphis noxia, populations virulent to resistance genes has recently 153 prompted the development of tolerant cultivars (e.g. “Sydney” and “Stoneham”) in an attempt to 154 reduce selection pressure on the aphid population, thus increasing the durability of genotypes 155 (Haley et al. 2004; Marimuthu & Smith 2012; Mornhinweg et al. 2009; Mornhinweg et al. 2012). 156 Despite its successful use in some crops, little is known about the mechanisms underlying 157 tolerance. 158 Tolerance is currently believed to be caused by six general physiological mechanisms: (i) 159 increased net photosynthetic rate after herbivory, (ii) high relative growth rates, (iii) increased 160 branching or tillering, (iv) pre-existing high levels of carbon storage in roots, (v) increased 161 resource allocation from root to shoot after damage (Strauss & Agrawal 1999), and (vi) up- 162 regulation of detoxification mechanisms to counteract deleterious effects of herbivory (Koch et 163 al. 2016). Possible morphological features of tolerance include protected meristems, number of 164 meristems, and developmental plasticity (Rosenthal & Kotanen 1994). At the molecular level, 165 only a few transcripts (e.g. SNF1-related kinases, peroxidases, and catalases) have been 166 identified as been involved in tolerance to herbivory through resource allocation (Schwachtje et 167 al. 2006) or reactive oxygen species (ROS) detoxification mechanisms (Ramm et al. 2013; Smith 168 et al. 2010). PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed Manuscript to be reviewed 192 to germplasm screening and selection. Nevertheless, understanding the genetics of plant 193 tolerance to herbivory, as with any other trait, requires both the capability to detect polymorphic 194 alleles and the recombination or segregation of these alleles. 192 to germplasm screening and selection. Nevertheless, understanding the genetics of plant 193 tolerance to herbivory, as with any other trait, requires both the capability to detect polymorphic 194 alleles and the recombination or segregation of these alleles. 192 to germplasm screening and selection. Nevertheless, understanding the genetics of plant 193 tolerance to herbivory, as with any other trait, requires both the capability to detect polymorphic 194 alleles and the recombination or segregation of these alleles. 193 tolerance to herbivory, as with any other trait, requires both the capability to detect polymorphic 194 alleles and the recombination or segregation of these alleles. 195 To meet these requirements, large breeding populations need to be developed and 196 screened. Lack of knowledge of the mechanisms underlying tolerance hinders the ability to 197 precisely phenotype plants and interferes with the capacity of detecting polymorphisms. Despite 198 the challenges, genetic variation in tolerance to herbivory has been demonstrated in crop and 199 non-crop species (Marimuthu & Smith 2012; Punnuri et al. 2013; Shen & Bach 1997). Similar to 200 antibiosis and antixenosis, tolerance seems to be mostly controlled by multiple loci and their 201 interactions. Though QTL associated with tolerance to herbivory have been identified, to our 202 knowledge, no gene has been cloned. Thus, further research should aim to enhance the genetic 203 resolution of target QTL, which ultimately may result in the identification and cloning of causal 204 genes. 205 Establishing high-throughput screening methods for large-scale phenotyping of tolerance is 206 difficult 207 One of the bottlenecks of breeding for insect tolerance is the difficulty in identifying 208 diagnostic traits that can be easily, precisely, and consistently quantified under natural and/or 209 imposed insect pressure. Screening methods that are laborious or time-consuming might be 210 adequate for research purposes, but are for the most part not useful for screening the large 211 number of lines regularly phenotyped in plant breeding programs. 212 For example, wheat tolerance to the bird cherry-oat aphid, Rhopalosiphum padi, can be 213 assessed using a diverse set of methods that target a variety of plant traits (e.g. Manuscript to be reviewed All of these 175 factors complicate the identification and characterization of tolerance mechanisms. Also, some 176 mechanisms are constitutively expressed while others are induced. Evaluation of germplasm 177 showing induced tolerance must be done in the presence of pest populations, which is often more 178 challenging due to seasonal variation in pest infestation at any given location. 169 It is important to note that mechanisms that contribute to tolerance may vary with 170 herbivore specialization (e.g. specialists, generalists) (Agrawal & Fishbein 2006; Delaney et al. 171 2008; Delaney et al. 2009; Delaney & Higley 2006; Foyer et al. 2015), feeding guild (e.g. 172 chewing, sucking) (Zhou et al. 2015), the plant’s symbiotic relationships (e.g. several milkweed 173 species show increased tolerance to herbivory when associated with arbuscular mycorrhizal 174 fungi) (Tao et al. 2016) and environmental conditions (Wise & Abrahamson 2007). All of these 175 factors complicate the identification and characterization of tolerance mechanisms. Also, some 176 mechanisms are constitutively expressed while others are induced. Evaluation of germplasm 177 showing induced tolerance must be done in the presence of pest populations, which is often more 178 challenging due to seasonal variation in pest infestation at any given location. 190 The ability to predict phenotypic characteristics based on plant genotype is key to 191 expediting the development of improved crops, mainly because it adds efficiency and precision 190 The ability to predict phenotypic characteristics based on plant genotype is key to 191 expediting the development of improved crops, mainly because it adds efficiency and precision PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 215 Lamb & MacKay 1995; Papp & Mesterházy 1993). However, not all methods allow for the 216 evaluation of thousands of plants in a timely manner (Dunn et al. 2011). Thus, the establishment 217 of high-throughput phenotyping methods that allow for the precise characterization of a large 218 number of lines will greatly contribute for the development of tolerant crop plants. Challenges 219 associated with implementing high-throughput phenotyping for plant breeding programs are 220 associated with costs of equipment, facilities, and software licenses (required for data analysis), 221 lack of personnel trained for manipulation of large data sets, and lack of standards for 222 experimental design and data analysis (Goggin et al. 2015). 215 Lamb & MacKay 1995; Papp & Mesterházy 1993). However, not all methods allow for the 216 evaluation of thousands of plants in a timely manner (Dunn et al. 2011). Thus, the establishment 217 of high-throughput phenotyping methods that allow for the precise characterization of a large 218 number of lines will greatly contribute for the development of tolerant crop plants. Challenges 219 associated with implementing high-throughput phenotyping for plant breeding programs are 220 associated with costs of equipment, facilities, and software licenses (required for data analysis), 221 lack of personnel trained for manipulation of large data sets, and lack of standards for 222 experimental design and data analysis (Goggin et al. 2015). Manuscript to be reviewed gain yield, 214 thousand kernel mass, biomass ratios, and development of roots and shoots) (Dunn et al. 2011; 195 To meet these requirements, large breeding populations need to be developed and 196 screened. Lack of knowledge of the mechanisms underlying tolerance hinders the ability to 197 precisely phenotype plants and interferes with the capacity of detecting polymorphisms. Despite 198 the challenges, genetic variation in tolerance to herbivory has been demonstrated in crop and 199 non-crop species (Marimuthu & Smith 2012; Punnuri et al. 2013; Shen & Bach 1997). Similar to 200 antibiosis and antixenosis, tolerance seems to be mostly controlled by multiple loci and their 201 interactions. Though QTL associated with tolerance to herbivory have been identified, to our 202 knowledge, no gene has been cloned. Thus, further research should aim to enhance the genetic 203 resolution of target QTL, which ultimately may result in the identification and cloning of causal 204 genes. 212 For example, wheat tolerance to the bird cherry-oat aphid, Rhopalosiphum padi, can be 213 assessed using a diverse set of methods that target a variety of plant traits (e.g. gain yield, 214 thousand kernel mass, biomass ratios, and development of roots and shoots) (Dunn et al. 2011; PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) 223 Entomologists lack substantial training in plant biology 224 Because entomologists have been the primary participants in research on plant resistance 225 to insects, entomocentric views have prevailed. Consequently, instead of concentrating on plant 226 responses to insect-induced injury, entomologists have often used the plant to deliver a control 227 tactic. This strategy reflects an inherent disadvantage in research training specialization 228 (overspecialization?) of contemporary scientists (Jacobs & Frickel 2009; Rhoten 2004; Welter 229 1989). Very few entomologists have had formal training in aspects of plant biology, such as 230 photosynthesis, metabolism, anatomy, and water relations. Entomologists trained to consider the 231 plant in insect-plant interactions potentially would improve research and development of tolerant 232 cultivars. Additionally, interdisciplinary research teams may be able to develop tolerant cultivars. 233 However, interdisciplinary research incorporating aspects of pest biology, plant physiology, and 234 agronomy is still in its infancy (Peterson 2001; Peterson & Higley 2001). 224 Because entomologists have been the primary participants in research on plant resistance 225 to insects, entomocentric views have prevailed. Consequently, instead of concentrating on plant 226 responses to insect-induced injury, entomologists have often used the plant to deliver a control 227 tactic. This strategy reflects an inherent disadvantage in research training specialization 228 (overspecialization?) of contemporary scientists (Jacobs & Frickel 2009; Rhoten 2004; Welter 229 1989). Very few entomologists have had formal training in aspects of plant biology, such as 230 photosynthesis, metabolism, anatomy, and water relations. Entomologists trained to consider the 231 plant in insect-plant interactions potentially would improve research and development of tolerant 232 cultivars. Additionally, interdisciplinary research teams may be able to develop tolerant cultivars. 233 However, interdisciplinary research incorporating aspects of pest biology, plant physiology, and 234 agronomy is still in its infancy (Peterson 2001; Peterson & Higley 2001). Manuscript to be reviewed 238 tactic of insecticide use common in the 1950s and early 1960s. Through antixenosis, and 239 especially antibiosis mechanisms, resistant cultivars essentially are suppressing insect 240 populations. Unlike insecticide use, the adverse environmental impacts of using resistant 241 cultivars are quite low. In this respect, resistant cultivars satisfy one objective of IPM: 242 minimizing detrimental environmental effects. However, cultivars with antibiotic mechanisms 243 place selection pressure on insect populations, potentially encouraging the development of 244 resistance. Although, resistant cultivars may represent a more desirable control tactic, they do no 245 necessarily represent a truly sustainable pest management practice. New approaches for 246 incorporating resistance in plants also will not be sustainable. For example, plants that are 247 engineered to produce the Bacillus thuringiensis (Bt) toxin have selected for resistance (even 248 when pest populations were not economic) (Tabashnik et al. 2008). 249 The issue of control versus management in IPM is a critical factor when attempting to 250 understand why tolerance is not as prominent in plant resistance. The terms "control" and 251 "management" as they relate to pest management have been discussed (Higley & Pedigo 1993; 252 Higley & Pedigo 1996; Menalled et al. 2016; Pedigo & Higley 1996; Pedigo & Rice 2009). 253 Briefly, “control” implies a program focused on the pests themselves, and, in particular killing 254 pests. Therefore, this often results in strong selection pressure for resistance. The focus on killing 255 pests includes the highly efficacious antibiotic tactic represented by Bt crops. In contrast, 256 “management” implies a program focused on the “judicious use of means to accomplish a 257 desired end” (Pedigo & Higley 1996). Tolerance, then, as a type of plant resistance, clearly fits 258 well with management. 259 Oth bi l i l f t 238 tactic of insecticide use common in the 1950s and early 1960s. Through antixenosis, and 239 especially antibiosis mechanisms, resistant cultivars essentially are suppressing insect 240 populations. Unlike insecticide use, the adverse environmental impacts of using resistant 241 cultivars are quite low. In this respect, resistant cultivars satisfy one objective of IPM: 242 minimizing detrimental environmental effects. However, cultivars with antibiotic mechanisms 243 place selection pressure on insect populations, potentially encouraging the development of 244 resistance. Although, resistant cultivars may represent a more desirable control tactic, they do not 245 necessarily represent a truly sustainable pest management practice. 35 Plant resistance efforts are targeted toward the control of pest population 236 We believe that plant resistance research, although overtly progressive and consistent 237 with IPM, has largely followed a unilateral approach to pest management, similar to the control 236 We believe that plant resistance research, although overtly progressive and consistent 237 with IPM, has largely followed a unilateral approach to pest management, similar to the control PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed New approaches for 246 incorporating resistance in plants also will not be sustainable. For example, plants that are 247 engineered to produce the Bacillus thuringiensis (Bt) toxin have selected for resistance (even 248 when pest populations were not economic) (Tabashnik et al. 2008). 238 tactic of insecticide use common in the 1950s and early 1960s. Through antixenosis, and 239 especially antibiosis mechanisms, resistant cultivars essentially are suppressing insect 240 populations. Unlike insecticide use, the adverse environmental impacts of using resistant 241 cultivars are quite low. In this respect, resistant cultivars satisfy one objective of IPM: 242 minimizing detrimental environmental effects. However, cultivars with antibiotic mechanisms 243 place selection pressure on insect populations, potentially encouraging the development of 244 resistance. Although, resistant cultivars may represent a more desirable control tactic, they do not 245 necessarily represent a truly sustainable pest management practice. New approaches for 246 incorporating resistance in plants also will not be sustainable. For example, plants that are 247 engineered to produce the Bacillus thuringiensis (Bt) toxin have selected for resistance (even 248 when pest populations were not economic) (Tabashnik et al. 2008). 249 The issue of control versus management in IPM is a critical factor when attempting to 250 understand why tolerance is not as prominent in plant resistance. The terms "control" and 251 "management" as they relate to pest management have been discussed (Higley & Pedigo 1993; 252 Higley & Pedigo 1996; Menalled et al. 2016; Pedigo & Higley 1996; Pedigo & Rice 2009). 253 Briefly, “control” implies a program focused on the pests themselves, and, in particular killing 254 pests. Therefore, this often results in strong selection pressure for resistance. The focus on killing 255 pests includes the highly efficacious antibiotic tactic represented by Bt crops. In contrast, 256 “management” implies a program focused on the “judicious use of means to accomplish a 257 desired end” (Pedigo & Higley 1996). Tolerance, then, as a type of plant resistance, clearly fits 258 well with management. 249 The issue of control versus management in IPM is a critical factor when attempting to 250 understand why tolerance is not as prominent in plant resistance. The terms "control" and 251 "management" as they relate to pest management have been discussed (Higley & Pedigo 1993; 252 Higley & Pedigo 1996; Menalled et al. 2016; Pedigo & Higley 1996; Pedigo & Rice 2009). Manuscript to be reviewed Having just one crop species in an area 272 tolerant to corn earworm injury may result in unacceptable populations for the other crop 273 species. 274 Socioeconomic factors Manuscript to be reviewed 260 Conceptually, tolerance has very attractive attributes for use in IPM programs. However, 261 because tolerance has been so poorly studied and understood, we do not know if or how much 262 specific interactions with the environment (such as drought or heat stress) will render the tolerant 263 variety completely susceptible to pest injury. This is especially relevant in the face of climate 264 change and the increase in drought-prone areas. In non-crop species for instance, drought has 265 been shown to limit a plant’s ability to tolerate herbivory (Atala & Gianoli 2009; Gonzáles et al. 266 2008). But even closely related species of plants may respond differently to herbivory under 267 drought conditions (Shibel & Heard 2016). Thus, the impact of environment on the plant’s 268 ability to tolerate insect herbivory might have to be assessed for each crop species and/or variety. 269 In several crop systems, some arthropod species move from one crop species to another 270 during their life cycle. For example, in North Carolina the corn earworm, Helicoverpa zea, may 271 injure corn, tobacco, wild hosts, soybean, and cotton. Having just one crop species in an area 272 tolerant to corn earworm injury may result in unacceptable populations for the other crop 273 species. 260 Conceptually, tolerance has very attractive attributes for use in IPM programs. However, 261 because tolerance has been so poorly studied and understood, we do not know if or how much 262 specific interactions with the environment (such as drought or heat stress) will render the tolerant 263 variety completely susceptible to pest injury. This is especially relevant in the face of climate 264 change and the increase in drought-prone areas. In non-crop species for instance, drought has 265 been shown to limit a plant’s ability to tolerate herbivory (Atala & Gianoli 2009; Gonzáles et al. 266 2008). But even closely related species of plants may respond differently to herbivory under 267 drought conditions (Shibel & Heard 2016). Thus, the impact of environment on the plant’s 268 ability to tolerate insect herbivory might have to be assessed for each crop species and/or variety. 269 In several crop systems, some arthropod species move from one crop species to another 270 during their life cycle. For example, in North Carolina the corn earworm, Helicoverpa zea, may 271 injure corn, tobacco, wild hosts, soybean, and cotton. 259 Other biological factors PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed Manuscript to be reviewed 283 Tolerant cultivars must be agronomically desirable. Nguessen and Quisenberry (1994) 284 identified several rice lines that are tolerant to rice weevil, Sitophilus oryzae, injury. However, 285 they were not agronomically desirable. This is a major limitation to incorporating tolerance into 286 crops and must be addressed by researchers. Another major limitation is that tolerant crops may 287 be more vulnerable to cosmetic damage than crops displaying other types of resistance. 288 Consumer preference for fruits and vegetables, for example, is influenced by product 289 appearance. Thus consumer preference for undamaged food products might limit the use of 290 tolerance in some crop species. 283 Tolerant cultivars must be agronomically desirable. Nguessen and Quisenberry (1994) 284 identified several rice lines that are tolerant to rice weevil, Sitophilus oryzae, injury. However, 285 they were not agronomically desirable. This is a major limitation to incorporating tolerance into 286 crops and must be addressed by researchers. Another major limitation is that tolerant crops may 287 be more vulnerable to cosmetic damage than crops displaying other types of resistance. 288 Consumer preference for fruits and vegetables, for example, is influenced by product 289 appearance. Thus consumer preference for undamaged food products might limit the use of 290 tolerance in some crop species. 274 Socioeconomic factors 275 In the U.S., growers attempt to control pests to avoid risk as much as, if not more, than to 276 optimize yields (Higley 2006). Understandably, then, growers may be uncomfortable with a 277 large number of pests feeding on their tolerant cultivar. In this case, the cultivar may be able to 278 tolerate the injury, but the grower cannot. The attitude that the "only good bug is a dead bug" is 279 undoubtedly alive and well in modern farming systems. Additionally, private companies may not 280 embrace tolerant cultivars because they do not want their customers to doubt that their varieties 281 are indeed resistant. Therefore, education about tolerance and tolerant cultivars must be a priority 282 if this resistance strategy is to be successful. 275 In the U.S., growers attempt to control pests to avoid risk as much as, if not more, than to 276 optimize yields (Higley 2006). Understandably, then, growers may be uncomfortable with a 277 large number of pests feeding on their tolerant cultivar. In this case, the cultivar may be able to 278 tolerate the injury, but the grower cannot. The attitude that the "only good bug is a dead bug" is 279 undoubtedly alive and well in modern farming systems. Additionally, private companies may not 280 embrace tolerant cultivars because they do not want their customers to doubt that their varieties 281 are indeed resistant. Therefore, education about tolerance and tolerant cultivars must be a priority 282 if this resistance strategy is to be successful. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 291 CONCLUSIONS AND RECOMMENDATIONS 291 CONCLUSIONS AND RECOMMENDATIONS 292 Although antixenosis and antibiosis may lessen or negate the need for pesticides applied 293 to the crop, they can produce selective pressures on insect populations that are similar to 294 pesticides. The management tactic may be more environmentally acceptable and therefore may 295 be more popular with policy makers and the public, but if sufficient selective pressure is placed 296 on the pest population the tactic is not sustainable in the long term (Kennedy et al. 1987; Tolmay 297 et al. 2007). Tolerance, as a resistance mechanism, is very appealing because it is a sustainable 298 tactic (Kennedy et al. 1987; Pedigo 1995; Pedigo & Rice 2009; Rausher 2001). By not placing 299 selective pressure on insect populations, it essentially factors the pest out of the equation. 301 varieties. Therefore, reduced pesticide inputs would result. Because of these factors, tolerance is 302 a more stabilizing management strategy for pests. 303 Velusamy and Heinrichs (1986) list three factors they believe are responsible for the lack 304 of attention to tolerance. They include: a lack of suitable techniques to identify and incorporate 305 tolerance into crops; the ability of tolerant cultivars to serve as reservoirs for insect vectors of PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) 333 References 334 Agrawal AA, and Fishbein M. 2006. Plant defense syndromes. Ecology 87:S132-S149. 335 Atala C, and Gianoli E. 2009. Effect of water availability on tolerance of leaf damage in tall 336 morning glory, Ipomoea purpurea. Acta Eocologica 35:236-424. 337 Barnes DK, Hanson CH, Ratcliffe RH, Busbice TH, Schillinger JA, Buss GR, Campbell WV, 338 Hemken RW, and Blickenstaff CC. 1970. The development and performance of Team 339 alfalfa. Washington, DC: U.S. Department of Agriculture Bulletin ARS 34-115. 340 Byrne DH, Guerrero JM, Bellotti AC, and Gracen VE. 1982. Yield and plant-growth responses 341 of Mononychellus mite resistant and susceptible cassava cultivars under protected vs 342 infested conditions. Crop Science 22:486-490. 343 Chamarthi S, Sharma H, Vijay P, and Narasu M. 2011. Leaf surface chemistry of sorghum 344 seedlings influencing expression of resistance to sorghum shoot fly, Atherigona soccata. 345 Journal of Plant Biochemistry and Biotechnology 20:211-216. 343 Chamarthi S, Sharma H, Vijay P, and Narasu M. 2011. Leaf surface chemistry of sorghum 344 seedlings influencing expression of resistance to sorghum shoot fly, Atherigona soccata. 345 Journal of Plant Biochemistry and Biotechnology 20:211-216. 346 Delaney KJ, Haile FJ, Peterson RKD, and Higley LG. 2008. Impairment of leaf photosynthesis 347 after insect herbivory or mechanical injury on common milkweed, Asclepias syriaca. 348 Environmental Entomology 37:1332-1343. 349 Delaney KJ, Haile FJ, Peterson RKD, and Higley LG. 2009. Seasonal patterns of leaf 350 photosynthesis after insect herbivory on common milkweed, Asclepias syriaca: 351 Reflection of a physiological cost of reproduction, not defense? American Midland 352 Naturalist 162:224-238. 353 Delaney KJ, and Higley LG. 2006. An insect countermeasure impacts plant physiology: midrib 354 vein cutting, defoliation and leaf photosynthesis. Plant, Cell and Environment 29:1245- 355 1258. 356 Du B, Zhang W, Liu B, Hu J, Wei Z, Shi Z, He R, Zhu L, Chen R, Han B, and He G. 2009. 357 Identification and characterization of Bph14, a gene conferring resistance to brown 358 planthopper in rice. Proceedings of the National Academy of Sciences 106:22163-22168. 359 Dunn B, Porter D, Baker C, and Carver B. 2011. Screening USDA-ARS wheat germplasm for 360 bird cherry-oat aphid tolerance. Journal of Crop Improvement 25:176-182. 361 Flinn M, Smith M, Reese J, and Gill B. 2001. Categories of resistance to greenbug (Homoptera: 362 Aphididae) biotype I in Agilops tauschii germplasm. Journal of Economic Entomology 363 94:558-563. 364 Foyer CH, Verrall SR, and Hancock RD. 2015. Manuscript to be reviewed 329 and molecular biologists; and, (4) progression of IPM theory to a true paradigm for managing 330 plant stress. Ultimately, to understand the conceptual importance of tolerance to plant resistance, 331 th i t f t l t IPM t b i t d 329 and molecular biologists; and, (4) progression of IPM theory to a true paradigm for managing 330 plant stress. Ultimately, to understand the conceptual importance of tolerance to plant resistance, 331 the importance of tolerance to IPM must be appreciated. Manuscript to be reviewed 306 viruses; and, the lack of basic information on the inheritance of tolerance. We believe they have 307 identified three factors that potentially constrain the development of tolerance. However, we 308 believe our factors are more encompassing, reflecting the biological, economic, and social 309 constraints on tolerance development. For example, the lack of suitable techniques to identify 310 tolerance is really a reflection of the lack of understanding about basic physiological mechanism 311 underlying tolerance. 312 Before substantial work on tolerance development can occur, we must conduct basic 313 research on the physiological and biochemical mechanisms of tolerance. This must involve 314 interdisciplinary research between plant scientists and entomologists. Beyond an 315 interdisciplinary focus, it is important that there is awareness from applied researchers about 316 research and findings from fundamental researchers and vice-versa. There are longstanding 317 issues of lack of communication between biologists, ecologists, and agricultural scientists 318 (Higley et al. 1993) and this must be addressed before tolerance can be appreciably advanced. 319 More generally, research on the physiological responses of plants to arthropod injury 320 (irrespective of tolerance) must progress beyond what is currently known. Higley et al. (1993) 321 argued that a focus on plant physiology provides a common language for characterizing plant 322 stress and is essential for integrating understanding of stress. Peterson and Higley (1993) and 323 Peterson (2001) discuss approaches for synthesizing plant responses to arthropod injury. 324 Based on the factors we have discussed above, we believe the development and use of 325 tolerance in plant resistance to arthropods can be hastened by achieving the following goals: (1) 326 research characterizing the physiological mechanisms underlying tolerance; (2) research 327 determining the physiological responses of plants to arthropod injury; (3) encouragement of PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 370 Goggin F, Lorence A, and Topp C. 2015. Applying high-throughput phenotyping to plant–insect 371 interactions: picturing more resistant crops. Current Opinion in Insect Science 9:69-76. 370 Goggin F, Lorence A, and Topp C. 2015. Applying high-throughput phenotyping to plant–insect 371 interactions: picturing more resistant crops. Current Opinion in Insect Science 9:69-76. 372 Gonzáles W, Suárez L, and Molina-Montenegro M. 2008. Water availability limits tolerance of 373 apical damage in the Chilean tarweed Madia sativa. Acta Oecologica 34:104-110. 374 Haley S, Quick J, Johnson J, Peairs F, Stromberger J, and Clayshulte S. 2004. Registration of 375 ‘Anchor’ wheat Crop Science 44:1025-1026 372 Gonzáles W, Suárez L, and Molina-Montenegro M. 2008. Water availability limits tolerance of 373 apical damage in the Chilean tarweed Madia sativa. Acta Oecologica 34:104-110. 373 apical damage in the Chilean tarweed Madia sativa. Acta Oecologica 34:104-110. 374 Haley S, Quick J, Johnson J, Peairs F, Stromberger J, and Clayshulte S. 2004. Registration of 375 ‘Anchor’ wheat. Crop Science 44:1025-1026. 374 Haley S, Quick J, Johnson J, Peairs F, Stromberger J, and Clayshulte S. 2004. Registration of 375 ‘Anchor’ wheat. Crop Science 44:1025-1026. 376 Higley LG. 2006. The devil and Leon Higley: an IPM story. In: Gray M, editor. Illinois Crop 377 Protection Technology Conference. Urbana, Illinois: University of Illinois Extension. p 378 132-134. 379 Higley LG, Browde JA, and Higley PM. 1993. Moving towards new understandings of biotic 380 stress and stress interactions. In: Buxton DR, Shibles R, Forsberg RA, Blad BL, Asay 381 KH, Paulsen GM, and Wilson RF, eds. International Crop Science I. Madison, 382 Wisconsin: Crop Science Society of America, 749-754. 383 Higley LG, and Pedigo LP. 1993. Economic Injury Level concepts and their use in sustaining 384 environmental quality. Agriculture, Ecosystems and Environment 46:233-243. 385 Higley LG, and Pedigo LP. 1996. Pest science at a crossroads. In: Higley L, and Pedigo L, eds. 386 Economic Thresholds for Integrated Pest Management. Lincoln, Nebraska: University of 387 Nebraska Press. 388 Jacobs J, and Frickel S. 2009. Interdisciplinarity: a critical assessment. Annual Review of 389 Sociology 35:43-65. 390 Jenkins J, and Parrott W. 1971. Effectiveness of frego bract as a boll weevil resistance character 391 in cotton. Crop Science 11:739-743. 392 Jindal V, and Dhaliwal G. 2011. Mechanisms of resistance in cotton to whitefly (Bemisia 393 tabaci): antixenosis. Phytoparasitica 39:129-136. 394 Kennedy GG, Gould F, Deponti OMB, and Stinner RE. 1987. 333 References Systematic analysis of phloem-feeding insect- 365 induced transcriptional reprogramming in Arabidopsis highlights common features and 366 reveals distinct responses to specialist and generalist insects. Journal of Experimental 367 Botany 66:495-512. 368 Gilbert JC, Chinn JT, and Tanaka JS. 1966. Spider mite tolerance in multiple disease resistant 369 tomatoes. Proceedings of the American Society for Horticultural Science 89:559-562. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed 414 Mornhinweg D, Bregitzae P, Porter D, Peairs F, Baltensperger D, Hein G, Randolph T, Koch M, 415 and Walker T. 2012. Registration of ‘Stoneham’ spring feed barley resistant to Russian 416 wheat aphid. Journal of Plant Registrations 6:1-5. 417 Nguessan FK, and Quisenberry SS. 1994. Screening selected rice lines for resistance to the rice 418 water weevil (Coleoptera: Curculionidae). Environmental Entomology 23:665-675. 419 Núñez-Farfán J, Fornoni J, and Valverde P. 2007. The evolution of resistance and tolerance to 420 herbivores. Review of Ecology, Evolution, and Systematics 38:541-566. 421 Painter R. 1951. Insect resistance in crop plants. Lawrence, Kansas: University of Kansas Press. 422 Papp M, and Mesterházy Á. 1993. Resistance to bird cherry-oat aphid (Rhopalosiphum padi L.) 423 in winter wheat varieties. Journal of Economic Entomology 89:1649-1657. 421 Painter R. 1951. Insect resistance in crop plants. Lawrence, Kansas: University of Kansas Press. 422 Papp M and Mesterházy Á 1993 Resistance to bird cherry-oat aphid (Rhopalosiphum padi L ) 422 Papp M, and Mesterházy Á. 1993. 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African Entomology 15:228-230. 495 Varella A, Weaver D, Sherman J, Black N, Heo H-Y, Kalous J, Chao J, Hofland M, Martin J, 496 Kephart K, and Talbert L. 2015. Association analysis of stem solidness and wheat stem 497 sawfly resistance in a panel of North American spring wheat germplasm. Crop Science 498 55:2046-2055. 499 Velusamy R, and Heinrichs E. 1986. Tolerance in crop plants to insect pests. Insect Science and 500 Application 7:689-696. 499 Velusamy R, and Heinrichs E. 1986. Tolerance in crop plants to insect pests. Insect Science and 500 Application 7:689-696. 501 War A, Paulraj M, Ahmad T, Buhroo A, Hussain B, Ignacimuthu S, and Sharma H. 2012. 502 Mechanisms of plant defense against insect herbivores. Plant Signaling and Behavior 503 7:1306-1320. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed Manuscript to be reviewed The damage curve relating intensity of injury to yield. The damage curve relating intensity of injury to yield. Manuscript to be reviewed 504 Weaver D, Buteler M, Hofland M, Runyon J, Nansen C, Talbert L, Lamb P, and Carlson G. 505 2009. Cultivar preferences of ovipositing wheat stem sawflies as influenced by the 506 amount of volatile attractant. Journal of Economic Entomology 102:1009-1017. 507 Webster J. 1990. Yellow sugarcane aphid (Homoptera: Aphididae): detection and mechanism of 508 resistance among Ethiopian sorghum lines. Journal of Economic Entomology 83:1053- 509 1057. 510 Webster J, Baker C, and Porter D. 1991. Detection and mechanisms of Russian wheat aphid 511 (Homoptera: Aphididae) resistance in barley. Journal of Economic Entomology 84:669- 512 673. 513 Welter SC. 1989. Arthropod impact on plant gas exchange. In: Bernays EA, ed. Plant-Insect 514 Interactions. Boca Raton: CRC, 135-150. 515 Wise M, and Abrahamson W. 2007. Effects of resource availability on tolerance of herbivory: a 516 review and assessment of three opposing models. American Naturalist 169:443-454. 517 Zhou S, Lou Y, Tzin V, and Jander G. 2015. Alteration of plant primary metabolism in response 518 to insect herbivory. Plant Physiology 169:1488-1498. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Figure 2 The damage curve showing different portions where tolerance can be expressed. a) shows extending the initial zero slope of the damage curve, i.e., no damage per unit injury may be expressed at higher levels of injury for tolerant plants than for nontolerant plants; b) shows that because this area is curvilinear (with a negative decreasing slope), tolerant plants may express less damage per unit injury; c) shows that the curvilinear portion may be extended into higher levels of injury; d) shows that the constant, negative slope (constant damage per unit injury) may have a less negative slope for tolerant plants; e) shows that the linear portion may be shorter; e) shows that desensitization and inherent impunity may occur at a higher yield; f) shows that overcompensation (increasing yield per unit injury), may be expressed by both tolerant plants and nontolerant plants, but tolerant plants may express a higher yield increase per unit injury. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) Manuscript to be reviewed Figure 3 The relationship between injury (often expressed as number of insects), time, and the economic injury level with and without tolerance. PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017) PeerJ reviewing PDF \| (2017:07:19220:1:0:CHECK 22 Sep 2017)
https://openalex.org/W2135999256	http://mdh.diva-portal.org/smash/get/diva2:571771/FULLTEXT02	English	null	Midwives’ views on factors that contribute to health care inequalities among immigrants in Sweden: a qualitative study	International journal for equity in health	2,012	cc-by	9,919	Mälardalen University Mälardalen University Abstract Introduction: Ethnic and socioeconomic inequalities in the Swedish health care system have increased. Most indicators suggest that immigrants have significantly poorer health than native Swedes. The purpose of this study was to explore the views of midwives on the factors that contribute to health care inequality among immigrants. Methods: Data were collected via semi-structured interviews with ten midwives. These were transcribed and related categories identified through content analysis. Results: The interview data were divided into three main categories and seven subcategories. The category “Communication” was divided into subcategories “The meeting”, “Cultural diversity and language barriers” and “Trust and confidence”. The category “Potential barriers to the use of health care services” contained two subcategories, “Seeking health care” and “Receiving equal treatment”. Finally, the category “Transcultural health care” had subcategories “Education on transcultural health care” and “The concept”. Conclusions: This study suggests that midwives believe that health care inequality among immigrants can be the result of miscommunication which may arise due to a shortage of meeting time, language barriers, different systems of cultural beliefs and practices and limited patient-caregiver trust. Midwives emphasized that education level, country of origin and length of stay in Sweden play a role when an immigrant seeks health care. Immigrants face more difficulties when seeking health care and in receiving adequate levels of care. However, different views among the midwives were also observed. Some midwives were sensitive to individual and intra-group differences, while some others viewed immigrants as a group of “others”. Midwives’ beliefs about subgroup-specific health services vs. integrating immigrants’ health care into mainstream health care services should be investigated further. Patients’ perspective should also be considered. Keywords: Immigrants, Midwives, Communication, Inequality, Transcultural health care RESEARCH Open Access © 2012 Akhavan; 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. Midwives’ views on factors that contribute to health care inequalities among immigrants in Sweden: a qualitative study Sharareh Akhavan* Correspondence: sharareh.akhavan@mdh.se Department of Public Health - University of Skövde & School of Health, Care and Social Welfare, University of Mälardalen, Mälardalen, Sweden Mälardalen University This is a published version of a paper published in International Journal for Equity in Health. Citation for the published paper: Akhavan, S. (2012) "Midwives’ views on factors that contribute to health care inequalities among immigrants in Sweden: a qualitative study." International Journal for Equity in Health, 11: article nr: 47 URL: http://dx.doi.org/10.1186/1475-9276-11-47 Access to the published version may require subscription. Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-16225 http://mdh.diva-portal.org This is a published version of a paper published in International Journal for Equity in Health. Citation for the published paper: Akhavan, S. (2012) "Midwives’ views on factors that contribute to health care inequalities among immigrants in Sweden: a qualitative study." International Journal for Equity in Health, 11: article nr: 47 URL: http://dx.doi.org/10.1186/1475-9276-11-47 Access to the published version may require subscription. Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-16225 http://mdh.diva-portal.org Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 varied. Lack of available information, communication diffi- culties [12] and lower levels of trust in the health care sys- tem [9,13] are some factors that have been discussed. Ethnic discrimination [14,15] and insufficient clinical follow-up treatments and/or fewer post-operative checkups [16] are other factors that have been mentioned in earlier research. not seek health care when they need to and do not re- ceive the treatment that they need when they seek care [4]. This study is one segment of a large study which has been conducted to explore factors that contribute to in- equalities in the provision of health care in Sweden. Midwives were chosen as the study group because the perinatal period is often the first contact that a newly arrived immigrant family has with the health care sys- tem, and that experience will affect future use of the sys- tem [6]. Furthermore, midwives are responsible for a high percentage of obstetrics care in Sweden [7]. As such, midwives play a crucial role as the representatives of the larger health care system for immigrants. The aim of this study is to explore the views of one group of health care professionals (midwives) on the fac- tors that contribute to health care inequality among immigrants. Participants Th id i The midwives or the superintendent of units in two mu- nicipalities in a city in western Sweden were informed about the study by telephone or via e-mail and appoint- ments were made with those who were interested in being interviewed. The municipalities were selected ran- domly from a group of 20 that had a higher number of immigrants. The municipalities with a higher number of immigrants were identified from the segregation index that was calculated for all municipalities in Sweden for the years 1997–2006 [19]. The criteria for being included in the study were that the midwives were professionally trained and had worked in the selected district for at least 12 months. Ten midwives, all native Swedes, were interviewed. Their mean age was 49.2 years, with a range of 35–57. Most of them had between 6–25 years of ex- perience in the field and worked often, or almost always, with immigrant women (Table 1). Immigrants in Sweden experience worse physical and psychological health compared with native Swedes [4,5]. There are differences in healthcare utilization. The Sta- tistics Central Board’s [4] study showed that 21 percent of immigrant women reported need of health care but had not sought it (self-reported), in comparison with 12 percent of native Swedish women. The study [4] showed that the rate of preventable mortality (death due to ill- nesses that the health care sector is equipped to address through the application of preventative or targeted med- ical treatment) is higher among immigrants. Immigrants are treated unequally within the Swedish health care sec- tor; the use of well-documented medical treatments, for example for heart attack, heart failure, stroke and chronic obstructive pulmonary disease is lower among immigrants than among native Swedes [11]. Methods Today, roughly 20 percent of the Swedish population are immigrants or descendants of immigrants, i.e., they were either born outside of Sweden or have at least one parent who was born outside of Sweden [8]. The term “immigrants” will therefore be used to refer to both groups throughout this paper. It cannot be ignored, however, that the term ‘immigrants’ encompasses a very diverse group comprising people from different coun- tries and with different socioeconomic backgrounds. Over the years there have been various patterns of migration to Sweden. During the 1950s and 1960s, labor migration resulted in an increased number of immi- grants from countries such as Italy, Greece and Turkey. During the 1970s and 1980s, war and the political situ- ation in countries such as Chile, Iran and Iraq resulted in refugees entering Sweden. The last two decades have been characterized by migration from countries such as Yugoslavia and Somalia, where civil war has threatened the life and health of people [9]. Most immigrants will primarily be from European countries outside of the European Union, Africa, Asia and Latin America [8]. What these people have in common is the experience of ethnic discrimination [10]. A qualitative approach was chosen to obtain a deeper understanding of the midwives' views on inequalities in the provision of health care due to immigrant status and cultural differences. Based on the objective, semi- structured interviews were considered to be the best method, with all interviewees being asked the same questions. The use of semi-structured interviews enables the researcher to prepare a number of questions in ad- vance. The interviewer may also ask spontaneous ques- tions and change the order of the set questions as the interview progresses. Semi-structured interviews also allow the interviewees to recount their experiences with as little guidance as possible from the interviewer [17]. The questions were open-response alternatives, creating equal opportunities for all midwives to share their views and experiences [18]. Introduction central government, regional, i.e., the municipalities and local, represented by the county councils. The county councils plan the development and organization of health care according to the needs of their residents, among others immigrants. However, asylum seekers and undocu- mented immigrants in Sweden have very restricted access to state subsidized health care [2,3]. The practice of health care in Sweden has encountered new challenges in recent decades as the immigrant popu- lation has increased. The goal of the Swedish health care system is to provide good care on equal terms to all people and in so doing, contribute to a more equitable spread of health [1]. Health care in Sweden is a public responsibility, financed primarily through taxes that are levied by county councils and municipalities. The Swedish health care sys- tem is structured on three levels: national, represented by Reports show that inequalities in the Swedish health care system have increased since the beginning of the 1990s. Most indicators suggest that immigrants have sig- nificantly poorer health than native Swedes [4,5]. Al- though the increasing disparity may have different causes, one may be due to the fact that immigrants do Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Page 2 of 10 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Data collection Each midwife was interviewed individually and in a quiet environment that the midwife selected. The interviews lasted between 50-60 minutes. Audio recordings were made of all interviews. The interviews were conducted The factors that contribute to health inequality due to immigrant status and cultural differences are complex and Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Page 3 of 10 Table 1 The interviewed midwives Participants Age Workplace Education Number of years in the profession Reported frequency of working with immigrant patients 1 55 Municipality 1 HealthCare College in Gothenburg (HCCG) 15 Often 2 45 Municipality 1 HCCG 8 Often 3 45 Municipality 2 Health Care College in Stockholm (HCCS) 9 Often 4 47 Municipality 2 HCCG 13 Often 5 55 Municipality 1 HCCG 18 Almost always 6 44 Municipality 2 HCCG 12 Almost always 7 55 Municipality 2 HCCG 14 Almost always 8 54 Municipality 1 HCCG 18 Almost always 9 57 Municipality 1 HCCG 25 Almost always 10 35 Municipality 2 HCCS 6 Often Table 1 The interviewed midwives between January 2009 and February 2010. The interviews were transcribed and translated from Swedish to English by the author and a research assistant. The questions posed were open-ended in order to obtain spontaneous in- formation on the study’s purpose. The research questions were prepared as Lofland & Lofland [20] suggested, by considering ‘Precisely what about this thing is puzzling me?’ They suggested that the puzzlement can be stimu- lated by various activities, such as discussions with collea- gues and studying existing literature on the topic. The research questions were: What happens during the meet- ing with an immigrant woman? What are your opinions on inequality in health care? How can inequality arise in the meeting with an immigrant woman? What are your thoughts on transcultural health care? to check that their interpretations were similar. The first step in the analytical process was to pick up meaning- bearing units, each related to the purpose of the study. A meaning-bearing unit is a paragraph or sentence that highlights the content of the material (Ibid). The next step was to shorten the chosen meaning-bearing units to con- densed units, i.e., to make the content more manageable but still maintain the parts that were considered to be of importance. Data collection The next step in the analytical process was to pick codes out of the condensed units. This was done to flag the contents for a higher level of analysis and to briefly describe the contents. The codes may be, as Granheim & Lundman [21] described them, discrete objects or phenomena that are related to the context. The author and her research assistant agreed upon the codes and the created subcategories and categories before pro- ceeding. The criteria for inclusion of a coding category were (1) how relevant the codes were to current study’s aim and (2) whether the code actually emerged in the text. Categories were initially kept as broad as possible without overlapping. Therefore few categories are chosen in the initial stages of the analysis. Then, as more data accumu- lated, the major categories were sorted into three categor- ies [22-24]. These three categories were compared with the entire body of interviews in order to verify their ori- ginal contexts. Furthermore, two external co-analyzers read the transcribed interviews and drew conclusions regarding the main content of each interview. Their find- ings were discussed with the author and their conclusions regarding the contents of the interviews agreed well with the authors’ coding. Finally, the analytical consistency was investigated by the author (Table 2). A Research Assistant with a Master’s degree in Public Health assisted in preparing the research questions, as well as with conducting and analyzing the interviews. This was to ensure that the analysis was conducted by two indivi- duals with diverse professional backgrounds, in order to balancing existing individual biases. The basic requirements of this study were that oral and written information be provided to participants and that written consent be obtained from them. The interviews were voluntary and informants were able to terminate the interview without justification. Privacy issues were consid- ered when noting the midwives' names. Participants will therefore remain anonymous. The study was approved by the Ethical Committee in Gothenburg (Dnr: 262–09). Data analysis A qualitative content analysis method [17] was used to analyze the midwives' views. Each interview was printed on paper and read through several times before and dur- ing the analytical process by the author and her research assistant, independently of each other. This was in order Results The interview data were divided into three main cat- egories and seven subcategories. The first category Page 4 of 10 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Table 2 Examples of meaning units, condensed meaning units and codes Meaning unit Condensed meaning unit Code Subcategory Category It is important to let the immigrant woman herself say what she needs and that the midwives then follow up on these needs and try to make the meeting a positive experience Important to let the immigrant woman herself say what she needs Listen, follow up and make the meeting a positive experience The meeting Communication midwives then follow up on these needs to make the meeting a positive experience ble 2 Examples of meaning units, condensed meaning units and codes eaning unit Condensed meaning unit Code Listen, follow up and make the meeting a positive experience It is important to let the immigrant woman herself say what she needs and that the midwives then follow up on these needs and try to make the meeting a positive experience It is important to let the immigrant woman herself say what she needs and that the midwives then follow up on these needs and try to make the meeting a positive experience to make the meeting a positive experience problems . . . It’s important to use professionally trained medical interpreters". It was not always feasible to use an interpreter. One midwife stated “It would be much better if the patient could speak Swedish". She added "Some- times even with an interpreter it becomes difficult to understand, because, we naturally use a great many dif- ficult words in health care". For some immigrant groups which came from countries with ethnical diversity and different languages and accents, the choice of interpreter was important. One midwife said that “the interpreters’ accents and ethnic identities can sometimes be problem- atic”. Other midwives said language should not be regarded as a factor that contributes to health care in- equality. One midwife said “It does not matter what a woman's cultural background is or what her skills in the Swedish language are . . . As a midwife I should provide good care”. Another one added “midwives should adapt their way of communicating”. “Communication” had three subcategories, “The meet- ing”, “Cultural diversity and language barriers” and “Trust and confidence”. The meeting According to the midwives there was a need for an "open" and welcoming meeting. By “open” they meant that it was necessary to listen and to consider the needs of immigrant women. “It is important to let the immi- grant woman herself say what she needs and that the midwives then follow up on these needs and try to make the meeting a positive experience”. Cultural differences and the response of health care staff to these differences were mentioned as another fac- tor that may lead to inequalities in health care provision. Differences in cultural beliefs, behaviors and expecta- tions may lead to misunderstanding and miscommunica- tion. Some midwives mentioned that there were some cultural collisions between immigrants and health care staff because of the patriarchal culture or religious beliefs, etc. One said “These kinds of beliefs can affect the immigrant men and women when making decisions, for example about abortion . . . we can only inform, we cannot contribute in any other way”. It was important to give the immigrant woman the feeling that she could choose, that she had control and that it was her decision. One midwife gave an example: “Different women from different cultures give birth in different positions and we try to understand and adapt . . . no way is wrong . . . the aim is to deliver a healthy baby and that the mother feels good”. Time was another aspect that had to be considered. The need for an advanced consultation might arise dur- ing the meeting, but the time allotted for a meeting was very limited and midwives were unable to extend the time available. The results showed that the midwives experienced inadequate time as a factor that might con- tribute to inequalities in healthcare. “A meeting with an immigrant woman demands more time; for example, more time to explain and to get confirmation that she understands. We have a set time for each patient and this cannot be extended”. Another midwife argued "It's obvious that everyone should get good care, but time lim- itations may restrict the provision of good care on equal terms. For example, we need a longer period of time when we use an interpreter. It's very important that we understand each other“. Communication The results from all the interviews showed that commu- nication has a central and significant role and may con- tribute to health inequality owing to ethnic and cultural differences. Results The second category “Potential barriers to the use of health services” had two subcat- egories, “Seeking health care” and “Receiving equal treat- ment”. Finally, the third category “Transcultural health care” had two subcategories, “Education on transcultural health care” and “The concept”. Trust and confidence The midwives all agreed that it takes time to establish trust and confidence in a meeting. In order to provide good care on equal terms, it was "important to under- stand and to trust in each other". According to the inter- viewed midwives, some policies might damage the establishment of trust and confidence between the care Seeking health care There were two different ways of thinking about re- ceiving equal treatment. Some midwives believed that it was the responsibility of the society and the health care services to be able to provide equal treatment to all citi- zens. One said "They need a better introduction to the society so that they know how it works. They should also get the opportunity to learn Swedish and to acquire good language skills so they can get better care”. According to one midwife “It is very important that society takes re- sponsibility and provides information. If you know what rights you have, and above all, have knowledge of what health care can help with . . . immigrants do not know what they can get help with". And another midwife said “It’s our responsibility to have better knowledge of differ- ent cultures in order to improve their chances for receiv- ing equal treatment". One midwife, however, expressed her confusion: “I don’t know if it depends on attitudes and prejudices in the Swedish health care system or on immigrants’ lack of knowledge of how the system works”. Indeed, some midwives believed that there could be dif- ferences in treatment and access to care, but ". . . It is The majority of the midwives observed no major differ- ences in the seeking of health care between immigrant and native Swedish women. However, a few midwives had another view. One said “some immigrant women are used to difficult conditions and seek health care when it may be too late". Generally, based on their experiences, midwives felt that a woman's level of education, country of origin and length of stay in Sweden could affect her views on how she uses the health care services. The mid- wives regarded level of education as a more important factor than cultural differences. One said “Education is more important than culture, the more educated (the woman is), the fewer the differences, but the woman is still shaped by her culture". Another midwife remarked: “Just because you are immigrants it doesn’t mean that your health care seeking behavior differs so much from that of native Swedes”. She continued, “some women are isolated, do not speak Swedish and have no contact with the Swedish society. They are newly arrived or have been here for a short time. . . Potential barriers to the use of health care services The interviewed midwives believed that inequality in health care could be more easily identified by investigat- ing health-seeking behavior and received treatment. Cultural diversity and language barriers According to some of the midwives language was an es- sential instrument for promoting effective communica- tion. Good language skills could reduce inequalities in the provision of health care. "There may be language Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Page 5 of 10 have serious problems like high blood pressure during pregnancy”. provider and the patient. An example of this was the Swedish health care guidelines to X-ray pregnant women from certain countries because of the risk of tubercu- losis. One midwife said “normally pregnant women should not be X-rayed, but in the case of immigrant women there is an exception and some immigrant women refuse to do it because of the pregnancy and they mistrust the system that has this policy”. Mistrust might also develop due to a lack of medical knowledge and language barriers. A midwife gave this example: “It is dif- ficult to give information about fetal diagnosis through an interpreter and talk about probability here and prob- ability there. These difficulties in communication can es- tablish mistrust”. Another midwife believed that trust could be established by allowing immigrant women to disclose their medical histories without fear of immigra- tion authorities. provider and the patient. An example of this was the Swedish health care guidelines to X-ray pregnant women from certain countries because of the risk of tubercu- losis. One midwife said “normally pregnant women should not be X-rayed, but in the case of immigrant women there is an exception and some immigrant women refuse to do it because of the pregnancy and they mistrust the system that has this policy”. Mistrust might also develop due to a lack of medical knowledge and language barriers. A midwife gave this example: “It is dif- ficult to give information about fetal diagnosis through an interpreter and talk about probability here and prob- ability there. These difficulties in communication can es- tablish mistrust”. Another midwife believed that trust could be established by allowing immigrant women to disclose their medical histories without fear of immigra- tion authorities. Receiving equal treatment The midwives all agreed that immigrant women’s status could affect how they are treated in the health care sys- tem. Furthermore, they assumed that immigrant women did not receive the same treatment and care as native Swedish women. One midwife gave an example: “newly arrived immigrant women may not have interpreters during the birthing process. This is terrible and can cre- ate lots of problems for care givers and for mothers”. An- other said ". . . I think there are big differences for those from other countries regarding how they are treated and how treatment works . . .perhaps due to ignorance or pre- judices". Another one added "I can imagine that a Swed- ish couple who is highly educated receives very different care and treatment in a hospital than a couple from a different culture who does not speak any Swedish". The reason that people are treated differently in the health care sector, according to the midwives, is that immi- grants cannot demand their rights. One midwife said “It is perhaps that it is hard to assert their rights for health care. One has to express oneself well. And in many cases, immigrants are not as good at it as the Swedes". Another midwife mentioned that "The vulnerable groups in soci- ety have more difficulties in getting adequate care . . . I believe that many people who come from other countries unfortunately count as a vulnerable group". One of the midwives mentioned that immigrants and native Swedes do not get the same care “because those born abroad have more difficulties in making their voices heard in the health care sector”. “For example, Somalian women may have children that are not their own, they just raise them as their chil- dren to save their lives, but for me as a midwife is im- portant to know how many children she has given birth to. If I can show that I am a health care staff and that I have no contact with the immigration authorities and if I give her a chance to narrate her history, listen and show understanding, then she will trust me”. Seeking health care for them, seeking health care when they need it is a problem, especially when they Page 6 of 10 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 and viewed immigrants as a group. One midwife said “I try to see and understand how they express themselves". Another explained ". . . we should learn how people from other cultures act . . . society must also have an under- standing of it. We come from different cultures and it has to be respected in order for everyone to feel welcome". The fact that immigrants were viewed as a homogenous group was emphasized by another midwife “For me it is like going abroad, I try to place myself in their culture and their world and to think with their brains . . .". Some midwives had a different view of transcultural health care; for them it was mostly about seeing the individual. One said “I try to see who I have in front of me and form my idea of what she reflects and expresses. I am not pro- grammed to run the same procedure for everyone”. not always the health care services that are the problem". They believed that is an individual’s (the immigrant patient’s) responsibility to know the system, to speak the language and be able to express herself. “An individual's ability to express herself and understand is critical to the standard of received treatment in the health care system". Education on transcultural health care All midwives expressed the opinion that there should be more on the subject of transcultural health care in their education and training in order to improve their com- munication skills and enable them to provide equal and good health care. The midwives said that they needed continuous training in cultural diversity. One said “The world is constantly changing and people are moving to Sweden for various reasons. Midwives would like to con- tinuously update their knowledge of different cultures”. One said "In the 1990s we had a lot of information, espe- cially when large groups came from Somalia. But now it's like you have to seek the information yourself". During their training they had no courses on cultural diversity or cultural sensitivity. Training in transcultural health care meant different things to different midwives. One midwife said “one cannot learn about all different cul- tures . . . cultural sensitivity training means to learn to accept, respect and be keen and open”. Another believed that health care staff needed training in ethnic and Euro- centric attitudes. ”I wish that we could learn about ethni- city and culture during our training. . . I want to learn how to meet culturally diverse people in the right way . . .we have to improve our ways of communication and our cultural competency”. All midwives agreed that having culturally diverse health care staff was an important resource for providing culturally sensitive health care, but they were all negative about the idea of ethnic health care services. One said “Then we will have even more segregation “. And another added “I think we can learn from each other. The native Swedish health care staff can learn from staff who are immigrants and vice versa. Employing immigrant health care staff will facilitate this”. According to the midwives, another negative aspect of ethnic health care services would be that they would provide low quality care be- cause they would get fewer resources and qualified health care staff would not work in such services. One midwife said of such a health care service: “Nothing will work, staff will leave, we must make it attractive to work with culturally diverse patients and not establish segre- gated health care services”. Another midwife stated that establishing ethnic health care services “will cement prejudices”. The concept The findings of this study show that midwives view com- munication as having a central role that may contribute to health inequalities. An open meeting in which the care provider (in this case the midwives participating in the study) allows for adequate time to listen to and con- sider the needs of the patient and a meeting in which the cultural and language differences do not lead to mis- understandings are factors that contribute to the provision of equitable health care. Midwives believe that the potential barriers to the use of health care services are immigrants’ health care seeking behavior and the way immigrants are treated in the health care system. Finally, the questions on transcultural health care shed light on two different perspectives on immigrant patients; they are either viewed as (a) individuals or (b) a group. Furthermore, all midwives agreed that having cul- turally diverse health care staff was an important re- source for providing culturally sensitive health care, but “Transcultural health care” was an unfamiliar expression to most of the midwives who were interviewed in the study. One said "not words we use, but we are caring in a cultural way, it means trying to be observant and try- ing to capture what is different". One added "I have never heard the term but I think different cultures have differ- ent beliefs and that is the only difference”. Although the expression was unfamiliar, the midwives had ideas about the concept of transcultural health care. One said “we live in a society which is culturally diverse and the health services should be more aware that people come from different cultures. It is something that must be accepted and respected”. To work transculturally, according to one midwife, meant “to adapt our know- ledge and experience to different cultures”. Another said “transcultural health care means to work beyond the bor- ders and norms”. Some midwives believed that transcul- tural health care was about "cultural communication” Page 7 of 10 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Page 7 of 10 patient/provider relationship. Research highlights their potential value in understanding the performance of health care systems [37]. According to the interviewed midwives, mistrust can be established because of health care policies (e.g. X-raying pregnant women from some countries). The concept Although health care workers in Sweden are not required to report immigration law violations [38], miscommunication can arise due to language and cul- tural barriers and patient circumstances (e.g. inaccurate registration of the children in some Somalian families). The ability to communicate correlates with levels of trust [39]. Miscommunication results in lower levels of trust in health care, a relationship that can cause costly problems for society [40]. Using the concept of public trust in health care, Straten et al. [41] combine consider- ation of inter-personal, organizational and system trust. The results of this study show that the midwives are working hard on establishing inter-personal trust; they try to act in the patients’ best interests [42]. With regards to patient-caregiver trust, the common issues in- clude the patient focus of the caregiver, caregiver com- petence and quality of care, communication and co- operation and supportive structures and resources. How- ever, the differences between countries, levels of educa- tion and the role of non-western medical traditions [43] might invalidate such an approach. It is well known that minority individuals report lower levels of trust than members of the majority [44,45]. they all responded negatively to the idea of ethnic health care services. Potential barriers to the use of health care services Generally, midwives noted that a woman’s level of edu- cation and whether she comes from an urban or a rural area can be more important than cultural norms in de- termining whether or not she seeks health care. Their assumption about the women’s socioeconomic back- ground and length of stay having an effect upon their health care behavior is in agreement with earlier re- search [46,47]. Another aspect of the study of health care inequality is to consider the provision of equal treatment and who is responsible for it. Some midwives believed that it was the society’s and the health care services’ responsibility to be able to provide equal treatment to all citizens; other midwives believed that it was an individual’s (the immigrant patient’s) responsibility to know the system, to speak the language and be able to express herself. Rundström [48] states that ideally, from the macro- sociological perspective, it is the staff who should obtain knowledge and so become skilled in the medical-cultural issues. The micro-sociological perspective focuses on the individual responsibility for health or individuals’ ability to learn the rules, norms and behaviors which exist and to adapt to them without feeling their integrity or cul- ture is violated, even if she/he is confronted with Communication The results of the interviews show that midwives believe that poor verbal communication or language skills may lead to miscommunication which in turn may contribute to inequalities in the provision of health care. In agree- ment with the results of this study, previous research articles [25,26] mention the quality of verbal communi- cation and language skills as factors that may contribute to inequality in health care. Fortier et al. [27] assert that a failure to ensure adequate communication between patient and provider “can lead to inappropriate or unnecessary testing, clinical inefficiency, misdiagnosis, negative outcomes and malpractice.” Previous research [28,29] indicates that language bar- riers can adversely affect the quality of care. Some researchers point out that when a patient does not speak the language of his or her health care provider, multiple adverse effects on the patient’s health may occur and lead to poor patient satisfaction, poor compliance and underuse of services [30,31]. Some interviewed midwives emphasized that as caregivers they should provide good care, regardless of whether their patient can speak the language or not. In other words, language should not be a barrier to providing equitable health care. Employing bilingual health care staff, using qualified interpreters or using community-based health navigators (CBHN) [32] and providing written information in different languages may facilitate communication, increase patient satisfac- tion and increase patient understanding. It would also help to avoid errors in diagnosis and treatment and avoid the costs of employing telephone interpreters [33,34]. Almost all communication between midwives and immigrant patients was conducted through an inter- preter, which meant that it took longer to communicate all of the information. The use of an interpreter could not be avoided; this was a tool that the midwives felt that they had to work with in order to provide good care on equal terms. According to the midwives, using pro- fessionally trained medical interpreters can provide a higher degree of accuracy and confidentiality and increased overall effectiveness. However, even this ap- proach is not without potential problems. For example, the information advantage is lost when health profes- sionals are not aware of how much information was translated by the interpreter [35] or when the interpreter is unable to mediate cultural, class and power differences between the patient and provider [36]. Trust and confidence Trust and confidence are crucial for obtaining equality in a health care system, as well as for fostering a good Page 8 of 10 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 something unexpected [49].The results of the interviews show that some midwives believe that the vulnerable groups (immigrants, among others) face more difficulties in getting adequate care. The vulnerable groups suffer because of the structural conditions in the society and health care system and not because of their inability to adapt to health care services. It is important that attempts to identify weaknesses in health care policies do not degenerate into a position that blames the victim. The ideology of individual responsibility for health tends to obscure the reality of the impact of social inequality on health and it views the individual as being essentially independent of his or her surroundings [50]. expressed the opinion that there should be more on the subject of transcultural care in their education and train- ing program. Previous research [56-58] recognizes the need for educating health care staff on transcultural health care issues. Some midwives regarded transcultural health care as “cultural communication” and viewed immigrants as a group of “others”1 to be studied and analyzed. The dan- ger of the “seeing immigrants as a group” approach is that it assigns everyone to a particular group with the same life experiences and the same cultural behaviors. Maintaining a focus on “others” may reinforce negative qualities and lead to stereotyping and discrimination [59]. Transcultural care is about providing culturally relevant care [57]. It emphasizes the requirement for the development of self-reflection on one’s own cultural identities as an individual and health professional and toward a greater focus on the patient as an individual [56]. It is about cultural awareness and openness [57] or as Campinha-Bacote’s model [60] explains, it is about embodying the following attributes: awareness of one’s own biases and prejudices toward other cultures, know- ledge about culture in general, the ability to conduct ac- curate cultural assessments and interpersonal skills in cross-cultural encounters. Another crucial issue related to transcultural health care that midwives raised in the interviews is the idea of ethnic health care services. The midwives were all negatively disposed to the idea. Cultural differences and transcultural health care Cultural differences and transcultural health care Cultural background, cultural beliefs and expectations were other contributing factors to inequalities in health care. Different systems of cultural beliefs and practices and different views and expectations may lead to con- flicts between immigrant women and their care givers [51,52]. The results of this study show that some mid- wives have developed an appropriate way to provide in- formation and to offer choices and let the immigrant women feel that they are in control of their own bodies and health care decisions, i.e., to see them as individuals and not as a group. Rice [51] argues that one of the fac- tors that may lead to miscommunication is that immi- grant women are not given information and allowed to make their own choices. They should be offered a choice and their individual needs should be considered [53]. As one of the interviewed midwives emphasized “It is im- portant to let the immigrant woman herself say what she needs and what she wants”. The interviewed midwives felt that “health care ser- vices should be more aware that people come from dif- ferent cultures”. Furthermore, they all agreed that having culturally diverse health care staff was important means through which to provide culturally sensitive health care. Previous research shows that receiving culturally appro- priate services from health care staff is more than simply a patient’s right; in reality, it is a key factor in the safety and quality of patient care and moves away from a “one size fits all” approach that negatively affects the quality of care for diverse patients [54]. Transcultural values may result in fewer communication problems because of language and cultural differences [55] and the employ- ment of bilingual and bicultural staff, especially in ob- stetric services, is recommended [33]. The results show that the midwives’ knowledge of the concept of transcul- tural health care was limited. However, midwives have a professional and culturally sensitive approach, thanks to their long experience and genuine interest in their work. The results also show that there is a need for continuous training in cultural diversity. The interviewed midwives Trust and confidence They believed that ethnic health care services would lead to increased segregation, reinforce prejudices and provide low quality care since patients would get fewer resources under such a system. They also believed that qualified health care staff would not want to work in such ser- vices. Kai [61] stressed that most people from diverse ethnic communities do not want ethnic services. Like everyone else they just desire good quality services. If regarding immigrants as a group is a form of ethnocen- trism and ethnic discrimination, then providing ethnic health care services would be the other side of the coin, i.e., it would be providing “culturally relativist” health care. Conclusions de los Reyes P, Kamali M: Bortom vi och dom - Teoretiska reflektioner om makt, integration och strukturell diskriminering. Stockholm: SOU, Edita Norstedts Tryckeri AB; 2005. No. 41. [Trans: Beyond we and them - theoretical reflections on power, integration and structural discrimination]. 11. National Board of Health and welfare: Hälso- och sjukvård - lägesrapporter 2007. Stockholm; 2008. [Trans: Health and health care, Current report for 2007]. http://www.socialstyrelsen.se/Lists/Artikelkatalog/Attachments/8864/ 2008-131-7_20081317_rev.pdf. 11. National Board of Health and welfare: Hälso- och sjukvård - lägesrapporter 2007. Stockholm; 2008. [Trans: Health and health care, Current report for 2007]. http://www.socialstyrelsen.se/Lists/Artikelkatalog/Attachments/8864/ 2008-131-7_20081317_rev.pdf. 12. Social Department: Hälso-och sjukvård inför 90-talet, Invandrarna i hälso-och sjukvården, HS 90. Stockholm: SOU, Socialstyrelsen; 1984. No. 45: [Trans: Health and healthcare in the 90s, Immigrants in health care]. 12. Social Department: Hälso-och sjukvård inför 90-talet, Invandrarna i hälso-och sjukvården, HS 90. Stockholm: SOU, Socialstyrelsen; 1984. No. 45: [Trans: Health and healthcare in the 90s, Immigrants in health care]. 13. Fagerli RA, Lien ME, Wandel M: Health worker style and trustworthiness as perceived by Pakistani–born persons with type 2 diabetes in Oslo, Norway. Health: An interdisciplinary Journal for the Social Study of Health, Illness and Medicine. 2007, 11(1):109–129. Conclusions Midwives believe that health care inequality among immigrants may be the result of miscommunication which may arise due to a shortage of meeting time, lan- guage barriers, different systems of cultural beliefs and practices and limited patient-caregiver trust. Immigrants face more difficulties in seeking health care and in re- ceiving adequate levels of care. The level of education, country of origin and length of stay in Sweden is believed to influence immigrants’ health care seeking be- havior. An interesting difference was observed among the midwives’ views; some midwives are sensitive to in- dividual and intra-group differences while other mid- wives view immigrants as a group of “others”. The findings of the study suggest that more research is needed about the potentials of educating health care staff on the provision of transcultural health care and regarding midwives’ attitudes toward subgroup-specific health care services. This might be a starting point in developing strategies for reducing ethnic inequalities in the health care system. 5. Akhavan S: The health and working conditions of female immigrants in Sweden, PHD thesis. Karolinska Institute: Public Health Department; 2006. 6. ACOG. American College of Obstetricians and Gynecologists, Committee on Health Care for Underserved Women: ACOG committee opinion: cultural competency in health care. Int J Gynecol Obstet 1998, 62:96–99. 7. The Swedish association of health professionals database: Örebro: SCB-Tryck; https://www.vardforbundet.se/In-English. 8. Statistics Central Board: Befolkningsstatistik i sammandrag 1960–2005. Stockholm: EO Print; 2005 [Trans: Population statistics between 1960 and 200]. 9. Hogstedt C, Backhans M, Bremberg S, Lundgren B, Törnell B, Wamala S: Välfärd, jämlikhet och folkhälsa – vetenskapligt underlag för begrepp, mått och indikationer.: Statensfolkhälsoinstitutet, EO Print; 2003. No.12. [Trans: Welfare, equality and public health – scientific basis for concept, measurement and indications (The Swedish Public Health Board)]. 9. Hogstedt C, Backhans M, Bremberg S, Lundgren B, Törnell B, Wamala S: Välfärd, jämlikhet och folkhälsa – vetenskapligt underlag för begrepp, mått och indikationer.: Statensfolkhälsoinstitutet, EO Print; 2003. No.12. [Trans: Welfare, equality and public health – scientific basis for concept, measurement and indications (The Swedish Public Health Board)]. 10. de los Reyes P, Kamali M: Bortom vi och dom - Teoretiska reflektioner om makt, integration och strukturell diskriminering. Stockholm: SOU, Edita Norstedts Tryckeri AB; 2005. No. 41. [Trans: Beyond we and them - theoretical reflections on power, integration and structural discrimination]. 10. Authors’ contributions SA is the only author of the manuscript and takes full responsibility for the manuscript. Methodological considerations One limitation of this study may be the limited number of interviewees used. However, the number of partici- pants was enough to attain adequate thematic saturation because of sample homogeneity: they were all female, midwives and worked with the same category of patients. Guest et al., [62] stated that the more similar participants in a sample are in their experiences with re- spect to the research domain the sooner we should ex- pect to reach saturation. Another limitation may be that the results may have suffered from selection bias, i.e., the sampling method may have affected the findings. Page 9 of 10 Page 9 of 10 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 Akhavan International Journal for Equity in Health 2012, 11:47 http://www.equityhealthj.com/content/11/1/47 This may have occurred due to the fact that the study participants were chosen from two municipalities in districts that had a higher number of immigrants. Differ- ent results may have been obtained if the study had also included interviews with midwives who work in districts with fewer immigrants. Such a selection might have improved the investigation of the role of immigrant patients’ socioeconomic situation. A well-selected and diversified sample is important. If the findings are based on the range of social settings that is likely to contribute to a particular experience, it strengthens the generalizability of the conclusions [63]. The interview location was planned according to the wishes of the interviewee, as the aim was to create a relaxed setting. The subjectivity of the researcher is another methodological issue that can be discussed. Morse [17] states that in order to conduct valid research it is imperative that the researcher be aware of personal bias or agenda. Research questions may not be value-free but may even reflect the researcher’s values. In this study, the questions about transcultural health care were based on the general discussion on transcultural health care in Sweden and the author’s previous research and knowledge in the field. They could therefore be seen as leading questions. Competing interests The author declared that he has no competing interest. Competing interests The author declared that he has no competing interest. Competing interests Authors’ information Department of Public Health – University of Skövde & School of Health, Care and Social Welfare – University of Mälardalen– Sweden. Sharareh Akhavan is Senior Lecturer in Public Health and working on several research projects related to immigrants’ health. References 1. The National Board of Health and welfare. The 2009 Swedish Health Care Report. Västerås: Edita Västra Aros; 2010. 1. The National Board of Health and welfare. The 2009 Swedish Health Care Report. Västerås: Edita Västra Aros; 2010. http://www.socialstyrelsen.se/publikationer2009/2009-9-18. Report. Västerås: Edita Västra Aros; 2010. http://www.socialstyrelsen.se/publikationer2009/2009-9-18. p http://www.socialstyrelsen.se/publikationer2009/2009-9-18. 2. The World Health Organization database; http://www.euro.who.int/__data/ assets/pdf_file/0010/96409/E88669.pdf. p _ p 3. Djurfeltd A, Huldt E: Immigrants and health care. A right-based utilitarian approach. Lund University: Department of political science; 2007. 4. Statistics Central Board: Ohälsa och sjukvård 1980–2005, Levnadsförhållandena; 2006. Rapport nr. 113. [Trans: Ill-health and health care between 1980 and 2005, Life conditions]. 13. Fagerli RA, Lien ME, Wandel M: Health worker style and trustworthiness as perceived by Pakistani–born persons with type 2 diabetes in Oslo, Norway. Health: An interdisciplinary Journal for the Social Study of Health, Illness and Medicine. 2007, 11(1):109–129. Acknowledgements h h h The author wishes to thank all participants in this study; without their contribution it would not have been possible to undertake the research. Thanks to Research Assistant Sabina Adamsson. The study was supported by the Research Center Skaraborgs Institutet. Received: 12 October 2011 Accepted: 13 August 2012 Published: 18 August 2012 14. 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https://openalex.org/W2905439464	http://sajie.journals.ac.za/pub/article/download/1947/890	English	null	EARLY FAULT DETECTION OF ELEVATORS USING REMOTE CONDITION MONITORING THROUGH IOT TECHNOLOGY	South African journal of industrial engineering	2,018	cc-by	9,709	South African Journal of Industrial Engineering December 2018 Vol 29(4), pp 17-32 South African Journal of Industrial Engineering December 2018 Vol 29(4), pp 17-32 ABSTRACT Remote condition monitoring (RCM) of machines seeks to enhance proactive maintenance through just-in-time responses to machine faults and process deterioration. This approach offers the benefit of reduced manning of machines and robust joint maintenance decisions, due to remote access to the machines’ condition. This paper employs a remote condition monitoring approach to two elevator parameters, vibration and machine room-temperature, using an Internet of Things (IoT) device for remote data acquisition and remote fault indication. A remote monitoring set-up was developed that uses augmented sensors, networked connections, and an Arduino Yun microcontroller installed on the elevator system to monitor remotely any deterioration in its working condition. The set-up was configured to monitor the conditions online remotely through an email application service. The data from the email were analysed, and notifications were generated at the machine’s severity level. The result showed that RCM enables faster repair and maintenance decisions, prevents the catastrophic breakdown of machines, and serves as a troubleshooting guide for fault diagnosis. Contact details * Corresponding author IsaacO@dut.ac.za Author affiliations 1 Department of Industrial Engineering, Durban University of Technology, South Africa OPSOMMING Afstand toestand monitering van masjiene poog om proaktiewe onderhoud te verbeter deur net-in-tyd reaksie op masjienfoute en prosesagteruitgang te bewerkstellig. Hierdie benadering bied die voordeel van verminderde toerusting bemanning en robuuste gesamentlike onderhoud besluite as gevolg van afgeleë toegang tot die toerusting se toestand. Hierdie artikel verskaf ’n afstand toestand moniteringbenadering van twee hysbak parameters, naamlik vibrasie en masjienkamertemperatuur deur ŉ internet-van- dinge toestel vir afstand dataverkryging en afstand fout aanduiding. ŉ Afstand moniteringopstelling, bestaande uit versterkte sensors netwerkverbindings en ŉ Arduino Yun mikro-beheerder, is geïnstalleer op die hysbakstelsel om enige agteruitgang in die werkende toestand te monitor. Die opstelling is gekonfigureer om die toestand oor ŉ afstand te monitor. Die opstelling is gekoppel deur ŉ e-pos toepassing. Die e-pos data is ontleed en kennisgewings gegenereer op grond van die erns van die toerusting se toestand. Die resultaat toon dat toestand monitering oor ŉ afstand vinniger herstel- en instandhoudings-besluite moontlik maak, katastrofiese faling van die toerusting voorkom en ook dien as ’n probleemoplossingsgids vir foutbespeuring. DOI http://dx.doi.org/10.7166/29-4-1947 EARLY FAULT DETECTION OF ELEVATORS USING REMOTE CONDITION MONITORING THROUGH IOT TECHNOLOGY I.O. Olalere1# & M. Dewa1† I.O. Olalere1# & M. Dewa1† ARTICLE INFO Article details Submitted by authors 19 Mar 2018 Accepted for publication 22 Aug 2018 Available online 10 Dec 2018 This paper is further work from a paper in the 28th SAIIE conference proceedings entitled ‘Remote condition monitoring for fault indication in elevators using IoT technology for improved maintenance’. Contact details * Corresponding author IsaacO@dut.ac.za Author affiliations 1 Department of Industrial Engineering, Durban University of Technology, South Africa # The author was enrolled for an M Eng (Industrial) degree in the Department of Industrial Engineering, Durban University of Technology, South Africa DOI http://dx.doi.org/10.7166/29-4-1947 ARTICLE INFO Article details Submitted by authors 19 Mar 2018 Accepted for publication 22 Aug 2018 Available online 10 Dec 2018 This paper is further work from a paper in the 28th SAIIE conference proceedings entitled ‘Remote condition monitoring for fault indication in elevators using IoT technology for improved maintenance’. ARTICLE INFO # The author was enrolled for an M Eng (Industrial) degree in the Department of Industrial Engineering, Durban University of Technology, South Africa DOI http://dx.doi.org/10.7166/29-4-1947 2 LITERATURE REVIEW Recently, intelligent elevator systems that can be remotely tracked for maintenance have been developed [3]. However, the need for an augmented maintenance monitoring system still cannot be ruled out, as a system’s interaction with its environment is peculiar and dynamic. This research seeks to adopt the remote condition monitoring of lifts and elevator systems for the earlier detection of abnormalities in their condition. Several remote monitoring approaches are now being adopted, which may include a self-test remote machine monitoring system or remotely monitoring conditions through augmented automation using IoT devices. Datta et al. [4] developed self-testing software that remotely analyses the industrial machine condition to detect abnormality during operation, and that generates a report on this analysis. The software was interfaced with a USB modem in order to send multimedia messages during any abnormality in the machine’s condition. This would assist in providing faster machine maintenance, compared with breakdown maintenance. In elevator systems, parameters such as the vibration of the elevator car, sound from the drive system, and temperature from the machine room could point to an impending fault in the elevator system [5]. Remote condition monitoring and a pre-alarm system are gradually being developed, based on the Internet of Things (IoT) and cloud computing (CC), achieved with the capability of real- time monitoring [6]. IoT technology and devices create the option of remote condition monitoring, diagnosis, and reporting, thus keeping the maintenance team abreast of the condition of the system. For example, vibration in the elevator car would indicate a malfunctioning drive system or the misalignment of the elevator car on the guide rails [7]. Vibration in the elevator car or lift car is a major condition that indicates the condition of the drive system, while the machine room temperature may indicate the functioning of the control system [8]. A malfunctioning drive system, guides, controls, elevator car, and hall equipment would generate a side sway, oscillation, or vibration of the elevator car [9]. Kwashaka and Mariani [10] performed vibration analysis on traction elevators using three axis accelerometer sensors, Endevco 66A11, to determine the excitation of the system, obtaining five experimental modal analyses. The vibration and temperature parameter is monitored remotely, and the data is logged on the cloud while evaluating the current conditions against the severity level of the parameter. 2 LITERATURE REVIEW An Arduino Yun IoT microcontroller is used as the principal component in the monitoring system; an SPF 3-AXIS Accelerometer ADXL335 BD vibration sensor is used to capture the vibration data; and an LM 35 temperature sensor is used for the machine’s room temperature. The data is captured remotely on the cloud using the email choreo, and an email alert is generated and sent for each severity level of any of the parameters. 1 INTRODUCTION Maintenance of machines and facilities is gaining more attention, as it helps to increase productivity and competitiveness through reduced machine downtime and maintenance costs. Augmented automation, alongside reduced manning and increased operating tempos in production machinery, 17 has given rise to a rapid increase in installed machinery sensors in order to repair it faster and to ensure that the equipment operates reliably for long periods [1]. Lifts should be subject to scheduled maintenance and minimum downtimes [2]. The research efforts focus on the construction of a remote monitoring device that applies emerging IoT technology to reporting the state of the elevator system and indicating the severity level of its condition, to support proactive maintenance and provide a deterioration pattern/history before breakdown maintenance is required. Since lift and elevator systems are not manned, the maintenance policy most often adopted is scheduled maintenance. The device was configured to monitor the machine room temperature and vibration parameters, which are instrumental in indicating the malfunction of a system. The monitoring device was installed on the elevator car’s roof to monitor the vibration of the elevator car and the drives, and also the machine room’s temperature for proper functioning of the controls. has given rise to a rapid increase in installed machinery sensors in order to repair it faster and to ensure that the equipment operates reliably for long periods [1]. Lifts should be subject to scheduled maintenance and minimum downtimes [2]. The research efforts focus on the construction of a remote monitoring device that applies emerging IoT technology to reporting the state of the elevator system and indicating the severity level of its condition, to support proactive maintenance and provide a deterioration pattern/history before breakdown maintenance is required. Since lift and elevator systems are not manned, the maintenance policy most often adopted is scheduled maintenance. The device was configured to monitor the machine room temperature and vibration parameters, which are instrumental in indicating the malfunction of a system. The monitoring device was installed on the elevator car’s roof to monitor the vibration of the elevator car and the drives, and also the machine room’s temperature for proper functioning of the controls. 3 SYSTEM ARCHITECTURE FOR REMOTE MACHINE CONDITION MONITORING A Spark Fun SPF 3 Axis Accelerometer ADXL 335 BD SEN-09269, from analog devices with low noise and power consumption of 32µA and a sensing range of +/-3g, is the sensor for measuring the vibration of the elevator car. The angle of inclination of the system, relative to the installation of the sensor, is also considered by the sensor. An LM 35 temperature sensor is used for measuring the temperature of the machine room. Figure 1: System architecture for machine remote condition monito Figure 1: System architecture for machine remote condition monitoring Microcontroller-enabled sensors: These measure the conditions of the machines for both the vibration parameter and the machine room temperature parameter. A Spark Fun SPF 3 Axis Accelerometer ADXL 335 BD SEN-09269, from analog devices with low noise and power consumption of 32µA and a sensing range of +/-3g, is the sensor for measuring the vibration of the elevator car. The angle of inclination of the system, relative to the installation of the sensor, is also considered by the sensor. An LM 35 temperature sensor is used for measuring the temperature of the machine room. Microcontroller-enabled sensors: These measure the conditions of the machines for both the vibration parameter and the machine room temperature parameter. A Spark Fun SPF 3 Axis Accelerometer ADXL 335 BD SEN-09269, from analog devices with low noise and power consumption of 32µA and a sensing range of +/-3g, is the sensor for measuring the vibration of the elevator car. The angle of inclination of the system, relative to the installation of the sensor, is also considered by the sensor. An LM 35 temperature sensor is used for measuring the temperature of the machine room. API/control unit: this unit monitors the signal from the sensor and converts it to a digital value that is readable by the operator. The major component in this unit is the microcontroller, which is an IoT-enabled microcontroller named Arduino Yun. The controller connects to the cloud through a third-party web application known as Temboo, through which the sensor data and notifications are sent using the email choreo application. The data is then transmitted to the cloud web service and stored in the cloud, where it can be accessed remotely. Cloud services/Web application: This is the Internet end of the whole set-up that provides the remote link for the data being monitored by the device. 3 SYSTEM ARCHITECTURE FOR REMOTE MACHINE CONDITION MONITORING A system architecture for remote machine condition monitoring meets the requirement of monitoring and measuring the condition of machines, processing the data from the instrument, and accessing the conditions remotely without a physical presence. Our approach adopts the emerging technology of the Internet of Things (IoT) and cloud computing in building the remote monitoring system. IoT concepts with cloud computing have found enough application in a smart home (SH) environment to enable the user to measure home conditions (such as temperature, humidity, luminosity), to manipulate the heating, ventilation, and air conditioning (HVAC) of the home, and to monitor the home security system [11]. The approach in this research makes use of micro- controller enabled sensors to measure the parameters of the elevator, the powered IoT-compliant 18 microcontroller for controlling the whole system connection, and the data transmission from the sensor to the cloud database, which in this case uses the email choreo. The microcontroller works as the control unit, carrying out some logical reasoning and arithmetic on the read sensor data for maintenance decision-making. The platform is therefore categorised into three layers: the sensors layers for data capturing, the API/data transmission layer, and the web application client. Figure 1 illustrates the system architecture for the remote condition monitoring maintenance for a machine, using IoT technology. It has microcontroller-enabled sensors, an API/control unit, and cloud services/Web application as its major components. microcontroller for controlling the whole system connection, and the data transmission from the sensor to the cloud database, which in this case uses the email choreo. The microcontroller works as the control unit, carrying out some logical reasoning and arithmetic on the read sensor data for maintenance decision-making. The platform is therefore categorised into three layers: the sensors layers for data capturing, the API/data transmission layer, and the web application client. Figure 1 illustrates the system architecture for the remote condition monitoring maintenance for a machine, using IoT technology. It has microcontroller-enabled sensors, an API/control unit, and cloud services/Web application as its major components. Figure 1: System architecture for machine remote condition monitoring Microcontroller-enabled sensors: These measure the conditions of the machines for both the vibration parameter and the machine room temperature parameter. 3 SYSTEM ARCHITECTURE FOR REMOTE MACHINE CONDITION MONITORING This serves as the database for storing the data from the sensors, and as a point for remote access to the data and medium of event notifications. Cloud hosting has significant benefits, such as improved reliability and physical security. The website is hosted on a virtual partition instead of being hosted on one single physical server; this feature spreads the risk, since the pooled cloud resource is drawn from multiple data centres in different locations. 4 DESIGN OF REMOTE CONDITION MONITORING SYSTEM FOR ELEVATOR PARAMETERS Machine room temperature: The machine room temperature is important because it houses the controls for the system, and yet it is usually poorly ventilated. Since high temperatures may cause a malfunction in the system, the temperature in the room must be measured and recorded. Figure 2: Geared drive system of a traction elevator Figure 2: Geared drive system of a traction elevator Vibration of the elevator car and the drive system: This vibration takes into consideration the guide rails, the drive sheave, the lubrication of the gear box, the braking system, and the deflection of the cable. Figure 2 shows the geared drive system of a traction elevator whose components are susceptible to vibration — the braking system due to wear and tear and shock, the gear due to friction and lubrication — and the drive sheave, which propels the cable. Vibration of the elevator car and the drive system: This vibration takes into consideration the guide rails, the drive sheave, the lubrication of the gear box, the braking system, and the deflection of the cable. Figure 2 shows the geared drive system of a traction elevator whose components are susceptible to vibration — the braking system due to wear and tear and shock, the gear due to friction and lubrication — and the drive sheave, which propels the cable. Sending the data to the cloud: As mentioned earlier, Temboo is used to send the data to the cloud. This web application links and configures the controller to the cloud for IoT devices. It consists of different internet applications called choreos, which include Google email, Twitter, SMS, Instagram, Yahoo, PayPal, Amazon, and more than 30 other apps. The Google email choreo, configured on Temboo, was run on the microcontroller to send the data to the cloud. The aim of this research is to reduce the downtime of the system, eliminating any delay in sending a breakdown notification by using just-in-time condition capturing and severity notifications to the remote monitoring device. This is achieved by developing the remote monitoring device, capturing the mean parameter signals from the elevator systems, coding the controllers with the range of normality for fault indication on the cloud, monitoring the system, and analysing the historical data to generate a fault diagnosis using cause-and-effect analysis. 4 DESIGN OF REMOTE CONDITION MONITORING SYSTEM FOR ELEVATOR PARAMETERS The system architecture was designed to relay the conditions of the elevator system remotely to the maintenance team for prompt proactive and preventive maintenance of the machinery. The elevator maintenance system was discussed at One World Trade Center (WTC) using the Microsoft Azure Intelligent system, which responds to faults proactively by sending service engineers real-time data so that a total breakdown of the elevator can be prevented by feeding the data into a dynamic 19 predictive model [12]. The approach used in this research, however, uses the intelligent ability of the microcontroller to carry out the logical operation of the read values against a predetermined severity condition level of the system while using its IoT capability in sending the data and notifications in the event of an abnormality in the system. Several technologies are emerging on industrial manufacturing machines for better efficiency and maintenance. Asset monitoring, which enables the remote tracking of machines, has emerged from the technology of machine-to-machine (M2M) communication to IoT configurations [13]. This has gradually involved the adoption of the IoT as an augmented automation of the remote condition monitoring maintenance of machines. The configured remote monitoring device was developed to measure the machine room temperature and the vibration of the elevator car and the drive system, and to send the data to the cloud using a third-party web service known as Temboo. predictive model [12]. The approach used in this research, however, uses the intelligent ability of the microcontroller to carry out the logical operation of the read values against a predetermined severity condition level of the system while using its IoT capability in sending the data and notifications in the event of an abnormality in the system. Several technologies are emerging on industrial manufacturing machines for better efficiency and maintenance. Asset monitoring, which enables the remote tracking of machines, has emerged from the technology of machine-to-machine (M2M) communication to IoT configurations [13]. This has gradually involved the adoption of the IoT as an augmented automation of the remote condition monitoring maintenance of machines. The configured remote monitoring device was developed to measure the machine room temperature and the vibration of the elevator car and the drive system, and to send the data to the cloud using a third-party web service known as Temboo. 4.1 Hardware components and connections The hardware used for the device is the sensors, the microcontroller, the LED, a 5V power source, and jumper wires. The two sensors used in this project are an LM 35 temperature sensor, and the ACM 3 Axis Accelerometer ADXL 345 vibration sensor. The vibration sensor is connected to the female-to-female and male-to-male jumper wires to extend the sensors to the part on the elevator that is sensitive to vibration signals. The microcontroller, the Arduino Yun board, is the most recent 20 IoT board from Arduino, with powerful accessories such as WIFI connectivity, ethernet, USB connectivity, and a micro-SD card port, making it dynamic and robust in usage and application. The board is powered by a 5V voltage source, and the 5V power pin on the board is connected to the power rail on the breadboard, as is the ground pin on the board. IoT board from Arduino, with powerful accessories such as WIFI connectivity, ethernet, USB connectivity, and a micro-SD card port, making it dynamic and robust in usage and application. The board is powered by a 5V voltage source, and the 5V power pin on the board is connected to the power rail on the breadboard, as is the ground pin on the board. 4.1.1 Figure 3: The connection of the temperature sensor to the controller Figure 3: The connection of the temperature sensor to the controller 4.1.1 Temperature sensor 4.1.1 Temperature sensor The LM 35 has three pins: the power, the ground, and the voltage at the common collector, VCC. The power is connected to the power rail, the ground to the ground rail, and the VCC to the analog pin A0. The schematic of the connection is shown in Figure 3. 4.1.2 The ACM 3 Axis Accelerometer ADXL 345 is connected to the microcontroller through the breadboard. Its eight pins and connections were fixed to the board using jumper wires, from the power rail to the power pin, from the ground rail to the ground pin, and three VCC pins for the three axial space dimensions to analog input pins 1, 2, and 3. The connections are shown in Figure 4 below. The ADXL 345 has an adjustable measuring range of ±16g and a high resolution of up to 13 bits, with a sensitivity of 40mg/LSB in all ranges, equivalent to an accuracy higher than 1ᵒ [14]. This enables events, such as the detection of fast strokes and vibrations, and the detection of 0-g free fall conditions. Figure 4: ACM 3 Axis Accelerometer connection Source: https://www.arduino.cc/en/Tutorial/ADXL3xx Figure 4: ACM 3 Axis Accelerometer connection Source: https://www.arduino.cc/en/Tutorial/ADXL3xx 21 Figure 5 : Set-up connections for the controller and sensors 4.2 Configuring the Arduino microcontroller The controller is a major component of the remote monitoring device, as it interprets the signals from the sensors and controls the actuators. The Arduino Yun microcontroller is configured for two settings: the network settings and the sensor data acquisition settings, which make remote condition monitoring possible, unlike site-based on-condition monitoring. The network settings on the microcontroller are configured using wi-fi connectivity or ethernet. Security is a concern for IoT devices, so the default password for the microcontroller and for the wi-fi network for internet access is changed, as is password access. These are imperative: security attacks are problematic for IoT because of the minimal capacity of the devices being used, and the physical accessibility to sensors and actuators together with the microcontrollers at the installation sites [15]. The router The connected power sources Figure 5 : Set-up connections for the controller and sensors The router The connected power sources Figure 5 : Set-up connections for the controller and sensors The router The connected power sources The router The connected power sources Figure 5 : Set-up connections for the controller and sensors 4.2 Configuring the Arduino microcontroller 4.2 Configuring the Arduino microcontroller The controller is a major component of the remote monitoring device, as it interprets the signals from the sensors and controls the actuators. 4.1.2 The Arduino Yun microcontroller is configured for two settings: the network settings and the sensor data acquisition settings, which make remote condition monitoring possible, unlike site-based on-condition monitoring. The network settings on the microcontroller are configured using wi-fi connectivity or ethernet. Security is a concern for IoT devices, so the default password for the microcontroller and for the wi-fi network for internet access is changed, as is password access. These are imperative: security attacks are problematic for IoT because of the minimal capacity of the devices being used, and the physical accessibility to sensors and actuators together with the microcontrollers at the installation sites [15]. Configuring the microcontroller to read the values from the sensors involves coding, using the Arduino IDE. The latest IDE is downloaded and installed on the PC, and the communication port is initialised with the MAC address of the microcontroller. The code is run on the sketch page; it consists of three parts — the declaration of variables, setting up the code, and comparing the current condition with the severity level. Considering the calibration of the variables, the variables being considered are the room temperature and the three-axial vibration of the elevator car. The temperature variable name for the read signal is ‘tempIn’, which is the sensor signal in varying conditions. The power input is 5V, and the output signal ‘tempIn’ is also in voltage, which is converted to a Celsius temperature unit. The temperature in Celsius is given by the equation below: Temperature(⁰C) = (500.0tempIn)/1024.0; (1) 4.3 Comparison of current condition with the severity level The on-condition monitoring maintenance policy of machines monitors the condition of a system for the early detection of deterioration, thereby preventing a critical breakdown of the system. Unlike reactive maintenance, where actions are taken whenever a system failure occurs, on-condition monitoring maintenance checks the condition of the system against the limit threshold, which is the severity limit of each condition of the machine. The limits are determined by the machine manufacturer or through machine data learning algorithms. The excitation and vibration signature during a normal, good working condition of a system can be used as the baseline for developing severity levels from events. The condition of the system is diagnosed by reconciling the current machine condition with the severity limits, and indicating a decision for proactive maintenance before the critical breakdown of the system. This is executed using the lines of code for the sketch that executes the events and logic operations on the controller. Since the range of vibration under normal working condition is ±5mg: 𝑅𝑋+ 5𝑚𝑔≥ 𝑅𝑋 ≥ 𝑅𝑋−5𝑚𝑔 𝑅𝑋+ 5𝑚𝑔≥ 𝑅𝑋 ≥ 𝑅𝑋−5𝑚𝑔 The controller is coded to send data and a notification. The pseudocode for notification is given by: If 𝑅𝑋−5𝑚𝑔≥ 𝑅𝑋 ≥ 𝑅𝑋+ 5𝑚𝑔 OR 𝑅𝑌−5𝑚𝑔≥ 𝑅𝑌 ≥ 𝑅𝑌+ 5𝑚𝑔 OR 𝑅𝑍−5𝑚𝑔≥ 𝑅𝑍 ≥ 𝑅𝑍+ 5𝑚𝑔 OR T°C > 35°C then; send notification, ‘The system is malfunctioning’; else Stand by; (meaning the system is okay) end If. If 𝑅𝑋−5𝑚𝑔≥ 𝑅𝑋 ≥ 𝑅𝑋+ 5𝑚𝑔 OR 𝑅𝑌−5𝑚𝑔≥ 𝑅𝑌 ≥ 𝑅𝑌+ 5𝑚𝑔 OR 𝑅𝑍−5𝑚𝑔≥ 𝑅𝑍 ≥ 𝑅𝑍+ 5𝑚𝑔 OR T°C > 35°C then; send notification, ‘The system is malfunctioning’; else Stand by; (meaning the system is okay) end If. The line of code executes a logic operation that compares the current condition with the severity level as indicated by the machine manufacturer — i.e., the excitation from the mean value of each parameter. Once the severity limit is reached, a notification is sent out. Temperature(⁰C) = (500.0tempIn)/1024.0; (1) (1) The vibration parameter is read using the 3-axial ADXL 345 accelerometer, which is an analog sensor that works with the analogue to digital converter (ADC) module of the microcontroller. The output value from the accelerometer can be calibrated to values in meters per second or G-force (g). The conversion is done using the equation below; 𝑅𝑥 = (𝐴𝑑𝑐𝑅𝑥∗ 𝑉𝑟𝑒𝑓 2𝑛−1 − 𝑉𝑍𝑒𝑟𝑜 𝐺) /𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 (2) (2) where 𝐴𝑑𝑐𝑅𝑥 is the signal value from the sensor, 𝑉𝑟𝑒𝑓 is the reference voltage, which is 5V, 𝑉𝑍𝑒𝑟𝑜 𝐺 is the voltage at zero gravity, n is the number of bits of the sensor and the sensitivity of the sensor. where 𝐴𝑑𝑐𝑅𝑥 is the signal value from the sensor, 𝑉𝑟𝑒𝑓 is the reference voltage, which is 5V, 𝑉𝑍𝑒𝑟𝑜 𝐺 is the voltage at zero gravity, n is the number of bits of the sensor and the sensitivity of the sensor. 𝑅𝑋= 𝑅𝑋1+𝑅𝑋2+⋯𝑅𝑋𝑛 𝑛 (3) (3) where 𝑅𝑋RY, 𝑅𝑌, 𝑅𝑍 is the mean vibration signal on the x, y, and z axes respectively. where 𝑅𝑋RY, 𝑅𝑌, 𝑅𝑍 is the mean vibration signal on the x, y, and z axes respectively. 22 This value is determined from the first set of captured data, or from the graph. The microcontroller reads the signal from the sensor, processes it through the codes, and sends the values out for condition monitoring analysis. The bridge H, which is the protocol that listens and connects the sensor values and also responds to REST API, calls for the sensor values from the controller [16]. 6 DATA CAPTURING Brittain [19] designed a data capturing engine with automatic target data location, extraction, and storage at an interval of 15 minutes; similarly, the set-up is configured to capture and send the data to the email every 15 minutes. In the case of severity in any of the machine parameters, the message notification is sent along with the machine data. The machine experts can then respond immediately to the fault indication through remote access to the data history on the cloud (via email). The temperature and the three-axial vibrations were sent to email from the set-up at 15-minute intervals. The temperature value is read out in degrees centigrade, as calibrated through the code, while the vibration data are also calibrated by the code run by the microcontroller on the signal values from the sensor chip. The serial monitors outputs the read data from the sensors. The ‘normal’ room temperature chosen by the sensor is 270C. However, the national elevator industry states that there is a level of temperature at which there is a high chance that the controls will malfunction [20]. Therefore, elevator manufacturers mostly specify temperature limits for the machine room — typically in the 85 to 950𝐹 (30 to 350) range — which must be maintained in the controller cabinets for the proper functioning of the solid-state devices used in the control system [21]. It was crucial to evaluate the severity of the data; and so the severity limit was set to 360C. Thus, the logic statement is used, which applies the expert knowledge for decision-making to fault and condition monitoring in the elevator system. Whenever the values indicate a deviation in the normal condition of the machine, they imply that the system is faulty — hence the need for maintenance before total breakdown. 5 PROGRAMMING THE INTERNET OF THINGS USING ARDUINO YUN Programming the internet configuration of the device makes the monitoring set-up unique and remotely accessible for monitoring purposes and fault diagnoses. This is because all IoT devices adopt a mechanism to send or receive data, whether wired or wireless, via Bluetooth or cellular network, or many other routes [17]. A wireless connection is established between the microcontroller and the internet, and the email cloud service is used. The data read is sent to the email cloud through Temboo, which assists in connecting Arduino micro-controllers and Arduino- compatible devices to a vast array of web-based resources and services [18]. The third-party service configuration is in two parts: the configuration on the web page, and the configuration on the sketch environment of the Arduino IDE. The google email account is created first. The most important settings for the Gmail account to connect to the Temboo web-account are to get the app-specific password. This is peculiar to the individual email account. To get the app-specific password, it is necessary to activate the two-step verification by turning it on. An account is created on the Temboo web application, registered with the email address that has been created. The Google option is chosen, and then the ‘send email’ option is chosen. On the same page, the board type (Arduino Yun) 23 is selected, the app-specific password of the email is typed into the Temboo account set-up, and the ‘run’ button is clicked. This automatically generates lines of code that are copied and pasted into the Arduino IDE. The code or sketch also has to have some input variables, such as the data to be sent to the mail, the email address to be sent to, and the variable declaration and inclusion. The email address, username, password, and message-body are replaced with the information peculiar to this research, the email is created, and the username and Temboo-data password are generated. The header file, which is also generated from the Temboo app configuration, is copied to a separate tab on the Arduino IDE sketch, and the sketch is compiled. 7 DATA PRESENTATION The data from the remote monitoring device is captured at 10-minute intervals to show the current condition of the system and to detect any deterioration in the machine’s condition. The vibration and temperature data from the elevator is captured from the email application service. The data is represented in Figures 6, 7, 8, and 9. The interval between each recorded data set was also recorded, to note the moment of deterioration in the condition of the system. The remote condition monitoring device is employed over the period of a month for maintenance decisions and fault detection in the elevator. During any abnormality in the condition of the elevator system, there is a gradual or a sudden change in the parameters being monitored, which is an excitation in the condition of the elevator, indicating an abnormal condition. The condition subsequently returns to a normal level, which most often shows that there is a breakdown in the working condition of the machine; hence the parameter values are repeatedly the same at subsequent time intervals. Each time the machine is diagnosed to be faulty (from the parameters from the elevator), it is checked by the maintenance team and repaired. 8 RESULTS ANALYSIS The data from the remote monitoring device was captured instantaneously on the email and checked for a notification of any abnormality in the condition of the system. The condition of the elevator system can be accessed by analysing the data with any statistical tool. The data from the elevator system was represented in graph form by plotting the parameters against time using MATLAB. The graph in Figure 6 shows the temperature condition of the system being remotely monitored. The range of the temperature data of the system is 220𝐶 to 270𝐶, which falls within the normal range for the working condition of the system. Therefore, the breakdown in the elevator system will 24 not be caused by the temperature parameter, since the severity limit of this parameter, as stated earlier, is 330𝐶 to 350𝐶. Figure 6: Temperature vs time graph The vibration against the time graph on each of the axes is considered for the diagnosis and analysis of the condition of the elevator system in order to detect faults and malfunctions early, with each notification. The graph of the X-axial vibration is represented in Figure 7, the Y-axial vibration in Figure 8, and the Z-axial vibration in Figure 9. Figure 6: Temperature vs time graph The vibration against the time graph on each of the axes is considered for the diagnosis and analysis of the condition of the elevator system in order to detect faults and malfunctions early, with each notification. The graph of the X-axial vibration is represented in Figure 7, the Y-axial vibration in Figure 8, and the Z-axial vibration in Figure 9. The vibration against the time graph on each of the axes is considered for the diagnosis and analysis of the condition of the elevator system in order to detect faults and malfunctions early, with each notification. The graph of the X-axial vibration is represented in Figure 7, the Y-axial vibration in Figure 8, and the Z-axial vibration in Figure 9. Figure 7: X-axial vibration against time duration Figure 7: X-axial vibration against time duration 25 25 Figure 8: Y-axial vibration against time duration Figure 9: Z-axial vibration against time duration The elevator’s manufacturer, Schindler, states that the vibration of the elevator car should be ±5mg to its mean vibration; therefore, the condition of the system based on vibration is diagnosed, based on this provision. 8 RESULTS ANALYSIS Each excitation is an indication of a malfunction in the system that indicates immediate proactive maintenance. Four different breakdowns experienced by the elevator systems were captured during a 30-day period. The first breakdown occurred at 220 minutes, with a downtime of 96 hours; the second breakdown occurred at 650 minutes, with a downtime of four hours; the third breakdown occurred at 980 minutes; and the final breakdown at 1580 minutes. The parameters considered were the temperature and the three-axial vibration, used to diagnose the state of the elevator system, and the nature of the damage initiation. The collection and analysis of vibration signals can be used to judge the degree and type of mechanical fault, which can provide effective evidence to detect the running state and fault diagnosis of elevators [22]. In this study, the vibration parameter is considered more than temperature in diagnosing the nature of the fault and breakdown in the elevator system. This is because the season of the year in which the study was carried out was winter, which automatically erodes the likelihood of having the temperature parameter exceeding the severity limit stipulated by the elevator manufacturer. The three-axial vibration graph is considered in order to diagnose the current state of the elevator system. In order to have an efficient maintenance system, each critical point on any of the axial vibrations is considered to be a potential damage initiation. The first notification of a critical point on the x-axial vibration occurred at 120 minutes of the condition monitoring system. The system’s x-axial vibration moved from -256 mg to -251mg, and further measurements indicated a malfunctioning system until 140 minutes from the beginning of the condition monitoring. In order to have a better understanding of the condition of the system, other axial vibrations were checked at this point in the condition monitoring (120 — 140 minutes). The graph of the excitation on each axis at that time (120 —140 minutes) is represented in Figure 10. The excitation on the vibration parameter of the elevator system at this point occurred on all three axes, which indicated a damage initiation or malfunctioning of the elevator system. It could also be deduced from the combined graph in Fgure 10 that the magnitude of the excitation from the mean position of the elevator system on each of the axes was high. 8 RESULTS ANALYSIS The three-axial vibration graph is considered concurrently for better diagnosis of h f h l Th i l ib i d 257 10 Figure 8: Y-axial vibration against time duration Figure 8: Y-axial vibration against time duration Figure 9: Z-axial vibration against time duration Figure 9: Z-axial vibration against time duration The elevator’s manufacturer, Schindler, states that the vibration of the elevator car should be ±5mg to its mean vibration; therefore, the condition of the system based on vibration is diagnosed, based on this provision. The three-axial vibration graph is considered concurrently for better diagnosis of the state of the elevator system. The mean axial vibrations, 𝑅𝑋RY, 𝑅𝑌, and 𝑅𝑍 are -257 mg, 10 mg, and 2.5 mg respectively, which are evaluated from the set of data before the remote monitoring begins, using Equation 3. The first notification of excitation in the vibration signature of the system beyond the acceptable limit occurred at between 100 and 200 minutes. The excitation in the vibration parameter of the elevator system on the x-axis is meant to be between -260 mg and -250 mg, on the y-axis between 5 mg and 15 mg, and on the z-axis between 0 mg and 10 mg. The malfunctioning of the system was indicated on each of the three axial vibration parameters. The corresponding excitation on the vibration parameter for all three axes at the same time shows that there is a damage initiation in the system’s condition. This has remotely diagnosed the elevator’s condition as being faulty; so proactive maintenance is needed to restore the faulty system to its normal working condition. Remote condition monitoring through the developed IoT configured device reduces the downtime of the elevator system, avoiding delayed breakdown notifications. The corresponding data after the excitation that went beyond the normal working condition also indicated that the system had broken down, which was affirmed on-site. A constant vibration parameter value on all three axes of vibrations using the three-axial vibration sensor installed on the elevator system may, therefore, indicate a breakdown in the elevator system, or an idle state of the system. 26 Further excitations beyond the normal limit occurred at 290 minutes, with an excitation of -269 mg; at 650 minutes, with an excitation of -279 mg; at 960 minutes, with an excitation of -269 mg; and at 1500 minutes, with an excitation of -249 mg. 8 RESULTS ANALYSIS This meant that the condition of the elevator system was severe, and that the system needed to be shut down for reactive maintenance. The condition of the system became stable after 200 minutes, and all the excitation on the three- axial vibration became normal. The elevator system was checked on-site 72 minutes after the damage initiation, which was at about 272 minutes of the condition monitoring, and the system had stopped working. It could then be explained that the system had stopped after the arbitrary excitation in the vibration parameter of the system, and the subsequent data after the breakdown of the system was just the resting position of the system. This was the first breakdown experienced during the remote condition monitoring of the elevator system. The response time of the damage initiation was one hour and twelve minutes, as indicated in Table 1. Table 1: Table of downtime of elevator after optimisation Response time (hours) Repair time (hours) 1st breakdown 1.2 96 2nd breakdown 2.5 4 3rd breakdown 2 48 4th breakdown 1.5 72 Table 1: Table of downtime of elevator after optimisation The response time of the damage initiation is the time taken for the maintenance team to note the breakdown in the system and to plan reactive maintenance. This has previously been a considerably long period because of the remoteness of the elevator system from the maintenance team, and the absence of an operator on the elevator system. The IoT monitoring system was therefore used to bridge the gap created by the absence of an operator and brought the benefit of instantaneous monitoring of the machine condition for malfunctioning and breakdown, reducing or eliminating the machine downtime thanks to the quick response time of the maintenance team. The first breakdown in the elevator system took the maintenance team 96 hours to fix, as indicated in Table 1. This was as a result of the breakdown in the system caused by an obstruction on the guide rail of the elevator car. The obstruction had caused the elevator car to vibrate beyond its normal functional range on all three axes, before eventually forcing the system to stop. The data 27 during the response time of the system’s breakdown and the repair time was discarded, and the remote monitoring system was activated after the repair of the first breakdown in the elevator system. 8 RESULTS ANALYSIS Figure 11: Broken chain drive The third breakdown notification experienced by the elevator system was as a result of the chain of the cabin and landing door drive breaking. Figure 11 shows the broken chain that drives both the cabin and landing door. This fault affected the whole elevator system, as the system had been programmed to shut down once the door drive or system was faulty; thus, it was brought to a stop. The repair time for this fault in the system was 48 hours, as also shown in Table 1. The repair in this case was to replace the damaged component. Broken chain of both the cabin and the landing door drive Broken chain of both the cabin and the landing door drive Figure 11: Broken chain drive The third breakdown notification experienced by the elevator system was as a result of the chain of the cabin and landing door drive breaking. Figure 11 shows the broken chain that drives both the cabin and landing door. This fault affected the whole elevator system, as the system had been programmed to shut down once the door drive or system was faulty; thus, it was brought to a stop. The repair time for this fault in the system was 48 hours, as also shown in Table 1. The repair in this case was to replace the damaged component. The third breakdown notification experienced by the elevator system was as a result of the chain of the cabin and landing door drive breaking. Figure 11 shows the broken chain that drives both the cabin and landing door. This fault affected the whole elevator system, as the system had been programmed to shut down once the door drive or system was faulty; thus, it was brought to a stop. The repair time for this fault in the system was 48 hours, as also shown in Table 1. The repair in this case was to replace the damaged component. The fourth breakdown notification in the system occurred at 960 minutes of the remote condition monitoring system. The vibration excitation occurred on the x-axis of the system from a magnitude of -258 mg to -269 mg. There was also a little excitation, between 10 mg and 7 mg, on the y-axis of the system, but this could not independently reveal the condition of the system. 8 RESULTS ANALYSIS Figure 10: Combined vibration excitation at 120 minutes he next excitation beyond the normal range of vibration on the x-axis occurred at 290 minutes e condition monitoring. The excitation on the x-axis at this point was -269 mg, which was outsi Figure 10: Combined vibration excitation at 120 minutes Figure 10: Combined vibration excitation at 120 minutes The next excitation beyond the normal range of vibration on the x-axis occurred at 290 minutes of the condition monitoring. The excitation on the x-axis at this point was -269 mg, which was outside The next excitation beyond the normal range of vibration on the x-axis occurred at 290 minutes of the condition monitoring. The excitation on the x-axis at this point was -269 mg, which was outside 28 28 the normal vibration range on the x-axis. To check the likely condition of the system further, the other axial vibration data showed that there was also a corresponding excitation in the vibration parameter of the y-axis, but none on the z-axis. Subsequently, after 310 minutes the data of the condition monitoring became normal. A breakdown in the system occurred and was noticed after about 150 minutes, which was about 460 minutes after beginning the remote condition monitoring. The breakdown in the elevator system had occurred after the sudden excitation in the vibration parameter on both the x-axis and the y-axis. The nature of the vibration signal had suggested that the system was still running well after the sudden excitation. The fault experienced by the system was a break in one of the fuses on the controls. The response time for the breakdown as a result of this fault in the system was 150 minutes, as shown in Table 1. The downtime of the system as a result of the repair of the fault was four hours, also shown in Table 1. The next excitation in the vibration parameter on the x-axis occurred at 650 minutes of the remote condition monitoring system. The vibration on the x-axis excited from 258 mg to -279 mg. The vibration of the elevator system on both the y-axis and the z-axis were still within the normal condition at 650 minutes of remote monitoring. The response time to this malfunction in the elevator’s condition was two hours, as indicated in Table 1. The fault indication in this case had also brought the system to a stop. 8 RESULTS ANALYSIS However, when considered alongside the excitation on the x-axis, it could suggest that the system was malfunctioning. The vibration of the system on the z-axis remained within the normal range. The elevator had been brought to a stop due to the faulty assembly of the cabin door drive. This caused the cabin door to jam, and the system was automatically brought to a stop. The cabin assembly is shown in Figure 12; it engages and disengages during the closing and opening of the landing door for access to the elevator car. 29 29 Figure 12: Landing door assembly Figure 12: Landing door assembly The response time for this breakdown in the system was 90 minutes, while the repair time for the fault in the system was 72 hours. Remote condition monitoring of the elevator system using a device with IoT technology helped to lower the response time for each breakdown in the elevator system because of the instantaneous notifications. Apart from reducing the response time of the maintenance team for reactive maintenance, the remote condition monitoring device captured data through the configured sensors, which also pointed to the nature of the fault on the system. The remotely acquired vibration signals and the room temperature from the elevator system could be used to learn the signals for a fault, and to diagnose the system by adopting the cause-and-effect principle. This is used in diagnosing the state of the system, and further reduces the downtime due to troubleshooting the fault on the system. Table 2: Cause-and-effect table for the system’s breakdown Table 2: Cause-and-effect table for the system’s breakdown CAUSE EFFECT  Broken chain for landing and cabin door drives Marginal excitation on x-axis (± 21 mg)  Obstruction on the guide rails, worn out rollers, or uneven guide rails Significant excitation on all axes  x-axis (± 26mg); y-axis (± 46mg); z-axis (± 34 mg) Faulty door assembly/blocked door rail  Major excitation on x-axis (± 11 mg), and minor corresponding excitation on y-axis (± 3 mg) Faulty electronic component, power surge  Excitation on x-axis (±9 mg) and a corresponding excitation on y-axis (± 5 mg) The cause-and-effect table can also be used to predict the likely cause of a malfunction in any subsequent breakdown, based on the data captured remotely by the IoT device installed on the machine. 8 RESULTS ANALYSIS Historical data from the remote monitoring device could be analysed using the cause-and-effect technique, as shown in Table 2, to diagnose the nature of the damage to the system, thus reducing the downtime by troubleshooting the fault and carrying out the repairs. The downtime due to the response time at damage initiation has been considerably reduced by adopting remote condition monitoring, and the downtime due to repair time is dependent on the nature of the damage to the elevator system. Historical data from the remote monitoring device could be analysed using the cause-and-effect technique, as shown in Table 2, to diagnose the nature of the damage to the system, thus reducing the downtime by troubleshooting the fault and carrying out the repairs. Breakdowns and faults in the machines can therefore be picked up by networked sensors for each separate parameter, such as temperature, vibration, and acoustics. When multi-sensors of different parameters are networked on machine components for condition monitoring, this will give a broader view in order to analyse the condition using the captured data. 8 RESULTS ANALYSIS The faults experienced by the elevator system include a damaged braking system, a broken door chain, a door rail obstruction, malfunctioning controls, etc. The faults often result in a high rate of downtime due to notification of a fault by the maintenance team. Table 1 shows the downtime data of the maintenance of the elevator system after optimising the condition used by the IoT monitoring device. The downtime of the elevator at each breakdown due to delayed notification is indicated by the response time of the maintenance team. Table 1 shows the response time and the repair time at each breakdown of the elevator system after optimising the system. The downtime due to the response time after each breakdown occurs is relatively lower than the repair time. In Figure 13, the dotted bar region represents the repair time, and the shaded bar region the response time by the maintenance team. It shows the downtime graph of the system’s breakdowns before 30 implementing remote condition monitoring, while Figure 13 shows the results after implementation. It reveals that optimising the system using remote condition monitoring techniques results in a reduction in the system’s downtime due to delayed breakdown notification. implementing remote condition monitoring, while Figure 13 shows the results after implementation. It reveals that optimising the system using remote condition monitoring techniques results in a reduction in the system’s downtime due to delayed breakdown notification. Figure 13: Downtime graph of elevator’s breakdowns The downtime due to the response time at damage initiation has been considerably reduced by adopting remote condition monitoring, and the downtime due to repair time is dependent on the nature of the damage to the elevator system. Historical data from the remote monitoring device could be analysed using the cause-and-effect technique, as shown in Table 2, to diagnose the nature of the damage to the system, thus reducing the downtime by troubleshooting the fault and carrying out the repairs. 0 20 40 60 80 100 120 140 1st breakdown 2nd breakdown 3rd breakdown 4th breakdown Downtime chart after optimisation Response time Repair time Figure 13: Downtime graph of elevator’s breakdowns The downtime due to the response time at damage initiation has been considerably reduced by adopting remote condition monitoring, and the downtime due to repair time is dependent on the nature of the damage to the elevator system. 9 CONCLUSION IoT is being increasingly applied in industries, from embedded systems to the augmented automation of machines. IoT technology has made robust applications possible that are suitable for remote machine condition monitoring at remote locations using a network of sensors. This paper has explored an approach to remote condition monitoring of an elevator system, sending fault notifications for early breakdown detection, without a physical presence. This research has aimed to reduce downtime of the elevator system due to delayed notifications of a breakdown in the system, and also to monitor the historical data of the damage initiation and fault diagnosis on the system. A remote monitoring device was therefore developed, comprising mostly sensors for temperature and vibration, an IoT-enabled microcontroller, and other electronic components. The device was installed on the system at a location on the hoistway on the outer cabin roof. This helped to reduce machine downtime due to delayed response times after breakdowns in the system by sending a notification, just in time, during damage initiation. Furthermore, since the data from the machine is stored in the cloud and can be assessed remotely, this provides a faster maintenance service, as the maintenance team can access the data online and study the deterioration pattern to work out the likely nature of the damage to the system. This approach could also be adopted to monitor components or machine parts located in remote locations in machines that are difficult to 31 access by an operator while the machine is in operation. The remote condition monitoring approach using IoT technology could also be adopted in monitoring systems or work operations in hazardous environments, thereby reducing the downtime of the machine due to breakdown, and avoiding the risks posed to human life. access by an operator while the machine is in operation. The remote condition monitoring approach using IoT technology could also be adopted in monitoring systems or work operations in hazardous environments, thereby reducing the downtime of the machine due to breakdown, and avoiding the risks posed to human life. REFERENCES [1] Khazraei, K., 2011. Design, organization and implementation of a methods pool and an application systematics for condition based maintenance. Verlag Praxiswissen. Ph.D Thesis, Technische Universitat Dortmund, Germany , y [2] Heyes, E. & Spearpoint, M. 2012. Lifts for evacuation: Human behaviour considerations. Fire and Materials, 36(4), pp. 297-308. [3] Chandran, A. & Chandranandan, T. 2016. Manual elevator for material handling. International Journal of Engineering Research, 5(4), pp. 279-280 [4] Datta, J., Bera, J. & Chowdhuri, S. 2016. Development of remote monitoring analysis and reporting system for industrial machines. 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[10] Kawasakia, R. & Nishimurac, Y.H.M. 2010. Inter Noise 2010: Noise and sustainability. Paper presented at the Inter noise 2010, Portugal [11] Fiorenza, J. & Mariani, A. 2015. Improving trigger action programming in smart buildings through suggestions based on behavioral graphs analysis. Ph.D, Politecnico Milano, Available: http://hdl.handle.net/10589/115082 (Accessed 20 March 2018) p ( ) [12] Petroski, H., 2018. Success through failure: The paradox of design. Princeton University Pre [12] Petroski, H., 2018. Success through failure: The paradox of design. Princeton University Press. [13] Höller, J., Boyle, D., Karnouskos, S., Avesand, S., Mulligan, C. and Tsiatsis, V., 2014. From machine-to- machine to the internet of things (pp. 1-331). Cambridge: Academic Press. [14] Bot-Shop. 2017. Retrieved from https://www.botshop.co.za/product/adxl345-3-axis-digital-gravity- sensor-acceleration-module-tilt- sensor/?gclid=Cj0KEQjww7zHBRCToPSj c WjZIBEiQAj8il5OB9mkNmv8gCbQtXMYYD5EeIDvsXAtgTT8HLjLcSs [14] Bot-Shop. 2017. Retrieved from https://www.botshop.co.za/product/adxl345-3-axis-digital-gravity- sensor-acceleration-module-tilt- sensor/?gclid=Cj0KEQjww7zHBRCToPSj_c_WjZIBEiQAj8il5OB9mkNmv8gCbQtXMYYD5EeIDvsXAtgTT8HLjLcSs E4aAiEs8P8HAQ (Accessed on: 04 November 2017) or/?gclid=Cj0KEQjww7zHBRCToPSj_c_WjZIBEiQAj8il5OB9mkNmv8gCbQtXMYYD5EeIDvsXAtgTT8HLjLcSs iEs8P8HAQ (Accessed on: 04 November 2017) [15] Stankovic, J.A. 2014. Research directions for the internet of things. IEEE Internet of Things Journal, 1(1), pp. 3-9. pp [16] Sandhya, P. REFERENCES & Kanth, G.K. 2016. Sensor network accessing cloud services for data collection and sharing using Arduino Yun. International Journal of Scientific research, vol. 4, issue 9, pp.660-663, Sept. 2015. [17] Javed, A., 2016. 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https://openalex.org/W2055494261	https://ccforum.biomedcentral.com/counter/pdf/10.1186/cc13849	English	null	Management of Pseudomonas aeruginosa pneumonia: one size does not fit all	Critical care	2,014	cc-by	1,943	Abstract In view of the mortality associated with Pseudomonas aeruginosa (PSA) ventilator-associated pneumonia (VAP) and the frequency of inadequate initial empiric therapy, recent findings underscore the need for a different management paradigm with effective anti-pseudomonal vaccines for prophylaxis of patients at risk. The association of virulence factors is a variable that splits PSA in two phenotypes, with the possibility of adjunctive immunomodulatory therapy for management of virulent strains. We comment on recent advances in and the state of the art of PSA-VAP management and discuss a new paradigm for tailored and optimal management. Pulsed-field electrophoresis analysis performed in an ICU with a high prevalence of PSA identified the geno- types of more than 1,700 isolates [7]. Interestingly, the most frequently isolated clones were responsible for gut or skin colonization, in addition to respiratory colonization, but were only rarely associated with pneumonia. When ventilator-associated pneumonia (VAP) was present, most patients achieved clinical resolution without major conse- quences. On the other hand, non-related clones suggestive of prior colonization were associated with a very high mortality rate [7]. Most clonally related isolates caused gastric colonization before skin or respiratory tract colonization, suggesting an association with the tap water used in the administration of medication. These findings emphasize that different risk factors may be implicated depending on whether the clone is due to exogenous contamination or or as endogenous colonization from being a carrier. Therefore, conventional identifica- tion provided by the microbiology laboratory results is insufficient for assessing the patient and effective management. In the previous issue of Critical Care, Lu and colleagues [1] reported a visionary study assessing the distribution of Pseudomonas aeruginosa (PSA) serotypes in patients with ICU pneumonia and suggested differences in out- comes depending on serotypes. In this report, serotype O6 predominated, being associated with better clinical outcomes than serotype 011, which were frequently pro- ducing toxins secreted by the type III secretion system (TTSS). These findings have important implications for both clinical practice and future studies. In an international study of over 1,200 ICUs in 75 countries, the risk of infections, including those due to Pseudomonas species, was found to increase with duration of ICU stay; in addition, infection was associated with an increased risk of mortality [2]. * Correspondence: jrello@crips.es 1Critical Care Department, Hospital Vall d’Hebron, Passeig de la Vall d’Hebron, 119-129, 08035 Barcelona, Spain 2CIBERES, Recinto Hospital Joan March, Carretera Soller Km 1207110, Mallorca, Bunyola, Illes Balears, Spain Full list of author information is available at the end of the article © Rello et al.; licensee BioMed Central Ltd. The licensee has exclusive rights to distribute this article, in any medium, for 6 months following its publication. After this time, the article is available 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. 2014 Management of Pseudomonas aeruginosa pneumonia: one size does not fit all information on Pseudomonas pneumonia management. We know that one effective agent is equivalent to two [3,4] but that initial combination followed by de- escalation improves survival by reducing the risk of delay in appropriate therapy. We know that resolution of episodes with appropriate therapy is similar to core pathogens [5] but that wrong initial therapy is associated with a resolution similar to that of methicillin-resistant Staphylococcus aureus [6]. Rello et al. Critical Care 2014, 18:136 http://ccforum.com/content/18/2/136 Abstract Rello J, Ricart M, Ausina V, Net A, Prats G: Pneumonia due to Haemophilus influenzae among mechanically ventilated patients. Incidence, outcome, and risk factors. Chest 1992, 102:1562–1565. 6. Vidaur L, Planas K, Sierra R, Dimopoulos G, Ramirez A, Lisboa T, Rello J: Ventilator-associated pneumonia: impact of organisms on outcomes and medical resources utilization. Chest 2008, 133:625–633. In view of the mortality associated with PSA-VAP [3,5,12] and the frequency of inadequate initial empiric therapy [13-15], these findings underscore the need for a different management paradigm with effective anti- pseudomonal vaccines for prophylaxis of patients at risk and the need for rapid diagnostic test methods and monoclonal-specific antibodies blocking virulence fac- tors in patients with VAP. 7. Vallés J, Mariscal D, Cortés P, Coll P, Villagrá A, Díaz E, Artigas A, Rello J: Patterns of colonization by Pseudomonas aeruginosa in intubated patients: a 3-year prospective study of 1,607 isolates using pulsed-field gel electrophoresis with implications for prevention of ventilator- associated pneumonia. Intensive Care Med 2004, 30:1768–1775. 8. Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998, 62:379–433. 8. Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998, 62:379–433. 8. Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998, 62:379–433. 9. Veesenmeyer JL, Hauser AR, Lisboa T, Rello J: Pseudomonas aeruginosa virulence and therapy: evolving translational strategies. Crit Care Med 2009, 37:1777–1786. animals and plants. Microbiol Mol Biol Rev 1998, 62:379 433. 9. Veesenmeyer JL, Hauser AR, Lisboa T, Rello J: Pseudomonas aeruginosa virulence and therapy: evolving translational strategies. Crit Care Med 2009, 37:1777–1786. We have also learned that association of virulence factors is a variable that splits P. aeruginosa in two phenotypes, with the possibility of adjunctive immunomodulatory therapy for management of virulent strains [16]. A com- bination of general risk factors and molecular diagnosis techniques may identify suitable candidates for inter- vention. As in invasive pneumococcal infections [17], further research is required to identify potential associa- tions of comorbidities and serotypes as well as of sero- types and specific complications. 10. Shime N, Sawa T, Fujimoto J, Faure K, Allmond LR, Karaca T, Swanson BL, Spack EG, Wiener-Kronish JP: Therapeutic administration of anti-PcrV F (ab’)(2) in sepsis associated with Pseudomonas aeruginosa. J Immunol 2001, 167:5880–5886. 11. Author details 1C i i l C D 17. Luján M, Burgos J, Gallego M, Falcó V, Bermudo G, Planes A, Fontanals D, Peghin M, Monsó E, Rello J: Effects of immunocompromise and comorbidities on pneumococcal serotypes causing invasive respiratory infection in adults: implications for vaccine strategies. Clin Infect Dis 2013 57:1722–1730. 1Critical Care Department, Hospital Vall d’Hebron, Passeig de la Vall d’Hebron, 119-129, 08035 Barcelona, Spain. 2CIBERES, Recinto Hospital Joan March, Carretera Soller Km 1207110, Mallorca, Bunyola, Illes Balears, Spain. 3Vall d’Hebron Institute of Research, Passeig de la Vall d’Hebron, 119-129, 08035 Barcelona, Spain. 4Universitat Autònoma de Barcelona, Plaça Cívica, Campus de la UAB,Sardañola del Vallés, 08193 Barcelona, Spain. Cite this article as: Rello et al.: Management of Pseudomonas aeruginosa pneumonia: one size does not fit all. Critical Care 10.1186/cc13849 2014, 18:136 Competing interests h d d JR has served on advisory boards or speakers bureau (or both) for Kenta Biotech (Zürich-Schlieren, Switzerland), Astellas (Tokyo, Japan), Pfizer Inc. (New York, NY, USA), KaloBios (South San Francisco, CA, USA), Clinigen (Burton-on-Trent, Staffordshire, UK), Roche (Basel, Switzerland), and Bayer (Leverkusen, Germany) and has received research grants from Sanofi Pasteur (Paris, France) and Cubist (Lexington, MA, USA). The other authors declare that they have no competing interests. 15. Rello J, Allegri C, Rodriguez A, Vidaur L, Sirgo G, Gomez F, Agbaht K, Pobo A, Diaz E: Risk factors for ventilator-associated pneumonia by Pseudomonas aeruginosa in presence of recent antibiotic exposure. Anesthesiology 2006, 105:709–714. 16. Van Delden C, Kohler T, Brunner-Ferber F, Francois B, Carlet J, Pechere JC: Azithromycin to prevent Pseudomonas aeruginosa ventilator- associated pneumonia by inhibition of quorum sensing: a randomized controlled trial. Intensive Care Med 2012, 38:1118–1125. Abbreviations PSA P d Abbreviations PSA: Pseudomonas aeruginosa; TTSS: Type III secretion system; VAP: Ventilator- associated pneumonia. 14. Hurley JC: Paradoxical ventilator-associated pneumonia incidences among selective digestive decontamination studies versus other studies of mechanically ventilated patients: benchmarking the evidence base. Crit Care 2011, 15:R7. PSA: Pseudomonas aeruginosa; TTSS: Type III secretion system; VAP: Ventilator- associated pneumonia. Abstract El Solh AA, Akinnusi ME, Wiener-Kronish JP, Lynch SV, Pineda LA, Szarpa K: Persistent infection with Pseudomonas aeruginosa in ventilator- associated pneumonia. Am J Respir Crit Care Med 2008, 178:513–519. 12. Crandon JL, Ariano RE, Zelenitsky SA, Nicasio AM, Kuti JL, Nicolau DP: Optimization of meropenem dosage in the critically ill population based on renal function. Intensive Care Med 2011, 37:632–638. 13. Venier AG, Gruson D, Lavigne T, Jarno P, L’Hériteau F, Coignard B, Savey A, Rogues AM, REA-RAISIN group: Identifying new risk factors for Pseudomonas aeruginosa pneumonia in intensive care units: experience of the French national surveillance, REA-RAISIN. J Hosp Infect 2011, 79:44–48. Abstract In 2014, at a time when multidrug-resistant clones are emerging and represent a strong risk of dissemination, we have much more Indeed, recent advances have demonstrated the im- portance of virulence factors in PSA infections. Al- though several different mechanisms such as quorum sensing and biofilm formation have been reported [8], the TTSS, encoded by PSA, has become one of the most important and widely studied virulence factors. After the microorganism has come into contact with the cell, the needle-like TTSS mechanism allows the bacteria to inject toxins directly into the cytoplasm of the host cell * Correspondence: jrello@crips.es 1Critical Care Department, Hospital Vall d’Hebron, Passeig de la Vall d’Hebron, 119-129, 08035 Barcelona, Spain 2CIBERES, Recinto Hospital Joan March, Carretera Soller Km 1207110, Mallorca, Bunyola, Illes Balears, Spain Full list of author information is available at the end of the article Page 2 of 2 Rello et al. Critical Care 2014, 18:136 http://ccforum.com/content/18/2/136 Rello et al. Critical Care 2014, 18:136 http://ccforum.com/content/18/2/136 [9], evading direct recognition by the host’s immune system [10]. Recent studies suggest that failure to eradi- cate PSA in patients with VAP may be linked to TTSS. Patients infected with Pseudomonas sp. strains which express at least one type of TTSS protein (TTSS+) at the onset of VAP are more likely to have recovered at day 8 post-VAP, whereas eradication is achieved in patients with undetectable levels of TTSS proteins [11]. The transfer of our knowledge of the virulence factors to the clinical setting is crucial in order to evaluate the poten- tial of virulence factor-directed therapies. International study of the prevalence and outcomes of infections in intensive care units. JAMA 2009, 302:2323–2329. International study of the prevalence and outcomes of infections in intensive care units. JAMA 2009, 302:2323–2329. International study of the prevalence and outcomes of infections in intensive care units. JAMA 2009, 302:2323–2329. 3. Tumbarello M, Sali M, Trecarichi EM, Leone F, Rossi M, Fiori B, De Pascale G, D’Inzeo T, Sanguinetti M, Fadda G, Cauda R, Spanu T: Clinical outcomes of Pseudomonas aeruginosa pneumonia in intensive care unit patients. Intensive Care Med 2013, 39:682–692. 4. Garnacho-Montero J, Sa-Borges M, Sole-Violan J, Barcenilla F, Escoresca- Ortega A, Ochoa M, Cayuela A, Rello J: Optimal management therapy for Pseudomonas aeruginosa ventilator-associated pneumonia: an observational, multicenter study comparing monotherapy with combination antibiotic therapy. Crit Care Med 2007, 35:1888–1895. 5. References 1. Lu Q, Eggimann P, Luyt CE, Wolf M, Tamm M, François B, Merceir E, Garbino J, Laterre PF, Koch H, Gafner V, Rudolf MP, Mus E, Perez A, Lazar H, Chastre J, Rouby JJ: Pseudomonas aeruginosa serotypes in nosocomial pneumonia: prevalence and clinical outcomes. Crit Care 2014, 18:R17. 2. Vincent JL, Rello J, Marshall J, Silva C, Anzueto A, Martin CD, Moreno R, Lipman J, Gomersall C, Sakr Y, Reinhart K, EPIC II Group of Investigators: 1. Lu Q, Eggimann P, Luyt CE, Wolf M, Tamm M, François B, Merceir E, Garbino J, Laterre PF, Koch H, Gafner V, Rudolf MP, Mus E, Perez A, Lazar H, Chastre J, Rouby JJ: Pseudomonas aeruginosa serotypes in nosocomial pneumonia: prevalence and clinical outcomes. Crit Care 2014, 18:R17. 2. Vincent JL, Rello J, Marshall J, Silva C, Anzueto A, Martin CD, Moreno R, Lipman J, Gomersall C, Sakr Y, Reinhart K, EPIC II Group of Investigators:
https://openalex.org/W2406508073	https://www.scientific.net/MSF.854.3.pdf	English	null	Effect of Parameters in the Physical Simulated Rough Rolling Stage on Microstructure Evaluation and Tensile Properties of a Bainitic Pipeline Steel	Materials science forum	2,016	cc-by	2,897	Effect of Parameters in the Physical Simulated Rough Rolling Stage on Microstructure Evaluation and Tensile Properties of a Bainitic Pipeline Steel Mohamed Soliman1, a *, Heinz Palkowski1,b 1 Institute of Metallurgy, Clausthal University of Technology, Robert-Koch-Straße 42, 38678 Clausthal-Zellerfeld, Germany amohamed.soliman@tu-clausthal.de, bheinz.palkowski@tu-clausthal.de Keywords: Bainitic pipeline steel; hot working parameters; thermo-mechanical simulation; microstructure evolution. Abstract. Microstructure evolution and tensile properties were studied in a bainitic pipeline steel grade by performing a number of physical simulations on samples machined out of an industrially produced transfer bar. In these simulations, the cooling interval between roughing and finishing stages (tV) was varied from 5 s to 180 s. The austenite status after this cooling interval, regarding the prior austenite grain size and precipitates, simulates the condition of austenite before entering the finishing mill. The finishing parameters and the subsequent cooling strategy were kept unchanged throughout all the applied simulation processes. The gradual increase in tV resulted in a gradual increase of the granular bainite phase on the expense of the aciculare ferrite. This resulted in an incremental decrease in ultimate tensile strength and yield strength with increasing tV. However, this behavior approached a steady state condition after which the tV has limited/insignificant effect on the ultimate- and yield strength. This saturating value of tV is process parameter dependent. This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0) Materials Science Forum Materials Science Forum Submitted: 2015-09-22 Revised: 2015-12-15 Accepted: 2015-12-16 Online: 2016-05-17 Submitted: 2015-09-22 Revised: 2015-12-15 Accepted: 2015-12-16 Online: 2016-05-17 ate ials Science o um ISSN: 1662-9752, Vol. 854, pp 3-8 , , pp i:10.4028/www.scientific.net/MSF.854.3 2016 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Experimental Procedure Material and Specimens Preparation. The current study is carried out on samples machined out of a transfer bar of API X80 pipeline steel. Salzgitter Flachstahl GmbH is acknowledged for providing the raw material. The chemical composition of the studied material is given in Table 1. Table 1. Chemical analysis of the studied material (wt. %) Table 1. Chemical analysis of the studied material (wt. %) C Si Mn P N Cr Mo Ti Nb S 0.055 0.3 1.84 0.014 0.006 0.18 0.259 0.0256 0.101 0.0008 The provided steel slab has a thickness of 52 mm. All the specimens were taken with their longitudinal axes parallel to the rolling direction of the transfer bar and their thicknesses parallel to its thickness. The thickness-center region is excepted during machining of the specimens that is to avoid the zone of central segregation of the slab. The dimensions of the flat compression samples are shown in Fig. 1. The thickness of the specimen in the testing-zone is 6.4 mm. The specimen has 42 mm shoulders for clamping in the tensile testing machine after thermomechanical processing. The two Ø 6 mm holes are for reducing the heat dissipation from the testing- zone to the shoulders. All the simulation specimens are taken with their longitudinal axes parallel to the rolling direction and their thicknesses parallel to the thickness of the transfer bar. Fig. 1. Dimensions of the flat compressing sample 10 Fig. 1. Dimensions of the flat compressing sample TM simulator TTS 820 is used for carrying out the simulation process. For this purpose, a thermocouple is spot welded on the specimen, and then the specimen is placed on two ceramic rollers and fixed from the upper side by two ceramic rods. Two deformation stamps upset the specimen in its center. A detailed description of the flat compression setup of TTS820 is given in [7]. Thermo-mechanical Simulation. The samples with a geometry shown in Fig. 1 are subjected to the TM schedule sketched in Fig. 2. In this schedule, specimens were heated up to the austenitization temperature (TA) and subjected to one deformation step with a true strain value of ϕv at TV. The austenite status at this stage - regarding the prior austenite grain size (PAGS) and precipitation - simulates the condition of austenite after the roughing process. Introduction The demand of the pipeline industry for a more cost-effective pipeline design has pushed the standard pipeline steel grade requirements. Critical to the design of these steels is a low carbon equivalent for good field weldability [1, 2]. In these steels, the carbon is reduced to below 0.09 wt.%. The strength loss due to the low C content is compensated through alloy design philosophy based on the advanced use of cost effective micro-alloying elements, such as Nb, Ti and B in conjunction with moderate levels of other alloying elements, such as Mn, Si, Cr, Mo and Cu [3]. The use of aforementioned combinations of micro-alloying and alloying elements in conjunction with thermo-mechanical controlled processing (TMCP) lead to the development of API X80, X100 and X120 which exhibit yield strengths from 550 MPa up to 825 MPa [4]. In these steels, the desired balance of mechanical properties at a given steel composition are achieved through suitably designed thermo-mechanical processing schedules [5], which commonly involve controlled rolling, followed by controlled accelerated cooling. The controlled rolling compresses two stages, namely roughing and finishing rolling. Roughing starts after the austenitization process. During the rough rolling the austenite grain size is refined due to repeated cycles of work hardening and the recrystallization process. The finishing rolling starts subsequent to the roughing. During the finishing rolling the austenite is deformed in the non-recrystallization temperature regime, which brings significant refinement to the final microstructure. The accelerated cooling step aims to suppress the formation of polygonal ferrite and, instead, encourage non-equilibrium, non-equiaxed ferrite microstructures to be formed. The latter transformation products are known to contribute to increasing strength, through both small effective grain sizes and increased dislocation densities, while maintaining a reasonable level of toughness [1, 5, 6]. Objective. It was shown in [7] that decreasing the delay-time between the roughing and finishing rolling stages (tV) from 180 s to 5 s resulted in pronounced improvement in both of ultimate tensile strength and proof stress. During the current work the same steel is processed under the same Production and Further Processing of Flat Products 4 thermo-mechanical (TM) processing parameters except that and intermediate values of tV between 5 s and 180 s are selected. The effect of the cooling time on the microstructure development and the mechanical properties is investigated. Introduction thermo-mechanical (TM) processing parameters except that and intermediate values of tV between 5 s and 180 s are selected. The effect of the cooling time on the microstructure development and the mechanical properties is investigated. Experimental Procedure The subsequent three deformation steps are to simulate the finishing rolling process, the time between roughing and finishing is designated in the figure by tV. The studied parameters are varied according to the values listed in Table 2. The finishing rolling parameters and the subsequent cooling strategy were kept unchanged throughout all the applied simulation processes. The parameters in Table 2 are considered for varying the austenite status before entering the finishing mill. Two values of tV were Materials Science Forum Vol. 854 5 investigated in reference [7], namely 5 s and 180 s. The results of this project showed a strong dependence of the ultimate and proof strength on tV. During the current study, additional intermediate values for tV were investigated, written in italic font-style in Table 2. investigated in reference [7], namely 5 s and 180 s. The results of this project showed a strong dependence of the ultimate and proof strength on tV. During the current study, additional intermediate values for tV were investigated, written in italic font-style in Table 2. Fig. 2. Schematic drawing of the applied thermo-mechanical schedule. 500 °C Time Temperature TA TV (920 °C, 0.33) Simulation of the finishing rolling 30 K/h ϕV 10 K/s tV 600 s (860 °C, 0.26) (840 °C, 0.16) 2 s 10 K/s (T, ϕ ) 5 K/s Simulation of the finishing rolling Fig. 2. Schematic drawing of the applied thermo-mechanical schedule. Table 2. Combination of parameters studied (Fig. 2) TA (°C) 1285 1185 TV (°C) 1000 1100 1000 ϕV (-) 0.3 0.5 0.3 0.5 0.3 0.5 tV (s) 5 30 60 120 180 5 60 180 5 60 180 5 60 180 5 60 180 5 60 180 Table 2. Combination of parameters studied (Fig. 2) Light Optical Microscopy. Light optical microscopic (LOM) analysis of the as-received samples as well as samples from various processing and conditions was performed by sectioning the samples parallel to the deformation direction, and cold mounting. The samples were rough polished using standard metallographic abrasive grinding papers ranging from coarse (180) to fine (1200). The final polishing was done using 1.0 µm and 0.05 µm alumina, respectively. After polishing, the samples were rinsed with ethyl alcohol and dried. The microstructure was developed by etching with 2 % Nital. Tensile Testing. Experimental Procedure The tensile tests were conducted in a computerized universal testing machine (UTS) with a 250 kN load cell using a crosshead speed of 5 mm/min. Results and Discussion Microstructure Evolution. Metallographic investigations using LOM were conducted to investigate the effect of tV on the microstructure development. TA = 1250 °C – TV = 1000 °C - ϕV = 0.3. The effects of tV on the microstructure for the samples austenitized at TA = 1250 °C and deformed at TV = 1000 °C with ϕV = 0.3 is shown in Fig. 3. The micrographs of Fig. 3 show that the (Nb, Ti) (C, N) precipitates are well distributed in all microstructures. The composition of these precipitates was investigated by using energy dispersive X-ray spectroscopy (EDX) [7]. TiN precipitates form at higher temperature in the austenite region. These precipitates serve as “cores” for the nucleation and epitaxial growth at lower temperatures of shell of NbCN [8]. The obtained precipitates have an average size of about 132 nm. The addition of Nb and Ti to pipeline steels effectively refines the austenite grain during the hot-rolling process because the precipitates retard austenite recrystallization and, in turn, refine the final microstructure. This microstructure refinement together with the existence of nano-size phase, which is the Production and Further Processing of Flat Products Production and Further Processing of Flat Products 6 6 precipitates themselves result in enhancing the mechanical properties of the pipeline steel. The microstructure is predominantly a mixture of acicular ferrite (AF) and granular bainite (GB). The microstructures for the samples having tV = 180 s (Fig. 3e) is dominated by the GB structure. For tV = 5 s (Fig. 3a), the microstructures show more AF and finer GB than that obtained for tV = 180 s. The domination of the GB structure is also observed in the microstructures of the samples with tV = 60 s and 120 s. The sample with tV = 30 s shows more or less similar microstructural features to that for tV = 5 s. The very tiny phase, e.g. the encircled phase in Fig. 3, is defined as a martensite/austenite (M/A) phase; this is confirmed by scanning electron microscopic investigations as shown in [7]. The occurrence of tiny martensite/austenite (M/A) phase is more pronounced for tV = 5 s and 30 s than for tV = 60 s, 120 s and 180 s. The shorter cooling time between the roughing and finishing resulted in finer and/or pancaked prior austenite grains which motivated the formation of both, AF and fine M/A phases. Materials Science Forum Vol. 854 7 Mechanical Properties. Fig. 5 shows the effect of tV on the stress-strain curves for different TM treatment conditions. The incremental increase in tV generally results in an incremental decrease in ultimate tensile strength (Rm) and yield strength (Rp). e.g. 840 MPa was the highest recorded Rm value of the steel with TA = 1250 °C - TV = 1000 °C - ϕV = 0.5 when tV = 5 s. The lowest Rm value of 692 MPa was recorded for the same condition but with tV = 180 s. However, it seems that this behavior has a saturation point after which tV has a limited/insignificant effect on Rm and Rp. The saturation point for large prior austenite grains (TA = 1250 °C) deformed at a low temperature (TV = 1000 °C) is not reached at tV = 60 s (Fig. 5a and 5b) but rather at tV = 120 s (Fig. 5a). On the other hand, a value of 60 s for tV was enough to attain the saturation point for large prior austenite grains deformed at high temperature (Fig. 5b and 5e). For smaller PAG (TA = 1150 °C), ϕV is deceive for the saturating tV; for ϕV = 0.3 a tV of 60 s was enough for attaining saturation in Rm and Rp values (Fig. 5c). Higher tV is required to attain this saturation for ϕV = 0.5 (Fig. 5f). Fig. 5 Stress-strain curves of samples processed under the prescribed TM treatment conditions. (c) (d) (e) (f) (a) (b) (c) (a) (b) (a) (a) (b) (c) (d) (a) (c) (f) (e) ( ) ( ) (e) (f) ( ) (f) (e) (d) Fig. 5 Stress-strain curves of samples processed under the prescribed TM treatment conditions. Results and Discussion TA = 1150 °C – TV = 1000 °C - ϕV = 0.3. A similar effect of tV on the phase distribution of AF and GB in the samples with TA = 1250 °C is observed in samples with TA = 1150 °C (see Fig. 4). Explicitly, increasing tV to 180 s resulted in the domination of the GB phase. Furthermore, for tV = 60 s, the samples showed similar features compared to samples with tV = 180 s rather than to that with tV = 5 s. Fig. 3. Effect of tV on the microstructure for samples austenitized at TA = 1250 °C and deformed at TV with ϕV = 0.3. Etchant: Nital. (a) 5 s GB AF GB (Nb, Ti) (C, N) (b) 30 s (c) 60 s (e) 180 s (d) 120 s GB AF GB (Nb, Ti) (C, N) (a) 60 s (a) (e) 180 s GB AF GB (Nb, Ti) (C, N) (d) AF Fig. 3. Effect of tV on the microstructure for samples austenitized at TA = 1250 °C and deformed at TV with ϕV = 0.3. Etchant: Nital. Fig. 4. Effect of tV on the microstructure for the samples austenitised at TA = 1150 °C and deformed at TV = 1000 °C with ϕV = 0.3. Etchant: Nital. (a) 5 s (b) 60 s (c) 180 s (b) 60 s (a) 5 s (a) 180 s (b) Fig. 4. Effect of tV on the microstructure for the samples austenitised at TA = 1150 °C and deformed at TV = 1000 °C with ϕV = 0.3. Etchant: Nital. Materials Science Forum Vol. 854 Summary Microstructure evolution and tensile properties were studied in a pipeline steel grade API-X80 by performing a number of physical simulations on samples machined out of an industrially produced transfer bar. In this physical simulation, specimens were heated up to the austenitization temperature (TA) and subjected to one deformation step having a true strain value of ϕV at TV. The cooling interval between roughing and finishing is designated by (tV). The austenite status after this cooling interval, regarding the prior austenite grain size (PAGS) and precipitates simulates the condition of austenite before entering the finishing mill. The finishing parameters and the subsequent cooling strategy were kept unchanged throughout all the applied simulation processes. The results showed a strong dependence of the Rm and Rp on tV. The gradual increase in tV results in a gradual increase of the granular bainite phase on the expense of the aciculare ferrite. This results in an incremental decrease in Rm and Rp with increasing tV. However, it seems that this behavior has a saturation point after which the tV has a limited/insignificant effect on the Rm and Rp. This saturating value of tV is process parameter dependent. Production and Further Processing of Flat Products 8 References [1] J.G. Williams, C.R. Killmore, F.J. Barbaro, A. Meta, L. Fletcher, Modern technology for ERW linepipe steel production (X60 to X80 and beyond), Proc. Int. Conf. Microalloying`95. Warrendale, USA (1995) 117-139. [2] P. Cizek, Transformation behaviour and microstructure of an API x80 line-pipe steel subjected to simulated thermomechanical processing, Metal 2001, Ostrava, Czech Republic (2001). [3] P. Suikkanen, Development and Processing of Low Carbon Bainitic Steels, Academic dissertation, Acta Univ. Oul., OulunYliopisto, Oulu 2009. [4] API Specification 5L: Specification for Linepipe, ISO 3183:2007 (Modified), petroleum and natural gas industries-Steel pipe for pipeline transportation systems, forty-fourth edition, October 2007 [5] I. Tamura, H Sekine., T. Tanaka, C.Ouchi, Thermomechanical Processing of High-Strength Low-Alloy Steels, Butterworth & Co. Ltd., London, 1988. [6] B.P. Wynne, P. Cizek, C.H.J. Davies, B.C. Muddle, P.D. Hodgson, Effects of processing parameters on the mechanical properties of low-carbon microalloyed steels, In Proc. Int. Conf. THERMEC`97. Warrendale, USA: TMS, 1997, 837-843. [7] M. Soliman, H. Palkowski, Influence of hot working parameters on microstructure evolution, tensile behavior and strain aging potential of bainitic pipeline steel, Materials & Design 87 (2015) 450–465. [8] M. Gomez, P. Valles, S.F. Medina, Evolution of microstructure and precipitation state during thermomechanical processing of a X80 microalloyed steel, Materials Science and Engineering, 2011, Vol. A 528, 4761-4773. [8] M. Gomez, P. Valles, S.F. Medina, Evolution of microstructure and precipitation state during thermomechanical processing of a X80 microalloyed steel, Materials Science and Engineering, 2011, Vol. A 528, 4761-4773.
https://openalex.org/W2738667316	https://air.unimi.it/bitstream/2434/548773/2/seven%20years.pdf	English	null	Seven Years Cognitive Functioning and Early Assessment in Extremely Low Birth Weight Children	Frontiers in psychology	2,017	cc-by	7,607	Abbreviations: AGA/SGA, adequate/small for gestational age; BPD, bronchopulmonary dysplasia; CI, conﬁdence interval; ELBW, extremely low birth weight; IQ, intelligence quotient; IVH, intraventricular hemorrhage; MRI, magnetic resonance imaging; OR, odds ratio; PVL, periventricular leukomalacia; ROP, retinopathy of prematurity; SD, standard deviation; WISC- III, Wechsler Intelligence Scale for Children Third Edition. Edited by: Nuno Barbosa Rocha, Health School - P. Porto, Portugal Reviewed by: Gianluca Lista, Ospedale dei Bambini Vittore Buzzi, Italy Nicola Pitchford, University of Nottingham, United Kingdom Correspondence: Chiara Squarza chiara.squarza@mangiagalli.it Edited by: Nuno Barbosa Rocha, Health School - P. Porto, Portugal Reviewed by: Gianluca Lista, Ospedale dei Bambini Vittore Buzzi, Italy Reviewed by: Gianluca Lista, Ospedale dei Bambini Vittore Buzzi, Italy Nicola Pitchford, University of Nottingham, United Kingdom Correspondence: Chiara Squarza chiara.squarza@mangiagalli.it Specialty section: This article was submitted to Clinical and Health Psychology, a section of the journal Frontiers in Psychology Keywords: extremely low birth weight, school outcome, early assessment, Grifﬁths Scales, cognitive functioning Chiara Squarza1, Odoardo Picciolini1, Laura Gardon1, Maura Ravasi1, Maria L. Giannì1, Matteo Porro1, Matteo Bonzini2, Silvana Gangi1 and Fabio Mosca1 Exclusion criteria were genetic abnormalities, severe neurofunctional impairment, and/or neurosensory disabilities. Ninety-nine children were assessed at 1 year of corrected age using the Grifﬁths Mental Development Scales Revised. The same children were re-assessed at school age through the Wechsler Intelligence Scale for Children. Children with impaired Grifﬁths General Quotient (i.e., <1 SD) at 1 year of corrected age showed a signiﬁcantly lower Full Scale Intelligence Quotient at 7 years of chronological age when compared to children who scored in the normal range at 1 year (p < 0.01). Considering the Grifﬁths Sub-quotients separately, a poor score in the Performance or in the Personal-Social Sub-quotients at 1 year was associated with signiﬁcantly worse cognitive outcomes both in the Verbal and in the Performance Intelligence Quotients at 7 years (p < 0.01 and p < 0.05, respectively). A score <1 SD in the Locomotor or in the Eye and Hand Coordination Sub-quotients were speciﬁcally associated with poorer Performance or Verbal Intelligence Quotients, respectively (p < 0.05). Our ﬁndings suggest that a poor score on the Grifﬁths Scales at 1 year is associated with a higher risk of cognitive impairment at school age. Larger conﬁrmation studies are needed. INTRODUCTION Received: 05 May 2017 Accepted: 10 July 2017 Published: 21 July 2017 Improvements in medical knowledge and techniques for high-risk infants have progressively reduced the rates of mortality and major sequelae in preterm infants, especially with ELBW (<1000 g) (Doyle et al., 2011; Latini et al., 2013). At the same time, an increased risk for long-term minor neurobehavioral and cognitive deﬁcits has been reported (Marlow et al., 2005). Keywords: extremely low birth weight, school outcome, early assessment, Grifﬁths Scales, cognitive functioning Chiara Squarza1, Odoardo Picciolini1, Laura Gardon1, Maura Ravasi1, Maria L. Giannì1, Matteo Porro1, Matteo Bonzini2, Silvana Gangi1 and Fabio Mosca1 Chiara Squarza1*, Odoardo Picciolini1, Laura Gardon1, Maura Ravasi1, Maria L. Giannì1, Matteo Porro1, Matteo Bonzini2, Silvana Gangi1 and Fabio Mosca1 1 Neonatal Intensive Care Unit, Department of Clinical Sciences and Community Health, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy, 2 Protection and Promotion of Workers Health Unit, Department of Clinical Sciences and Community Health, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Infants born preterm are at high risk for the onset of cognitive dysfunctions at school age. The aim of this study was to investigate the association between early neurodevelopmental assessment and the risk of adverse cognitive outcome in extremely low birth weight children. We enrolled all newborns (January 2002 – April 2007) consecutively admitted to our Institution, with a birthweight < 1000 g. Exclusion criteria were genetic abnormalities, severe neurofunctional impairment, and/or neurosensory disabilities. Ninety-nine children were assessed at 1 year of corrected age using the Grifﬁths Mental Development Scales Revised. The same children were re-assessed at school age through the Wechsler Intelligence Scale for Children. Children with impaired Grifﬁths General Quotient (i.e., <1 SD) at 1 year of corrected age showed a signiﬁcantly lower Full Scale Intelligence Quotient at 7 years of chronological age when compared to children who scored in the normal range at 1 year (p < 0.01). Considering the Grifﬁths Sub-quotients separately, a poor score in the Performance or in the Personal-Social Sub-quotients at 1 year was associated with signiﬁcantly worse cognitive outcomes both in the Verbal and in the Performance Intelligence Quotients at 7 years (p < 0.01 and p < 0.05, respectively). A score <1 SD in the Locomotor or in the Eye and Hand Coordination Sub-quotients were speciﬁcally associated with poorer Performance or Verbal Intelligence Quotients, respectively (p < 0.05). Our ﬁndings suggest that a poor score on the Grifﬁths Scales at 1 year is associated with a higher risk of cognitive impairment at school age. Larger conﬁrmation studies are needed. Infants born preterm are at high risk for the onset of cognitive dysfunctions at school age. The aim of this study was to investigate the association between early neurodevelopmental assessment and the risk of adverse cognitive outcome in extremely low birth weight children. We enrolled all newborns (January 2002 – April 2007) consecutively admitted to our Institution, with a birthweight < 1000 g. ORIGINAL RESEARCH published: 21 July 2017 doi: 10.3389/fpsyg.2017.01257 Citation: Squarza C, Picciolini O, Gardon L, Ravasi M, Gianní ML, Porro M, Bonzini M, Gangi S and Mosca F (2017) Seven Years Cognitive Functioning and Early Assessment in Extremely Low Birth Weight Children. Front. Psychol. 8:1257. doi: 10.3389/fpsyg.2017.01257 July 2017 \| Volume 8 \| Article 1257 1 Frontiers in Psychology \| www.frontiersin.org Early Assessment and Cognitive Outcome Squarza et al. Very preterm birth (i.e., born < 32 weeks’ gestational age) and ELBW are most often co-occurring conditions. prevention and rehabilitation (Orton et al., 2009). However, few studies have addressed the association between early neurodevelopmental quotients and cognitive outcomes at school age. Very preterm infants with ELBW may experience a disruption of important processes involved in early brain development, as the last trimester of pregnancy is an essential period for the creation and organization of neuronal connections, underlying thinking, learning, and feeling (deRegnier, 2008). In this context, our study focuses on the association between multiple neurodevelopmental skills assessed at 1 year and separate domains of intelligence at 7 years, thus suggesting some insight about the complex integration of speciﬁc developmental abilities and their longitudinal association. Pioneering brain-scanning studies support the idea that altered networks play a part in cognitive problems of preterm and ELBW infants. Hüppi (2010) found that, compared with children born at term, the premature children’s neuronal tracts were organized less eﬃciently, often taking a more meandering path. These changes in organization were correlated with reduced social and cognitive skills. In current literature, there is mixed evidence about the predictive validity of the Griﬃths Mental Developmental Scales (Griﬃths and Huntley, 1996), a neurodevelopmental assessment tool commonly administered in clinical and research practice. Sutcliﬀe et al. (2010), considering a sample of infants born at term and with normal birth-weight, reported that the Griﬃths Scales, completed at 17 months, had a signiﬁcant predictive power for Performance IQ and Full Scale IQ but not for the Verbal IQ at age 5. Other studies suggest that the General Quotients obtained at 2 and 3 years of age with the Griﬃths Scales strongly correlate with intellectual ability at 5 years assessed through the Stanford Binet in a cohort of ELBW infants (Bowen et al., 1996). The validity of the Griﬃths Scales is also suggested by its strong agreement with the Bayley Scales (Picciolini et al., 2015). Citation: An estimated 40–70% of preterm ELBW children have been identiﬁed with minor impairments, such as borderline-to-low average IQ, mild motor problems, and poor adaptive behavior during preschool and school years (Allen, 2008), while the expected rates of mild impairment in a population of normal birth weight children would be around 16%. Citation: In addition, the premature birth triggers an emotional crisis in parents, mainly as a result of their concern for the infant’s survival, the frequent invasive treatment in the NICU, and the protracted separation from the child. The alteration of the parents’ role can aﬀect the way they perceive their child and respond to him (Holditch-Davis and Miles, 1997; Allen et al., 2004). Several studies suggest that the challenge of premature birth struggles the maternal responsiveness, thus reducing social skills and cognitive development among preterm ELBW children (Landry et al., 1997). The lengthy hospital stay and the separation from their parents hinders the parent–infant bond, reducing infant self-regulation (Bhutta et al., 2002; Clark et al., 2008; Wolke et al., 2014). The aim of this study was to investigate the extent to which Griﬃths’ neurodevelopmental quotients at 1 year of corrected age are associated with cognitive functioning at 7 years of chronological age in a cohort of children born very preterm with ELBW. These factors must be taken into account as they might have damaging repercussions on the global development of the preterm ELBW infant. We speculate that the deprivation of a critical period of rapid intrauterine growth, the altered neuronal connections and the clinical, emotional, and environmental challenges cited above (in particular, the lack of social and motor experiences due to the lengthy hospitalization and the protracted separation from parents) might signiﬁcantly inﬂuence the early neurodevelopmental pathways of premature infants with ELBW. In this perspective, we hypothesize that motor and emotional skills could be the most aﬀected domains during the ﬁrst year of life. Longitudinal studies demonstrate that children born extremely preterm and/or ELBW are at increased risk for neurobehavioral impairments, including cognitive deﬁcits, learning disabilities, and behavioral and emotional problems at school age (Hack et al., 2009; Anderson and Doyle, 2003). Diﬃculties in these areas have been related with academic struggles and higher rates of special education support (Msall, 2012). Indeed, self-regulation is generally considered an integrated ability of cognitive skills, that reﬂects functional performance (Howe et al., 2016). Secondly given the deep integration among intellectual and neuropsychological skills in early infancy, we hypothesize that these early impairments should have long term eﬀects on the cognitive maturation of these high-risk infants. Frontiers in Psychology \| www.frontiersin.org MATERIALS AND METHODS We performed a single-center longitudinal cohort study. The study design was approved by the Ethics Committee of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and written informed consent was obtained from all the infants’ parents. A complex set of factors have been associated to developmental outcomes of preterm ELBW children. Among the strongest predictors of later cognitive outcome for preterm ELBW children without severe disabilities, medical complications, maternal education, and early developmental assessment are often mentioned. The inﬂuence of perinatal risk factors tends to decrease over time as environmental factors become more important (Linsell et al., 2015). Inclusion criteria were having a weight < 1000 g and a gestational age < 32 weeks at birth. Exclusion criteria were the multiple birth, the presence of genetic abnormalities, severe neurofunctional impairment (deﬁned as Neurofunctional Assessment > 2, Picciolini et al., 2006; Gianní et al., 2007), and/or neurosensory disabilities (blindness, deafness). We decided such The identiﬁcation of early developmental markers that may be predictive of long term cognitive outcome is essential for July 2017 \| Volume 8 \| Article 1257 2 Early Assessment and Cognitive Outcome Squarza et al. FIGURE 1 \| Flow chart of the study. FIGURE 1 \| Flow chart of the study. Vries et al. (1992), ROP of stage 3 or higher, according to the International Classiﬁcation of Retinopathy of Prematurity (Committee for the Classiﬁcation of Retinopathy of Prematurity, 1984), and BPD, deﬁned as oxygen supplementation at 36 weeks postmenstrual age (Jobe and Bancalari, 2001), were also collected. IVH and PVL were detected by brain MRI examination at 40 weeks postmenstrual age. Corrected age was calculated up to 24 months of life, from the chronological age adjusting for gestational age. Mothers’ nationality and education were also recorded. Mothers’ educational level was used as a measure of socioeconomic status and classiﬁed using a 3-point scale, where 1 indicates primary or intermediate school education (≤8 years), 2 secondary school education (9–13 years), and 3 university degree (>13 years). exclusion criteria because having ﬁne motor control and sensory orientation was a prerequisite for undergoing the cognitive assessment we administered. All the ELBW babies admitted to NICU were enrolled after discharge in the follow-up program provided at authors’ Institution and were scheduled to be prospectively followed up to 7 years of chronological age. MATERIALS AND METHODS Infants were enrolled in the study at 1 year of corrected age in occasion of the neurodevelopmental assessment and were re-assessed at 7 years to evaluate their cognitive outcome. All the children of our sample underwent the same follow- up procedures, as part of a standard follow-up program. Children with severe neurofunctional impairment at 1 year of corrected age were enrolled in speciﬁc rehabilitation programs (i.e., neuromotor developmental therapy, and/or neuropsychological interventions), while children with milder signs of impairment were accurately observed step by step along with the follow-up visits, activating a parental counseling and creating a clinical network with local pediatricians. Statistical Analyses Neurodevelopmental and cognitive results for children, respectively, at 1 year and 7 years, were summarized by mean, SD, and range. For each scale we identiﬁed low performing children when score was lower than 1 SD below the mean. Dividing low performing and normal performing children at the Griﬃths Scales, we compared mean values of IQ scores at 7 years, testing diﬀerences with Student’s t-test. Then, we designed a multivariate logistic regression model, including potential confounders (selected a priori based on literature search, in details: gender, AGA/SGA, BPD, MRI, and maternal education), to calculate relative risk (expressed as adjusted ORs) of resulting low performing at WISC-III IQ scores after scoring low performing at the Griﬃths Scales at 1 year. The mean Griﬃths General Quotient and Sub-quotients at 1 year of corrected age and mean WISC-III IQs at 7 years of chronological age were in the average range (Table 1). The Griﬃths Locomotor Sub-quotient had the lowest mean score. No diﬀerences were found between participants and dropouts considering the mean Griﬃths General Quotient and Sub-quotients. As displayed in Table 2, children who gained a score < 1 SD at the Griﬃths General Quotient at 1 year of corrected age showed a signiﬁcantly lower Full Scale IQ at 7 years of chronological age than children who scored in the normal range at 1 year (p < 0.01). y A p-value < 0.05 was considered as statistically signiﬁcant and relative CIs at 95% of conﬁdence were calculated for all ORs. All data analyses were performed using software Stata (Stata Corp, Austin, TX, United States). Considering the Griﬃths Sub-quotients separately, a score < 1 SD in the Personal-Social or in the Performance Sub-quotients at 1 year was associated with signiﬁcantly worse cognitive outcomes both in the Verbal and in the Performance IQs at 7 years (p < 0.05 and p < 0.01). A score < 1 SD in the Locomotor or in the Eye and Hand Coordination Sub-quotients at 1 year was speciﬁcally associated with poorer Performance or Verbal IQs, respectively, at 7 years (p < 0.05). TABLE 1 \| Grifﬁths and WISC-III mean quotients at 1 and 7 years. Cognitive Assessment The cognitive functioning at 7 years of age was assessed by the Italian version of the WISC-III (Wechsler, 1991). This test consists of 13 subtests that are combined into three IQ scores (mean 100, SD 15); Full Scale IQ, Verbal IQ, and Performance IQ. Accordingly, children were classiﬁed as having a normal intelligence if their IQ scores were 85 or more while they were classiﬁed as having a developmental delay if their IQ scores were 84 or lower. The cognitive testing was performed by a specially trained psychologist, blind to the child’s performance at the Griﬃths Scales. The mean gestational age at birth for our very preterm ELBW cohort was 27.7 weeks (SD: 2.3) and mean birth weight was 769.7 g (SD: 165.5). Of the 99 infants enrolled, 43 (43%) were males, 58 (59%) were SGA and 15 (15%) were multiple births. 88 (90%) infants were born by cesarean delivery and mean hospitalization was 97.1 days (SD: 32.7). BPD was diagnosed for 42 (42%) infants and grade III/IV ROP for 10 (10%) infants. 6 (6%) infants had PVL, while 5 (5%) had IVH grade III/IV. Mothers’ mean age was 33.7 years (SD: 4.5), 56 of them (57%) had a high school education and 20 (20%) a university degree. RESULTS were 83 or lower. Because normative data of the Griﬃths Mental Development Scales Revised are not available in our country, we referred to the 1996 United Kingdom norms (Griﬃths and Huntley, 1996). A total of 229 very preterm ELBW infants were admitted to NICU Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, between January 2002 and April 2007. Among them, 180 (79%) were discharged home alive and enrolled in the follow- up program provided by the Authors’ Institution. Of these, 162 (90%) returned for the 1-year follow-up visit and 99 (55%) were assessed at 7 years and entered the study. No diﬀerences in the neonatal and growth variables were observed between infants who were lost to follow up and those who were evaluated. The ﬂow chart of the study is shown in Figure 1. Neurodevelopmental Assessment Neurodevelopmental Assessment According to our Follow-up program, neurodevelopmental outcome at 1 year was assessed using the validated Italian translation of the Griﬃths Mental Development Scales Revised (Battaglia and Savoini, 2007), administered by two trained examiners. This tool comprises a set of ﬁve subscales, speciﬁcally investigating neurodevelopment in the Locomotor, Personal-Social, Hearing and Language, Eye and Hand Coordination and Performance domains. The Scale yields standardized Sub-quotients for each domain (mean 100, SD 16) and a composite General Quotient (mean 100, SD 12). In accordance with this, children were classiﬁed as having typical development if their General Quotient was 88 or more and their Sub-quotients were 84 or more and they were classiﬁed as having a developmental delay if their General Quotient was 87 or lower and their Sub-quotients A number of neonatal characteristics, including gender, birth weight, being adequate or small for gestational age, mode of delivery, multiple birth, duration of hospital stay, were collected from the infants’ computerized medical charts. Gestational age was based on the last menstrual period and early ultrasound examination; infants with birth weight ≥10th centile or < 10th centile for gestational age, according to the Fenton Growth Chart (Fenton, 2003), were classiﬁed, respectively, as AGA/SGA. The occurrence of IVH grade 3 or higher, according to the Papile classiﬁcation scheme (Papile et al., 1978), PVL of grade 2 or higher, according to de July 2017 \| Volume 8 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 3 Early Assessment and Cognitive Outcome Squarza et al. Frontiers in Psychology \| www.frontiersin.org Statistical Analyses N = 99 Mean (SD) Range Number (%) < 1 SD Number (%) < 2 SD Grifﬁths Scales 1 year General Quotient 94.2 (9.5) 65−113 21 (21.2) 4 (4.1) Locomotor 88.5 (11.1) 58−108 29 (29.3) 6 (6.1) Personal-Social 92.7 (10.5) 60−113 17 (17.2) 3 (3.0) Language 98.5 (9.2) 78−122 6 (6.1) – Eye and Hand Coordination 92.9 (10.8) 60−117 20 (20.2) 1 (1.0) Performance 94.2 (12.5) 55−118 17 (17.2) 3 (3.0) WISC-III 7 years Full Scale IQ 103.0 (15.7) 52−139 11 (11.1) 3 (3.0) Verbal IQ 104.8 (15.9) 51−137 10 (10.1) 4 (4.0) Performance IQ 100.3 (14.4) 62−148 10 (10.1) 2 (2.0) TABLE 1 \| Grifﬁths and WISC-III mean quotients at 1 and 7 years. Unadjusted and adjusted results from regression analysis are reported in Tables 3, 4, respectively. A score < 1 SD at the Griﬃths General Quotient at 1 year moderately predicted subsequent impairment on WISC-III Full Scale IQ, after adjustment for a number of biological and social factors (Table 4). Three of the Griﬃths Sub-quotients were strongly associated with an increased risk of impairment at the WISC-III Verbal IQ, the Performance Sub-quotient (OR 62.30, 95% CI [5.43; 714.58]), the Eye and Hand Coordination Sub- quotient (OR 9.22, 95% CI [1.66; 51.22]) and the Personal- Social Sub-quotient (OR 7.45, 95% CI [1.29; 42.97]), with no prediction from the Language Sub-quotient (OR 0.83, 95% CI [0.04; 15.39]). July 2017 \| Volume 8 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 4 Early Assessment and Cognitive Outcome Squarza et al. Squarza et al. were shown between Personal-Social, Eye–Hand Coordination and Performance subscales at 1 year and Verbal IQ at 7 years and between Locomotor, Language, and Performance subscales at 1 year and Performance IQ at 7 years. Considering the Performance IQ, the Griﬃths Performance Sub-quotient (OR 9.13, 95% CI [1.77; 46.91]), the Language Sub- quotient (OR 4.64, 95% CI [0.62; 34.59]) and the Locomotor Sub-quotient (OR 4.26, 95% CI [0.88; 20.59]) were the most powerfully associated. Our ﬁndings suggest that a score < 1 SD on the Griﬃths General Quotient at 1 year of corrected age is associated with a higher risk of impairment on subsequent IQ scores at school age. A low score on the Performance Sub-quotient is the most associated with school-age cognitive impairments, especially for the Verbal IQ. Eye and Hand Coordination, Personal-Social and Locomotor Sub-quotients also show an association with later cognitive functioning. DISCUSSION Our ﬁndings suggest that a Griﬃths General Quotient < 1 SD at 1 year of corrected age increases the odds of low IQ scores at 7 years by 4.24 (95% CI [0.95; 18.99]), adjusting for biological, neonatal, and family factors. The highest associations TABLE 2 \| Differences on mean IQs at 7 years based on neurodevelopmental assessment at 1 year. Grifﬁths Scales 1 year (N = 99) WISC-III 7 years (N = 99) Full Scale IQ Mean (SD) p-value General Quotient normal range n = 78 105.6 (14.1) 0.005 General Quotient < 1 SD n = 21 95.0 (17.5) Verbal IQ Performance IQ Mean (SD) p-value Mean (SD) p-value Locomotor normal range n = 70 106.8 (13.4) 0.114 102.7 (12.0) 0.024 Locomotor < 1 SD n = 29 101.3 (19.8) 95.6 (18.0) Personal-Social normal range n = 82 107.0 (14.3) 0.010 101.9 (13.7) 0.044 Personal-Social < 1 SD n = 17 96.4 (19.2) 94.2 (15.7) Language normal range n = 93 105.7 (15.0) 0.171 101.2 (13.9) 0.116 Language < 1 SD n = 6 96.7 (24.3) 91.7 (19.2) Eye–Hand Coordination normal range n = 79 107.1 (14.1) 0.018 101.9 (13.4) 0.067 Eye–Hand Coordination < 1 SD n = 20 97.9 (19.5) 95.4 (16.8) Performance normal range n = 82 107.7 (13.0) 0.000 102.4 (12.8) 0.004 Performance < 1 SD n = 17 93.3 (21.5) 91.6 (17.9) Student’s t-test on mean WISC-III IQs at 7 years, comparing low performing (scores < 1 SD) and normal performing (scores in the normal range) children at the Grifﬁths Scales at 1 year. TABLE 3 \| Odds of cognitive impairment at 7 years based on neurodevelopmental assessment at 1 year. Grifﬁths Scales 1 year (N = 99) WISC-III 7 years (N = 99) Total IQ Odds ratio 95% CI General Quotient 4.5 1.16; 17.4 Verbal IQ Performance IQ Odds ratio 95% CI Odds ratio 95% CI Locomotor 3.39 0.84; 13.67 5.74 1.33; 24.83 Personal-Social 8.02 1.89; 34.15 2.68 0.59; 11.99 Language 2.1 0.22; 20.25 6.07 0.94; 39.16 Eye and Hand Coordination 10.71 2.39; 47.96 3.65 0.88; 15.13 Performance 27.65 5.03; 151.9 8.02 1.88; 34.15 Odds ratio of cognitive impairment (<1 SD) at 7 years based on Grifﬁths General Quotient and subscale scores < 1 SD at 1 year. DISCUSSION Frontiers in Psychology \| www.frontiersin.org 5 July 2017 \| Volume 8 \| Article 1257 TABLE 2 \| Differences on mean IQs at 7 years based on neurodevelopmental assessment at 1 year. Grifﬁths Scales 1 year (N = 99) July 2017 \| Volume 8 \| Article 1257 Early Assessment and Cognitive Outcome Squarza et al. aﬀect cognitive functions and academic achievements in preterm infants (Gianní et al., 2006; Aarnoudse-Moens et al., 2009; Pugliese et al., 2013). These results may be explained by the fact that early neurodevelopmental impairments emerging during the ﬁrst year of life represent the basis for the onset of later cognitive underachievement. Speciﬁcally, a diﬃculty in manipulating materials or in organizing visual-spatial skills, as assessed by the Griﬃths Performance subscale, reveal a lack of cognitive ﬂexibility aﬀecting long term intellectual outcome (Squarza et al., 2016). The predictive validity of early developmental assessment for long term cognitive outcomes has been questioned since neurodevelopmental evaluation in the ﬁrst 2 years of life may not reliably predict the full spectrum of disability at school age, particularly for less severe impairments (Aylward, 2002). According to the EPICURE study (Marlow et al., 2005), very early neurodevelopmental assessments at 6 to 12 months may not accurately predict neurodevelopmental impairment at school age in 25% of children born before 26 weeks of gestational age. Hack et al. (2005) found that infant testing at 20 months of corrected age in ELBW is more predictive among children with severe handicaps and/or subnormal functioning. Actually, the particularly strong association between the Performance subscale at 1 year and the cognitive functioning at 7 years might be explained considering that this subscale speciﬁcally focuses on a variety of intellectual skills that are strongly related with later cognitive functioning. To enhance the maturation of early cognitive skills, motor experiences and activities with objects are essential. Accordingly, a low score on the Locomotor and Eye–Hand Coordination Sub- quotients at 1 year was signiﬁcantly associated with long term cognitive impairments. Early motor impairments, hindering the child in discovering the environment and taking contact with objects and persons, may interfere with the maturation and organization of higher cognitive functions. Indeed, in literature there is evidence of a strong association between early motor development and later intellectual functions within the normal population (Murray et al., 2006). DISCUSSION In contrast to these ﬁndings, in the present study we found a strong association between early developmental assessment and long term cognitive outcome in children with only a mild impairment (<1 SD). The discrepancy between previous ﬁndings and ours may be almost partly related to the diﬀerent assessment tool. Both these studies referred to the Bayley Scales for the early neurodevelopmental assessment. Although the Griﬃths Scales have shown a good concordance with the Bayley Scales (Picciolini et al., 2015), they are a more complex and integrated instrument, assessing not only cognition, language, and motor abilities but also personal-social skills, thus providing a more comprehensive evaluation of the child, perhaps more associated with long term outcome. At the same time, the association between Eye-Hand Coordination Sub-quotient and Verbal IQ at school age may be related to the fact that an active exploration of objects and activities, such as showing and oﬀering objects to adults, may enable infants to extend periods of social interactions and to enhance the emergence of symbolization abilities. This provides opportunities for infants to expand language and learn rules of interaction, as reported in previous studies (Karasik et al., 2011). At the same time, the association between Eye-Hand Coordination Sub-quotient and Verbal IQ at school age may be related to the fact that an active exploration of objects and activities, such as showing and oﬀering objects to adults, may enable infants to extend periods of social interactions and to enhance the emergence of symbolization abilities. This provides opportunities for infants to expand language and learn rules of interaction, as reported in previous studies (Karasik et al., 2011). With regard to the Personal-Social domain, the importance of emotional regulation for an adequate cognitive functioning is widely recognized in literature. Follow-up studies conﬁrm that behavioral and social-emotional immaturity may negatively Consistently with our results, other studies revealed that early neurodevelopmental assessments of preterm infants are good predictors of school readiness. Patrianakos-Hoobler et al. (2010) found that 2-year neurodevelopmental delay (MDI or PDI < 70) predicted the need for special education services at the age of 5 years, while children with MDI or PDI scores lower than 85 (1 SD below the mean) had an increased risk of not being ready for school. Barnett et al. DISCUSSION (2004) examined a cohort of term infants With regard to the Personal-Social domain, the importance of emotional regulation for an adequate cognitive functioning is widely recognized in literature. Follow-up studies conﬁrm that behavioral and social-emotional immaturity may negatively TABLE 4 \| Odds of cognitive impairment at 7 years based on neurodevelopmental assessment at 1 year after adjustment for potential confounders∗. Grifﬁths Scales 1 year (N = 99) WISC-III 7 years (N = 99) Total IQ Odds ratio 95% CI General Quotient 4.24 0.95; 18.99 Verbal IQ Performance IQ Odds ratio 95% CI Odds ratio 95% CI Locomotor 2.63 0.49; 14.10 4.26 0.88; 20.59 Personal-Social 7.45 1.29; 42.97 2.38 0.46; 12.12 Language 0.83 0.04; 15.39 4.64 0.62; 34.59 Eye and Hand Coordination 9.22 1.66; 51.22 3.51 0.75; 16.35 Performance 62.30 5.43; 714.58 9.13 1.77; 46.91 Odds ratio of cognitive impairment (<1 SD) at 7 years based on Grifﬁths General Quotient and subscale scores < 1 SD at 1 year. ∗Adjusted for gender, AGA/SGA, BPD, MRI 40 weeks, maternal education. July 2017 \| Volume 8 \| Article 1257 6 Early Assessment and Cognitive Outcome Squarza et al. with neonatal encephalopathy and found that an abnormal score (<1 SD) on the Griﬃths Sub-quotients at 1 and/or 2 years was very likely to be associated with poor performance at school age. Although this study was performed on a cohort of infants born at term, it could yet be considered a population of infants at risk, which is the same target of our study, aiming at identifying early markers of risk for long term cognitive impairment. with neonatal encephalopathy and found that an abnormal score (<1 SD) on the Griﬃths Sub-quotients at 1 and/or 2 years was very likely to be associated with poor performance at school age. Although this study was performed on a cohort of infants born at term, it could yet be considered a population of infants at risk, which is the same target of our study, aiming at identifying early markers of risk for long term cognitive impairment. Further studies including a normal weight control group are required in order to conﬁrm if the neurodevelopmental pathways we observed are preterm and ELBW speciﬁc and to verify if the association with long term cognitive outcome could be eﬀective also for the normal population. DISCUSSION Moreover, the relatively small sample size with consequent large uncertainty in our risk estimates (i.e., 95% CI) deserves caution in interpreting our results. Moreover, it is possible that the weak association between the Language subscale at 1 year and the Verbal IQ at 7 years might be partially due to the translation of the Griﬃths Scales from the original standardization language to Italian. Another limitation is the relatively high rate of children lost to follow up, which may limit the interpretation of our ﬁndings. However, we found no diﬀerences in the neonatal and growth variables between infants who were lost to follow up and those who were evaluated. Studying a cohort of VLBW infants, Breeman et al. (2015) found that cognitive function in adulthood could be fairly well estimated from age 20 months and that IQ scores were most stable for VLBW individuals who had severe cognitive impairment in adulthood. Breeman et al. (2015) also found that the Griﬃths Mental Development Scales did not reliably predict cognitive development at 5 months, probably because of the faster state ﬂuctuation of cognitive function in younger infants. In our study, the association with long term cognitive outcome is found at an earlier age (1 year) and is strong also considering infants with only a mild impairment. Future long-term studies will need to include larger populations and to assess not only cognitive functioning, but also behavioral and emotional outcomes. Nevertheless, a strength of our study is that it is a longitudinal study that provides information on the long term cognitive outcome of a population of high risk children. Speciﬁcally, our ﬁndings strengthen the evidence of a deep integration among cognitive functions, in particular between early motor and later cognitive skills and between verbal and performance domains. In our study, the rates of ELBW infants with a developmental impairment at 1 year stand between 6.1–23.2% for the 1 SD cut- oﬀand between 0.0–6.1% for the 2 SD cut-oﬀ. At 7 years the rates of mild and severe impairment are around 10.1–11.1% and 2.0–4.0%, respectively. These rates are not higher than those expected from a normal birth weight population (23.7–30.2% and 3.2–4.4%, respectively for the Griﬃths Scales at 1 year and around 16% for the WISC-III at 7 years). CONCLUSION In our perspective, this is not surprising given that syndromic and severely impaired children were excluded from our study. Moreover, all the children of our sample were assessed at 1 year through the Neurofunctional Assessment, which has been demonstrated to correlate with later neurodevelopmental outcome, probably limiting the rates of impairment at 1 year and at 7 years in our sample (Picciolini et al., 2016). In summary, our study suggests that early neurodevelopmental assessment is associated with school-age cognitive achievement in a cohort of very preterm ELBW children. According to our ﬁndings, even a mild neurodevelopmental impairment at 1 year of corrected age seem to be associated with long term cognitive deﬁcits. These results have important implications for clinical services and follow-up programs since provision of timely intervention is dependent upon accurate, early identiﬁcation of infants at risk for adverse long-term outcomes. Our ﬁndings recommend timely activation of intervention programs so that early impairments at 1 year do not lead to more stable cognitive diﬃculties at later ages. Based on our ﬁndings, we would recommend starting a rehabilitation program for those children who scored < 1 SD at the Griﬃths Scales at 1 year. However, the particularly low rates of impairment in the Language subscale at 1 year (6.1% < 1 SD) is remarkable. This could be explained considering that at 1 year the early communication skills are still emerging and that speciﬁc language delays will arise later, during the second year of life, with the expansion of the vocabulary and the inﬂuence of emotional maturation (speciﬁcally the separation-individuation process). Our study has several limitations. The lack of a normal weight control group represents a major issue and prevents from clearly attributing the outcomes observed in our sample to any speciﬁcities in the developmental pathways of very preterm ELBW children, or to the developmental trajectories that characterize a normal birth weight population. 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AUTHOR CONTRIBUTIONS CS, OP, LG, and MR conceptualized and designed the study, drafted the initial manuscript, and approved the ﬁnal manuscript as submitted. MG, MP, and MB designed the data collection instruments, carried out the initial analyses, reviewed and revised the manuscript, and approved the ﬁnal manuscript as submitted. FM and SG coordinated and supervised data collection, critically reviewed the manuscript, and approved the ﬁnal manuscript as submitted. All authors approved the ﬁnal manuscript as submitted and agree to be accountable for the content of the work. Actually, our study shows an association between early neurodevelopmental assessment and the risk of adverse cognitive outcome at 7 years. In particular, our ﬁndings suggest speciﬁc areas of impairment in the developmental proﬁles at 1 year that seem very speciﬁc in comparison with a normal distribution based on normative data (e.g., locomotor abilities). These ﬁndings are particularly useful because the timely identiﬁcation of neurodevelopmental impairments associated with later cognitive outcome is essential to plan rehabilitative programs. July 2017 \| Volume 8 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 7 Early Assessment and Cognitive Outcome Squarza et al. ACKNOWLEDGMENTS Andrea Frigerio, Neuropsychomotor therapist, Marta Macchi, MD, and Paola Ajmone, MD, members of the preterms’ follow- up research group at NICU, Department of Clinical Sciences and Community Health, for their competent and experienced assistance throughout the research. We are grateful to the children and families who participated in the study. Thank you also to the nurses of the preterms’ Follow-up Clinic for their contribution. A special thanks to REFERENCES An international classiﬁcation of retinopathy of prematurity. Arch. Ophthalmol. 102, 1130–1134. Msall, M. E. (2012). 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Very preterm/very low birthweight infants’ attachment: infant and maternal characteristics. Arch. Dis. Child. Fetal Neonatal Ed. 99, F70–F75. doi: 10.1136/archdischild- 2013303788 Picciolini, O., Squarza, C., Fontana, C., Gianní, M. L., Cortinovis, I., Gangi, S., et al. (2015). Neurodevelopmental outcome of extremely low birth weight infants at 24 months corrected age: a comparison between Griﬃths and Bayley Scales. BMC Pediatr. 15, 139. doi: 10.1186/s12887-015-0457-x Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Pugliese, M., Rossi, C., Guidotti, I., Gallo, C., Della Casa, E., Bertoncelli, N., et al. (2013). Preterm birth and developmental problems in infancy and preschool age Part II: cognitive, neuropsychological and behavioural outcomes. J. Matern. 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https://openalex.org/W4280648957	https://www.preprints.org/manuscript/202204.0084/v1/download	English	null	How to Heal the Gut’s Brain: Regeneration of the Enteric Nervous System	International journal of molecular sciences	2,022	cc-by	7,064	Helen Rueckert 1,2 and Julia Ganz 1,* 1 Department of Integrative Biology, Michigan State University, East Lansing, MI, 48824; 2 Curent address: Department of Cell Biology, Duke University, Durham, NC 27710 * Correspondence: ganz@msu.edu 1 Department of Integrative Biology, Michigan State University, East Lansing, MI, 48824; 2 Curent address: Department of Cell Biology, Duke University, Durham, NC 27710 Abstract: The neural-crest derived enteric nervous system (ENS) is the intrinsic nerv- ous system of the gastrointestinal (GI) tract and controls all gut functions, including motility. Lack of ENS neurons causes various ENS disorders such as Hirschsprung Disease. One treatment option for ENS disorders includes the activation of resident stem cells to regenerate ENS neurons. Regeneration in the ENS has mainly been stud- ied in mammalian species using surgical or chemically-induced injury methods. These mammalian studies showed a variety of regenerative responses with generally limited regeneration of ENS neurons, but (partial) regrowth and functional recovery of nerve fibers. Several aspects might contribute to the variety in regenerative re- sponses, including observation time after injury, species, and gut region targeted. Zebrafish have recently emerged as a promising model system to study ENS regen- eration as larvae possess the ability to generate new neurons after ablation. As the next steps in ENS regeneration research, we need a detailed understanding of how regeneration is regulated on a cellular and molecular level both in animal models with high and low regenerative capacity. Understanding the regulatory programs necessary for robust ENS regeneration will pave the way for using neural regenera- tion as a therapeutic approach to treating ENS disorders. Keywords: enteric progenitor cell; zebrafish; inflammation; Hirschsprung Disease; neural crest cell; ENS neuropathies Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 1. Introduction The enteric nervous system (ENS) is derived from the neural crest cell lineage and provides the intrinsic innervation of the gastrointestinal (GI) tract forming a complex network of different types of neurons and glial cells [1-3]. It is the largest division of the peripheral nervous system – the ENS contains approximately the same number of neurons as the adult spinal cord [4-6]. The mammalian ENS consists of two enteric plexi, the myenteric plexus, and the submucosal plexus. ENS neurons are found in ganglia, which are connected by a network of nerve fibers [5]. As the intrinsic nervous sys- tem of the gut, the ENS regulates many essential intestinal functions such as GI motility, absorption of nutrients, secretion, fluid exchange, regulation of blood flow, epithelial barrier function, immune modulation, and microbiota colonization and composition [5, 7-9]. The ENS regulates intestinal functions along the entire length of the gut - in mammals, the GI tract can be subdi- vided into upper and lower parts. The upper GI tract contains the stomach and small intestine (duodenum, jejunum, and ileum), the lower GI tract the cecum, colon, and rectum (Figure 1). © Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 Figure 1. Subdivisions of the mammalian gastrointestinal tract. In mammals, the gastrointestinal (GI) tract, here exemplified in mice, is divided into an upper (green) and a lower GI tract (blue). The upper GI tract consist of duodenum, jejunum, and ileum. The lower GI tract consists of the cecum, colon, and rectum. A. modified from [10]. Figure 1. Subdivisions of the mammalian gastrointestinal tract. In mammals, the gastrointestinal (GI) tract, here exemplified in mice, is divided into an upper (green) and a lower GI tract (blue). The upper GI tract consist of duodenum, jejunum, and ileum. The lower GI tract consists of the cecum, colon, and rectum. A. modified from [10]. Each of these gut regions is exposed to daily abrasive and potentially harmful and toxic compounds which in turn subject the ENS to many stress- ors and mechanical forces. As the ENS directly overlays the intestinal smooth muscle layer it changes its shape during the gastrointestinal contractions and relaxations that accompany intestinal motility [11]. In aging animals, ENS neuron numbers are considerably reduced [11-13]. In addition, acute and chronic gut disorders can also impact ENS cells. 1. Introduction Deficits in ENS neuron abundance and composition cause severe GI dysfunction that occur in con- genital ENS disorders such as Hirschsprung disease, inflammatory gut dis- eases like inflammatory bowel syndrome, and neurodegenerative diseases, such as Parkinson’s Disease [14-19]. ENS defects in these various disorders range from a complete deficit of neurons in a gut subdivision to a lack of specific neuronal subtypes. For example, in Hirschsprung Disease, ENS neu- rons are lacking in the distal gut resulting in difficulties in passing stool. The length of the aganglionic part varies from distal colon to the entire colon and stretches of the small intestine [15]. 1.3. The mammalian ENS has limited regenerative ability Starting in the 1950s, regeneration studies have been performed in the ENS in a variety of mammalian research organisms, including adult guinea pigs, rats, mice, and dogs. Very few studies have so far been performed in non-mammalian species. In mammals, neurons or nerve fibers are generated in some but not all experimental settings after injury. Generally, neuron numbers are not restored to control levels and nerve fiber regeneration is often not complete. Additionally, within the injured area, significant struc- tural changes remain. Several aspects might contribute to this variety in the regenerative responses: type of injury, observation time after injury, animal model, and which gut region has been injured. In the next section, we will discuss the main injury models used to study ENS regeneration and how the variability in experimental parameters might impact the regenerative response. The injury models that we discuss here are surgical or mechanical methods of injury, chemical-induced injury, and in- fection with pathogens. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084 doi:10.20944/preprints202204.0084.v1 based therapeutic approaches have not been translated into the clinic and practical limitations remain [27]. As this review focuses on ENS regenera- tion, an in-depth discussion of stem cell-based therapies is out of its scope. Additional information on ENSCs, their origins, in vitro/in vivo experiments, and their therapeutic potential can be found in several excellent recent re- views [15, 24, 27-29]. The other avenue for treating insufficient or injured ENS neurons in pa- tients is to stimulate resident stem cells to regenerate the missing/damaged enteric neurons. For this approach to be successful, a detailed understanding of the extent, potential, and cellular and molecular mechanisms underlying ENS regeneration is necessary. In this review, we will discuss the ability of the ENS to regenerate and why ENS regeneration might be limited in mam- mals. We will also discuss the high regenerative capacity of the ENS in the zebrafish model system and close with an outlook on the open questions and future directions for ENS regeneration research. 1.2. What constitutes nervous system regeneration? Nervous system regeneration is generally defined as either the repair or the new generation of neurons damagd by injury or disease. This can occur at two different levels: First, the regrowth of just neuronal axons when the neuronal cell body is not damaged. Second, “large scale” regeneration, where new neurons are generated and have to connect to the existing neural circuitry or build a new circuit that then gets wired into the larger neural circuit [30]. Complete regeneration is viewed as the full restoration of lost neurons or neuronal function, whereas partial regeneration includes the gen- eration of new neurons or nerve fibers without the complete restoration of lost neurons or neuronal function. 1.1. Therapeutic approaches to treat ENS disorders At present, ENS disorders are treated symptomatically or require surgi- cal removal of the area with ENS neuron deficits [15, 18, 20, 21]. Because of the prevalence of ENS disorders and their strong impact on the patient’s quality of life, there has been an increasing interest in finding therapeutic approaches for restoring lost ENS neurons or glial cells. There are two main avenues for treating such ENS disorders: ENS stem- cell-based treatment or stimulation of resident stem cells to regenerate miss- ing ENS cells [20]. Stem-cell-based therapeutic approaches aim to transplant ENS stem cells into a patient’s gut where they then can differentiate into ENS neurons and/or ENS glia cells [20, 22]. In mammals including humans, en- teric neuronal stem cell (ENSC) populations are present into adulthood [22, 23]. When isolated and induced in culture, ENSCs can be amplified and dif- ferentiated into many enteric neuronal and glial subtypes [22, 24]. Addition- ally, recent work has generated different types of ENS neurons and glia cells from induced pluripotent stem cells (iPSCs) [25, 26]. Transplantation of EN- SCs in mouse models of congenital ENS disorders shows their ability to mi- grate and colonize mammalian guts [22, 24, 27]. Yet, so far, such stem cell- 1.4. ENS regeneration after surgical/mechanical injury in mammals Transection and reanastomosis and (partial) stenosis are the main types of surgical injury that have been used to study ENS regeneration in various mammalian animal models (Figure 2). Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 Figure 2. Common ENS injury approaches in mammals. Common models using sur- gical (grey boxes) or chemically-induced (red) injury methods to study ENS regener- ation in mammals: (1) transection & reanastomosis: the targeted part of the gut is transected (red line) with subsequent end-to-end anastomosis (purple). (2) For steno- sis, a ring (magenta) is placed around the gut, which causes partial obstruction. (3) Benzalkonium chloride (BAC) treatment comprises the treatment of a small gut seg- ment with BAC, which leads to a loss of ENS neurons in the treated area. Figure 2. Common ENS injury approaches in mammals. Common models using sur- gical (grey boxes) or chemically-induced (red) injury methods to study ENS regener- ation in mammals: (1) transection & reanastomosis: the targeted part of the gut is transected (red line) with subsequent end-to-end anastomosis (purple). (2) For steno- sis, a ring (magenta) is placed around the gut, which causes partial obstruction. (3) Benzalkonium chloride (BAC) treatment comprises the treatment of a small gut seg- ment with BAC, which leads to a loss of ENS neurons in the treated area. Transection and reanastomosis in the small intestine in dogs, guinea pigs, or rats resulted in nerve fiber regeneration across the injury site with accompanying functional recovery. In dogs, transection and reanastomosis led to an initial loss of migratory motor complex (MMC) propagations after surgery [31, 32]. MMCs are cyclic sweeping gut movements in the small in- testine during fasting [33]. After 40-60 days post-surgery, MMCs started to be coupled again between the two segments indicating functional recovery of gut movements across the injury site. By approximately 100 days post- surgery, MMCs were fully recovered suggesting regeneration of nerve fibers across the injury site [31, 32]. j y Analysis of structural changes after surgical injury in the ENS in rats or guinea pigs showed that neurons degenerated in the injury site between 1-2 months after transection and reanastomosis in the small intestine [34-36]. At 6 weeks post-surgery, there were significantly fewer neurons up to about 5mm from the surgical site. Farther away, no difference in neuron numbers was seen. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 Analysis of structural changes after transection and reanastomosis in the lower GI tract in guinea pigs had contrasting results [38-40]. Surgery in the colon showed substantial, and long-lasting disruption of neuron num- bers and neuronal pathways at the lesion site [38]. Neurons degenerated at the injury site and failed to regenerate after 8-24 weeks. Nerve fiber regener- ation occurred, but in this model, regrowth of nerve fibers occurred prefer- entially in an oral to anal direction, no regrowth of fibers anal to oral was observed between 10-24 weeks after surgery [38]. In contrast, transection and reanastomosis in the guinea pig rectum led to nerve fiber regrowth across the lesion site accompanied by recovery of rectal contractions between 2-8 weeks [39, 40]. In addition, new neurons were present in the injured site, but no ganglia were formed [39, 40]. The difference in neuron and nerve fiber regeneration between the different gut regions suggests that each gut subdi- vision may have different abilities to restore neuronal function after surgical injury. Injury models using (partial) stenosis focused on the small intestine [41- 45]. Between 1-2 weeks after stenosis in rats, there was an increase in the numbers and cell volume of neurons per ganglia accompanied by thymidine incorporation or expression of the proliferation marker PCNA in neurons upstream of the point of stenosis. This suggests that neurons either activated the cell cycle or were undergoing unscheduled DNA synthesis due to DNA repair [45]. It was not tested if the increase in neurons resulted in a full res- toration of lost neurons. After 4 weeks, there was no evidence of proliferating cells, but the nuclear volume of neurons was still increased [41-43]. In a guinea pig model of stenosis, there was no evidence of an increase in neuron numbers after 3-5 weeks [44], but the earlier time points where cell cycle ac- tivation in neurons was observed in the rat models were not analyzed in the guinea pig. Thus, parallel conclusions are unable to be made. In summary, surgical injuries result in partial nerve fiber regeneration with functional recovery, some generation of neurons, but no full recovery of neuron numbers after surgery. There is some evidence that gut regions and species differ in their regenerative ability and response, but more exten- sive research needs to be done in comparing the regenerative ability of dif- ferent species and gut regions. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 Notably, regenerative processes take a long time, which is relevant for the experimental setup to study ENS regeneration. 1.4. ENS regeneration after surgical/mechanical injury in mammals Even at 12 months, the surgical site did not contain any new neu- rons [34, 35]. The area close to the surgical site showed an increase in large extra-ganglionic neurons forming clusters, but these additional neurons did not restore neuron numbers to control levels. A little farther away, in the area that had not seen a reduction in neuron numbers, large extra-ganglionic neu- rons emerged at 6 months and continued to increase in numbers until reach- ing a plateau at 1-year post-surgery [34]. These extra-ganglionic neurons were found in higher numbers than controls, forming clusters and connect- ing to surrounding enteric ganglia [34]. Interestingly, in mice, extra-gangli- onic neurons that move into enteric ganglia after 24 weeks were observed in the small intestine and colon after experimental treatment with a 5-HT4 ago- nist to stimulate neurogenesis [37]. This indicates a potential connection be- tween the process of adult neurogenesis and regeneration. p g g After 2-4 weeks, regenerating nerve fibers appeared in the lesion site both from the oral and aboral sides. These fibers extended further after 8 weeks, suggesting that nerve fiber regeneration across the lesion site occurs also in guinea pigs and rats [34]. Yet, structural changes in neuronal fiber patterns were visible even after 1 year post-surgery indicating that nerve fi- bers were not fully restored across the lesion site [34]. Together, these studies showed that transection and reanastomosis in the small intestine led to dis- tinct structural changes in neurons and nerve fibers depending on the dis- tance from the surgical site and time after surgery, but never fully restore structural patterns as in the uninjured gut. 1.6.1. Time required for full regeneration Regeneration events generally took a long time and positive signs of re- generation were often not observed until months after injury. The reporting period of many studies was significantly shorter and may not cover a long enough time to determine if neuron regeneration might still happen. Thus, results determining no or limited regeneration capacity may be limited by the timeframe of observation. 1.6. What aspects might impact the regenerative process in mammals? The regeneration studies in mammals have shown that even though there are instances of nerve fiber regeneration and partial regeneration of neurons, complete regeneration with restoration of neuron numbers to con- trol levels does not take place. In this section, we will discuss three aspects that may impact the regenerative ability in the mammalian ENS: Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 adjacent to the area of injury) [48]. In the rat small intestine, nerve fiber re- growth took even longer – at 15 days, no neuron fibers were present. By 45 days, some neuronal fibers had regrown but fibers showed marked struc- tural differences to the control ENS [51]. In conclusion, injury of the ENS using chemical treatment results in de- nervation of the injured area. However, depending on the animal model and gut region the timing of regrowth of nerve fibers and the regenerative ability differs. As with the surgical injury method, regeneration in the colon seems to be impaired compared to the small intestine, for which some, limited re- generation of nerve fibers and ENS neurons was observed. 1.6. What aspects might impact the regenerative process in mammals? 1.5. ENS regeneration after chemically-induced injury in mammals A chemically-induced injury method commonly used to study ENS re- generation is the treatment with benzalkonium chloride (BAC), which leads to a loss of ENS neurons in the treated area (Figure 2, [46, 47]). In rat or mouse animal models, treatment with BAC of the small intestine or colon resulted in neuron degeneration and denervation within 2-5 days after treatment [48- 50]. In the small intestine of rats, there was a significant increase in new neu- rons at the lesion site at 30-60 days, which suggests a partial regenerative response with ~25-30% of control neuron numbers present 60 days post-in- jury [50]. In the treated mouse colon, in contrast, no consistent generation of new neurons was observed. Here, nerve fibers grew into the denervated area between 7-14 days post-treatment. Nerve fiber density increased until 60 days post-treatment to 35-60% of the control density. Occasionally new neu- rons were present along the nerve fiber bundles, but neuron numbers did not increase significantly [49]. Genetic lineage tracing in the small intestine in mice also found that neuronal projections grew into the injured area along with newly generated enteric glia cells, but at 3 months post-ablation, new neurons were not present within the injured area (though they were found Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 1.6.3. Ability of mammalian adult neurogenesis in the ENS In the CNS, the ability to regenerate neurons in the adult has been con- nected to the extent of adult neurogenesis, that is to say – high levels of adult neurogenesis correspond to the ability to regenerate the nervous system after injury. Low to no adult neurogenesis is generally correlated with low or no ability to regenerate neurons in the adult [53, 61, 62]. In the ENS, the extent of adult neurogenesis in mammals beyond postnatal stages has been debated in the field. The predominant view on neurogenesis in the mammalian ENS has been that the majority of enteric neurons are generated during embryo- genesis and early postnatal stages with no continuous generation of neurons throughout adult life [43]. Only in specific experimental conditions, for ex- ample, induction of inflammation with DSS treatments or the treatment with 5-HT4 agonists, has the generation of new neurons been observed in the adult [37, 57]. Treatment with 5-HT4 agonists translates to improved neural regen- eration, as nerve fiber regeneration and reflex recovery were accelerated in resection and anastomosis in the rectum of guinea pigs. Also, the generation of new neurons after DSS treatment was abolished by treatment with a 5-HT4 antagonist [39, 40, 57]. This suggests that signals that support adult neuro- genesis, also promote neural regeneration in the ENS. Recently, the view of little to no adult neurogenesis in the ENS has been challenged by the study of Kulkarni et al. (2016) which observed high levels of adult neurogenesis in the gut with a steady turnover of ENS neurons in homeostatic conditions [63]. Despite the lack of clarity regarding the extent of adult neurogenesis in the mammalian ENS, ENSCs can be isolated from the gut into adulthood and differentiate into ENS neurons in vitro [22, 24]. This shows that stem cells, which can generate new neurons are still present in the adult mammalian ENS, but work is still needed to determine how these cells can be re-acti- vated. 1.6.2. Impact of inflammation In the mammalian central nervous system (CNS), neuroinflammation is both detrimental as well as positive for a regenerative response. In zebrafish both detrimental as well as positive for a regenerative response. In zebrafish – a research organism with a remarkable ability to regenerate nervous sys- tem injury – inflammatory responses support brain, retina, and spinal cord regeneration [52-54]. The gut contains a large number of immune cells and intestinal inflammation results in structural changes in the ENS including hyperplasia or loss of neurons [55, 56]. Inflammatory responses have been evoked by different types of treatments, for example with trinitrobenzene sulfonic acid (TSA), dextran sulfate sodium (DSS) [57, 58], or by infection with the nematode Nippostrongylus brasiliensis [59]. Treatment with TSA re- sults in an increase in proliferating cells at 3 and 7d post-treatment, but the study did not investigate if this supported the generation of new neurons later [58]. DSS treatment in the colon led to a significant increase in the num- ber of ENS neurons 2 days after treatment. In this treatment setting, a subset of enteric glia cells was suggested to give rise to ENS neurons [57, 60]. Infec- tion with Nippostrongylus brasiliensis resulted in a strong inflammatory re- sponse and led to a significant degeneration of nerve fiber densities until 21 days post-infection (dpi) [59]. Reinnervation peaked at 18 dpi and experi- mental guts showed higher fiber density than in control animals [59]. To- gether, new neurons can be generated in an inflammatory setting. This sug- gests that inflammation might have positive effects on neural regeneration in the ENS. Even though inflammatory responses have been documented af- ter ENS injury, many experimental settings/studies did not report if there was inflammation or not. Also, it has not directly been tested if an inflamma- tory response impacts the regenerative processes as it does in the CNS. Thus, further study of the role of inflammation for ENS regeneration is necessary specifically in the context of different types of injury. 1.7. The zebrafish ENS regenerates neurons after focal ablation Recent studies have found that zebrafish generate new neurons after focal ablation of a small fraction of ENS neurons (Figure 3A, [64, 65]). This is consistent with the continuous generation of new ENS neurons into adult- hood in zebrafish [66]. After laser-ablation of a small number of neurons in the distal zebrafish larval gut, new neurons are generated at the site of abla- tion, and neuron numbers increase 5 and 10 days post-ablation (dpa) at a significantly higher rate than in the control (Figure 3B). It remains to be de- termined if neuron numbers completely catch up to control levels and if these neurons restore gut function. The study by Ohno et al. (2021) also shed light on potential cellular mechanisms that drive neuronal regeneration in the zebrafish ENS (Figure 3B). Nerve fibers first enter the ablated area at 1 dpa forming a bridge across the ablated area. At 1 dpa, sox10 positive enteric progenitor cells (EPCs) seem to enter the area of ablation via these nerve fiber bridges (Figure 3B). These EPCs proliferate indicating that progenitor cell proliferation drives ENS regeneration in zebrafish [64]. These data suggest that zebrafish can generate new neurons after injury, but the occurrence of regeneration in adult zebrafish remains to be tested. Thus, zebrafish are a promising new research model to study the cellular and molecular mecha- nisms underlying ENS regeneration. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 Figure 3. Regenerative processes in the zebrafish ENS after cell ablation. (A) Exper- imental setup of focal laser ablation (red) of a small set of ENS neurons in the zebrafish larvae. (B) Steps of regenerative processes in the zebrafish ENS in the ab- lated area marked by lines: (1) formation of nerve fiber bridge across the ablated area. (2) sox10 positive enteric progenitor cells (EPCs, blue) appear in the ablated area. (3) new neurons (green) are generated in the ablated area. A-B. modified from [64]. Figure 3. Regenerative processes in the zebrafish ENS after cell ablation. (A) Exper- imental setup of focal laser ablation (red) of a small set of ENS neurons in the zebrafish larvae. (B) Steps of regenerative processes in the zebrafish ENS in the ab- lated area marked by lines: (1) formation of nerve fiber bridge across the ablated area. (2) sox10 positive enteric progenitor cells (EPCs, blue) appear in the ablated area. 1.7. The zebrafish ENS regenerates neurons after focal ablation (3) new neurons (green) are generated in the ablated area. A-B. modified from [64]. References 1. Ganz, J., Gut feelings: Studying enteric nervous system development, function, and disease in the zebrafish model system. Dev Dyn 2018, 247, (2), 268-278. 2. Heanue, T. A.; Shepherd, I. T.; Burns, A. J., Enteric nervous system development in avian and zebrafi Dev Biol 2016, 417, (2), 129-38. 3. Rao, M.; Gershon, M. D., Enteric nervous system development: what could possibly go wrong? Nat Rev Neurosci 2018, 19, (9), 552-565. 4. Belkind-Gerson, J.; Graeme-Cook, F.; Winter, H., Enteric nervous system disease and recovery, plasticity, and regeneration. J Pediatr Gastroenterol Nutr 2006, 42, (4), 343-50. 5. Furness, J. B., The enteric nervous system. Blackwell Publishing: Oxford, 2006. 6. Furness, J. B.; Callaghan, B. P.; Rivera, L. R.; Cho, H. J., The enteric nervous system and gastrointestinal innervation: integrated local and central control. Adv Exp Med Biol 2014, 817, 39-71. g 7. Rolig, A. S.; Mittge, E. K.; Ganz, J.; Troll, J. V.; Melancon, E.; Wiles, T. J.; Alligood, K.; Stephens, W. Z.; Eisen, J. S.; Guillemin, K., The enteric nervous system promotes intestinal health by constraining microbiota composition. PLoS Biol 2017, 15, (2), e2000689. 8. Wiles, T. J.; Jemielita, M.; Baker, R. P.; Schlomann, B. H.; Logan, S. L.; Ganz, J.; Melancon, E.; Eisen, J. S.; Guillemin, K.; Parthasarathy, R., Host Gut Motility Promotes Competitive Exclusion within a Model Intestinal Microbiota. PLoS Biol 2016, 14, (7), e1002517. 9. Neunlist, M.; Van Landeghem, L.; Mahe, M. M.; Derkinderen, P.; des Varannes, S. B.; Rolli-Derkinderen, M., The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease. Nat Rev Gastroenterol Hepatol 2013, 10, (2), 90-100. ( ) 10. Nishiyama, K.; Sugiyama, M.; Mukai, T., Adhesion Properties of Lactic Acid Bacteria on Intestinal Mucin. Microorganisms 2016, 4, (3). abella, G., On the plasticity of form and structure of enteric ganglia. J Auton Nerv Syst 1990, 30 Suppl, S59-6 affrey, M. J., Cellular changes in the enteric nervous system during ageing. Dev Biol 2013, 382, (1), 344-55. 11. Gabella, G., On the plasticity of form and structure of enteric ganglia. J Auton Nerv Syst 1990, 30 Suppl, S59-66. 12. Saffrey, M. J., Cellular changes in the enteric nervous system during ageing. Dev Biol 2013, 382, (1), 344-55. 12. Saffrey, M. J., Cellular changes in the enteric nervous system during ageing. Dev Biol 2013, 382, (1), 344-55. 13. Saffrey, M. J., Aging of the mammalian gastrointestinal tract: a complex organ system. Age (Dordr) 20 9603. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084.v1 doi:10.20944/preprints202204.0084.v1 Funding: This research was funded by Funds from Michigan State University to J.G, by the American Neurogastroenterology and Motility Society Research Grant to J.G and by the NSF CAREER grant # 2143267 to J.G. Conflicts of Interest: The authors declare no conflict of interest 2. Outlook Previous studies have shown that ENS regeneration in mammals is lim- ited. Zebrafish have been put forward as a new model organism to study ENS regeneration. The zebrafish model has the potential to answer the ques- tions of how a robust regeneration response is controlled on a cellular and molecular level. It will be important to determine if a more extensive loss of ENS neurons still results in robust regeneration in zebrafish. Also, full func- tional recovery after neuron loss needs to be determined using approaches such as intestinal transit assay, or gut motility imaging [67, 68]. Important questions remain before regenerative approaches can be used to treat ENS disorders. The regenerative ability of mammalian models needs to be tested further with longer observation periods and a detailed analysis of functional recovery. This will also reveal important differences in the regenerative ability between ENS neuron types, gut regions, and species. g y yp g g p The field also needs a detailed understanding of how ENS regeneration is regulated on a cellular and molecular level both in animal models with robust vs. limited regenerative capacity. This will allow for the determina- tion of 1) which cell types are present during ENS regeneration, 2) which molecular changes take place, and 3) which cell lineages generate new neu- rons. For example, ENS glia cells generate neurons during adult ENS neuro- genesis [66, 69] and ENS glia cells have been suggested as a potential pro- genitor cell source in some injury settings [57, 60]. Also, the regulatory pro- grams underlying ENS regeneration need to be determined. This infor- mation will provide the critical framework of cell biological processes and molecular-genetic factors essential for successful regeneration in the ENS. These are necessary steps for identifying differences in the cellular and mo- lecular composition between model systems with high vs. low regenerative capacity in the ENS. Understanding these aspects of ENS regeneration will pave the way for using regenerative processes as a therapeutic approach to treating ENS disorders. Author Contributions: Conceptualization, H.R., J.G.; Writing – Original Draft Prep- aration, H.R., J.G.; Writing – Review & Editing, H.R., J.G.; Supervision, J.G.; Funding Acquisition, J.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Funds from Michigan State University to J.G, by the American Neurogastroenterology and Motility Society Research Grant to J.G and by the NSF CAREER grant # 2143267 to J.G. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 9 April 2022 doi:10.20944/preprints202204.0084 H.; Wang, J.; Hen, R.; Gershon, M. D., 5-HT4 receptor-mediated neuroprotection and neurogenesis in the enteric nervous system of adult mice. J Neurosci 2009, 29, (31), 9683-99. 38. Brookes, S. J.; Lam, T. C.; Lubowski, D. Z.; Costa, M.; King, D. W., Regeneration of nerve fibres across a colonic anastomosis in the guinea-pig. J Gastroenterol Hepatol 1996, 11, (4), 325-34. 39. Matsuyoshi, H.; Kuniyasu, H.; Okumura, M.; Misawa, H.; Katsui, R.; Zhang, G. X.; Obata, K.; Takaki, M., A 5- HT(4)-receptor activation-induced neural plasticity enhances in vivo reconstructs of enteric nerve circuit insult. 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https://openalex.org/W4396615813	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/82EBED5632BF11E5A629262CFF01BE2D/S0007123423000728a.pdf/div-class-title-mind-the-gap-why-wealthy-voters-support-brexit-div.pdf	English	null	Mind the Gap: Why Wealthy Voters Support Brexit	British journal of political science	2,024	cc-by	14,211	Mind the Gap: Why Wealthy Voters Support Brexit Jane Green1 and Raluca L. Pahontu2 Jane Green1 and Raluca L. Pahontu2 Jane Green1 and Raluca L. Pahontu2 J 1Department of Politics and International Relations, Nuffield College, University of Oxford, Oxford, UK and 2Department of Political Economy, King’s College London, London, UK Corresponding author: Jane Green; Email: jane.green@nuffield.ox.ac.uk (Received 1 December 2021; revised 28 April 2023; accepted 20 December 2023) 1Department of Politics and International Relations, Nuffield College, University of Oxford, Oxford, UK and 2Department of Political Economy, King’s College London, London, UK Corresponding author: Jane Green; Email: jane.green@nuffield.ox.ac.uk (Received 1 December 2021; revised 28 April 2023; accepted 20 December 2023) (Received 1 December 2021; revised 28 April 2023; accepted 20 December 2023) (Received 1 December 2021; revised 28 April 2023; accepted 20 December 2023) British Journal of Political Science (2024), page 1 of 21 doi:10.1017/S0007123423000728 © The Author(s), 2024. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. 1In a risky situation one can assign probabilities to the various possible outcomes, whereas in a case of uncertainty one is unable to identify the relevant probabilities. Similar to Baderin and Barnes (2020), we use risk in this paper to denote inter- mediate cases, that are neither instances of pure risk nor of pure uncertainty. © The Author(s), 2024. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. © The Author(s), 2024. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative 1In a risky situation one can assign probabilities to the various possible outcomes, whereas in a case of uncertainty one is unable to identify the relevant probabilities. Similar to Baderin and Barnes (2020), we use risk in this paper to denote inter- mediate cases, that are neither instances of pure risk nor of pure uncertainty. Abstract Wealth provides self-insurance against financial risk, reducing risk aversion. We apply this insurance mechanism to electoral behaviour, arguing that a voter who desires a change to the status quo and who is wealthy is more likely to vote for change than a voter who lacks the same self-insurance. We apply this argument to the case of Brexit in the UK, which has been widely characterized as a vote by the ‘economically left-behind’. Our results show that individuals who lacked wealth are less likely to sup- port leaving the EU, explaining why so many Brexit voters were wealthy, in terms of their property wealth. We corroborate our theory using two panel surveys, accounting for unobserved individual-level hetero- geneity, and by using a survey experiment. The findings have implications for the potential broader role of wealth-as-insurance in electoral behaviour and for understanding the Brexit case. Keywords: Brexit; wealth; insurance; risk; voting behaviour https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Keywords: Brexit; wealth; insurance; risk; voting behaviour Yet, ‘not all voters who are disenchanted with the status quo take a chance on the less known alternative’ (Morgenstern and Zechmeister 2001). As status quo changes pose potential economic risks to individuals, voters’ decisions are likely affected by their tolerance to that risk (Morisi 2018). Risk aversion is often linked to a stable personality trait, but a growing body of literature docu- ments its variation with wealth. In particular, the accumulation of wealth is associated with a higher tolerance for risk (Donkers, Melenberg, and Van Soest 2001; Pahontu 2020; Zanetti 2014). This happens because wealth provides insurance for individuals (Ansell 2014) in case of income shocks or economic risks (Hariri, Jensen, and Lassen 2020; Tertytchnaya and De Vries 2019). This should be highly relevant to electoral choice where risk aversion is a central compo- nent, such as in status quo votes (Liñeira and Henderson 2021; Morisi 2018; Steenbergen and Siczek 2017). We focus on the role of wealth in informing risk aversion and its effect on decision- making to study why some individuals refrain from supporting status quo changes despite want- ing to do so. We apply this framework to the case of Brexit, a high-stakes referendum that posited the eco- nomic, cultural, and sovereignty costs and benefits of leaving the European Union against the risks of fundamentally changing the UK’s relationship with its largest trading partner, the EU. The dominant explanation has been a focus on those ‘left behind’ from the economic and cultural benefits of globalization, either in the form of direct localized economic grievances (Carreras 2019; Colantone and Stanig 2018; Fetzer 2019) or through the relationship between economic and immigration concerns (Carreras, Irepoglu Carreras, and Bowler 2019; Green, Hellwig, and Fieldhouse 2022; Sobolewska and Ford 2020). In reality, the Brexit vote was heterogeneous. According to British Election Study (BES) data collected immediately after the 2016 EU referendum (Fieldhouse et al. 2016), 73 per cent of Leave voters in the referendum were homeowners (71 per cent for Remain voters); 60 per cent of Leave voters were ‘very unlikely’ or ‘unlikely’ to become unemployed (53 per cent for Remain voters); and 23 per cent of Leave voters were ‘very unlikely’ or ‘likely’ to be at risk of poverty (28 per cent for Remain voters). Older voters in Britain were substantially more likely to have voted Leave. Keywords: Brexit; wealth; insurance; risk; voting behaviour Many choices in political behaviour concern trade-offs between the net benefits a choice may bring and its potential risks. This is especially true in high-stakes elections that usher in major changes to the status quo: ‘in democracies, citizens can be called upon to make decisions that have profound and irreversible consequences, yet the environment in which they make these decisions is inherently uncertain, and sometimes hazardous’ (Nadeau, Martin, and Blais 1999, 523). Political actors compete over alternative versions of the likely costs and benefits of such a contentious electoral choice. However, this discord increases the risk to a voter who has to decide between the two sides of a consequential decision (Alvarez and Franklin 1994; Franklin 1991).1 Referendums provide such high-stakes and risky decisions (De Vries 2018; Hobolt 2009). Voters are asked to choose between a familiar status quo and the much less certain outcome of a fundamental change. When faced with such choices, voters tend to be biased toward the sta- tus quo (Masatlioglu and Ok 2005; Samuelson and Zeckhauser 1988) – for example, as seen in the cases of independence for Quebec, Catalonia, and Scotland (Hierro and Queralt 2021). Status quo votes tend to be irreversible in the foreseeable future. They relate to profound pol- itical, economic, cultural and constitutional change and, by definition, tend to lack local or recent precedent. However, voters may perceive benefits from changing the status quo, such as greater sovereignty, economic independence, and appeals to national identity. This was the experience of former Soviet republics, the unification of East and West Germany, and decisions to join, or 1In a risky situation one can assign probabilities to the various possible outcomes, whereas in a case of uncertainty one is unable to identify the relevant probabilities. Similar to Baderin and Barnes (2020), we use risk in this paper to denote inter- mediate cases, that are neither instances of pure risk nor of pure uncertainty. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Jane Green and Raluca L. Pahontu 2 indeed leave, the European Union’s economic area and monetary union (Abadie, Diamond, and Hainmueller 2015; Atikcan, Nadeau, and Belanger 2020; De Vries 2018; Hierro and Queralt 2021; Hobolt 2009; 2016). indeed leave, the European Union’s economic area and monetary union (Abadie, Diamond, and Hainmueller 2015; Atikcan, Nadeau, and Belanger 2020; De Vries 2018; Hierro and Queralt 2021; Hobolt 2009; 2016). https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Keywords: Brexit; wealth; insurance; risk; voting behaviour They were also more likely to have wealth through assets and savings and more likely to have higher economic security as a result (Chrisp and Pearce 2019; Green and de Geus 2022). There were also more Eurosceptics in the British population than voters who opted to leave the EU, suggesting that a sizeable proportion of voters might have seen greater downsides of membership of the EU but did not take the risk of voting Leave. p g We argue that this pattern can be understood through a self-insurance perspective. More voters in poorer areas of the UK saw benefits to Brexit than in prosperous ones (Adler and Ansell 2020; Ansell 2019; Carreras 2019). But within those areas, we show that individuals lacking self-insurance in the form of personal wealth were less likely to vote to leave the EU than those within the same areas who had assets. To identify this effect, we use a variety of individual-level data sources on wealth and public opinion, including two nationally representative panel surveys and a survey experiment. It is not common for detailed wealth measures to be combined with detailed political variables, so we took advantage of new datasets that made this possible. We present multiple sets of evidence in sup- port of our core expectation that wealth (at the individual level) increases support for Brexit and that this effect is due to its role in decreasing risk aversion. With little or no insurance, poorer Britons are less likely to support a status quo change and vote for Brexit. We also find that this effect holds once we study these individual-level effects within areas classified as above and below local median wealth levels. At the aggregate level, we replicate the patterns highlighted by Ansell (2019), Adler and Ansell (2020), and Carreras (2019), showing that wealthier areas British Journal of Political Science British Journal of Political Science 3 exhibited higher support for Brexit overall. But within those areas, wealth was associated with higher support for Leave. g The implications of our analysis may be instructive for contexts other than referendums con- cerning a risky (economic) choice. g g ( ) 3A further distinction can be made between the economic security afforded by liquid wealth in the form of savings and illiquid wealth in the form of assets. Liquid wealth, readily available savings (net of debts), may compensate for short-term income losses, smoothing consumption in the short term. This logic is illustrated by Hariri, Jensen, and Lassen (2020), who 2Savings and home ownership ensure forms of wealth. This is in contrast to higher-risk market speculation, which is far more uncommon (Alan 2006). Market speculation is more likely for those who have secure forms of wealth to fall back on, such that individuals with greater wealth are insured against risks (Vestman 2019). Keywords: Brexit; wealth; insurance; risk; voting behaviour While others have documented the importance of wealth as a source of economic voting (Lewis-Beck and Nadeau 2011; Nadeau, Foucault, and Lewis-Beck 2011; Nadeau, Lewis-Beck, and Foucault 2019), the application of the wealth-as-insurance logic might explain why, when choosing between an incumbent and uncertain hypothetical future under a challenger (Fiorina 1977; Fiorina 1981), some voters opt for the more predictable status quo. This might be especially important when an electoral choice could trigger a period of dramatic economic change or instability. That certainly applies to other secession referenda and may apply to electoral choices where a voter prefers certain goals (such as sovereignty, immigra- tion, environmental protection, and public spending), but the change itself will result in eco- nomic instability. A wealthier individual may be more likely to support a radical economic change associated with, for example, rapid decarbonization if they are insured against economic disruption. This logic expands the idea that economic inequalities drive people’s political prefer- ences by showing that they also alter people’s ability to act on those preferences. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Wealth as Insurance People care about risk and are likely to demand insurance against those risks (Moene and Wallerstein 2001). Insurance typically refers to welfare state provisions that provide a last-resort financial cushion against severe misfortune. As a result of its insuring role, support for welfare spending tends to be higher among individuals who rely on social insurance, as well as those faced with higher risks of unemployment, sickness, or exposure to crime (Hacker 2019; O’Grady 2019; Rehm, Hacker, and Schlesinger 2012; Rueda and Stegmueller 2016). However, many have increasingly documented the importance of ‘self-insurance’, which takes the form of an individual’s wealth (Ansell 2014; Busemeyer and Iversen 2020; Hilt and Rahn 2020; Tertytchnaya and De Vries 2019). Wealth provides an income buffer to individuals and their families in case of misfortune, such as job loss, inflationary pressures, and other market fluc- tuations. A lack of wealth in the form of savings or assets is an extremely important form of eco- nomic insecurity, as shown in a range of studies and applications (Ansell 2014; Conley and Gifford 2006; Ehrlich and Becker 1972; Pahontu 2020; Tertytchnaya and De Vries 2019). Those who lack wealth are more likely to support social insurance policies to insulate against income shocks (Hariri, Jensen, and Lassen 2020). Wealthier individuals are also found to be sig- nificantly less risk averse in light of their increased economic security (Donkers, Melenberg, and Van Soest 2001; Guiso and Paiella 2008; Malmendier and Nagel 2011; Paravisini, Rappoport, and Ravina 2017; Zanetti 2014).2 Wealth provides a permanent stock of financial means to buffer against risk. The same cannot be said of a person’s income absent wealth. If high incomes provide a buffer, they do so through the accumulation of wealth made possible by savings and asset holding. Due to its transitory nature, income does not offer the same type of economic security as wealth. This is because indi- viduals experiencing a drop in income, in the absence of wealth, cannot smooth consumption. We note that there is only a weak empirical correlation between wealth and income (Ansell 2019), which supports the importance of focusing on both wealth and income to understand peo- ple’s economic interests.3 As Hariri, Jensen, and Lassen (2020, 893) state, ‘economic vulnerability, https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Jane Green and Raluca L. Wealth as Insurance Pahontu 4 measured by a lack of access to economic buffers, is also common among middle-class and rich households and, thus, largely unrelated to current income’. Many high-income, middle-class households lack the economic security provided by wealth. On the other hand, consistent with the life cycle model (Ando and Modigliani 1963), wealth is especially high among older (retired) generations who otherwise have lower incomes, as is the case in the UK (Chrisp and Pearce 2019; Green and de Geus 2022). This happens partly because these individuals also have lower debt levels, particularly housing debt (Wolff 2010). p y g Home ownership, in particular, increases an individual’s sense of economic security (Ronald and Doling 2012; Weller 2007; Williams 2014). People buy homes for a sense of agency and as a long-term economic investment, both of which are sources of personal and economic security. Dupuis and Thorns (1998) argue that home ownership can provide a sense of ontological security in a world that is otherwise experienced as threatening and uncontrollable. In addition, asset ownership is associated strongly with the ability to borrow, and borrowing ability provides add- itional economic security against short-term income losses (Aladangady 2017). Home ownership is increasingly out of reach for people with unreliable income sources, unsecured debts, poor credit ratings, and without other borrowing potential, such as through family. Therefore, asset ownership reflects higher economic security, both as a proxy for greater access to potential short- term consumption smoothing through borrowing and as a source of longer-term household eco- nomic and psychological security. p y g y The security afforded through wealth should depend on its availability, its reliability as a form of insurance against economic shocks, and how different forms of wealth might ‘de-risk’ particu- lar electoral choices. While standard economic theory expects individuals to smooth consump- tion and savings over their lifetime (Browning and Crossley 2001), few individuals actually accumulate substantial savings in practice. Property wealth is typically substantially higher than financial wealth.4 In the UK, this is due, at least in part, to a series of government-induced incentives to take up home ownership (Lewis-Beck, Nadeau, and Foucault 2013). The appreci- ation of house prices has been far higher over a longer period than interest rates on savings or returns from the British stock market (Chrisp and Pearce 2019). Moreover, UK property wealth has been remarkably buoyant in response to major economic downturns. show that a lack of savings, as opposed to holding assets, explains support for social insurance policies in Denmark. More generally, however, asset-based and liquid wealth have been shown to offer a considerable psychological benefit to an indi- vidual (Kendall, Nguyen, and Ong 2019). 4For the UK, see Banks, Blundell, and Smith (2002); Crawford, Innes, and O’Dea (2016). See Causa, Woloszko, and Leite (2019) for comparative evidence. The mean (median) UK property wealth is £85,000 (£50,000), in contrast to £28,000 (£4,000) in savings (Crawford, Innes, and O’Dea 2016). The Brexit Case The Brexit cost-benefit calculation turned on a combination of the cultural, economic and pol- itical benefits of EU independence or the opposite costs of exiting the EU (Clarke, Goodwin, and Whiteley 2017; Evans and Menon 2017; Green, Hellwig, and Fieldhouse 2022; Hobolt 2016; Iakhnis et al. 2018). Much of the explanation for the Brexit vote draws on wider insights into the drivers of populism (Autor et al. 2019; Ford and Goodwin 2014; Mudde 2010; Van Hauwaert and Van Kessel 2018) and mirrors the interpretation of the election of Donald Trump in the United States (Bobo 2017; Mutz 2018; Norris and Inglehart 2019; Schaffner, MacWilliams, and Nteta 2018). In the UK, researchers pointed to the combination of cultural and economic grievances that motivated support for greater independence from the EU. The degree to which these motives are economic has been the source of considerable debate, with immigration concerns, national identity, and sovereignty being strongly related to voting Leave or Remain (Clarke, Goodwin, and Whiteley 2017; Hobolt 2016; Iakhnis et al. 2018; Norris and Inglehart 2019; Sobolewska and Ford 2020). Others have argued for the importance of local economics, either directly or via a connection between localized economic decline and immigration concerns, point- ing to local factors such as import shocks (Colantone and Stanig 2018), austerity (Fetzer 2019), and long-term relative local economic decline (Carreras, Irepoglu Carreras, and Bowler 2019). g p g These studies provide information on the sources of people’s Brexit preferences but not the risk calculation associated with a vote against the status quo. A status quo bias was evident in other referendums where the anti-EU vote was presented as entailing costly economic conse- quences (Born et al. 2019; Breinlich et al. 2017; Dhingra et al. 2017; Hobolt 2016). ‘People will only be expected to risk voting for Brexit when they perceive that their country could do as well, or even better outside’ (De Vries 2018, 156). Among those who have incorporated risk in the case of Brexit, researchers have pointed to three possible answers for why Britons voted by a majority to leave the EU. The first is that Leave voters and supporters of populist movements are generally less risk averse (Morisi 2018; Steenbergen and Siczek 2017) and less likely to be dissuaded by the risks associated with a depart- ure from the status quo. 5Note that our analysis shows that Leavers are not wealthier (or significantly poorer) than Remain voters. We demonstrate this in Fig. C.2. Wealth as Insurance Ansell (2014) shows how property wealth in the UK rebounded strongly after the 2008 global financial crisis, whereas the same period has been much more detrimental to savers. This property wealth accumulation offered homeowners economic security and appreciation in a protracted period of economic downturn, historically low interest rates on savings, and austerity. Moreover, post-2008 financing regulations meant that UK mortgages were far less likely to suffer from negative equity as afford- ability criteria were made more stringent. Those owning a home in the UK, post-2008–2009, were more likely to hold a secure asset that would remain secure against short-term income shocks, longer-term national-level recessions, or weak growth. Our key point is that the political and economic context should determine which type of wealth is most insuring. We expect property wealth, at least within the UK, to offer considerable economic security. This should be especially true for the case we explore in this paper – Brexit – where the short-term economic shock of leaving the EU might have favoured a focus on liquid assets, but the economic debate centred on the much longer-term economic consequences of Britain’s new trading partnerships and economic costs and benefits, and long-term projections about an overall loss of GDP. In this context, housing wealth gives individual households a https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press British Journal of Political Science British Journal of Political Science 5 5 reliable return on their assets and savings, and their borrowing ability helps smooth consumption in the shorter term. We expect wealth to alter support for the status quo in addition to a standard cost-benefit calculation. Despite having similar preferences, a wealthier individual is more likely to sup- port a status quo change than her less wealthy counterpart. This happens because wealth cushions individuals against risks, making wealthier individuals less risk averse. Incorporating risk aversion into the cost-benefit calculation enhances the understanding of status quo support. When calculating net benefits, a more risk averse individual should put higher weight on costs rather than benefits, dampening their decision to change the status quo. In brief, for a given set of preferences, wealthier individuals are expected to be more likely to support a change to the status quo. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press The Brexit Case For example, Morisi (2018) shows that levels of risk aversion are espe- cially important for less-informed voters and applies this finding to the EU and Scottish independence referenda. This expectation could help point to the importance of lower risk aver- sion via higher wealth among Leave voters, which is consistent with our expectations.5 The second is that Leave voters were persuaded against the risks associated with leaving the EU; that is to say, the Leave campaign successfully ‘de-risked’ the question of Brexit (Atikcan, Nadeau, and Belanger 2020). However, it was also the case that the Remain vote was much higher than the proportion of Britons who held a prior Eurosceptic preference; and economic concerns https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Jane Green and Raluca L. Pahontu 6 were their main reported motivation.6 This suggests that many Remain voters might have voted for Brexit had many not been convinced of the risks associated with an exit with the EU. Figure 1 shows the relationship between Euroscepticism before the referendum (in Wave 8 of the British Figure 1. Euroscepticism distribution among Remain voters. Figure 1. Euroscepticism distribution among Remain voters. were their main reported motivation.6 This suggests that many Remain voters might have voted for Brexit had many not been convinced of the risks associated with an exit with the EU. Figure 1 shows the relationship between Euroscepticism before the referendum (in Wave 8 of the British Election Study (BES) internet panel) and voting Leave in the referendum (Wave 9 of the BES panel). Many people who voted Remain in the referendum held strong preferences for greater independence from the EU. were their main reported motivation.6 This suggests that many Remain voters might have voted for Brexit had many not been convinced of the risks associated with an exit with the EU. Figure 1 shows the relationship between Euroscepticism before the referendum (in Wave 8 of the British Election Study (BES) internet panel) and voting Leave in the referendum (Wave 9 of the BES panel). Many people who voted Remain in the referendum held strong preferences for greater independence from the EU. p The third explanation, consistent with prospect theory, is that voters who resided in areas that had experienced economic decline were willing to risk Brexit since, relatively speaking, they thought the gains to the national economy would be higher (Carreras 2019). 6John Curtice (2016 ) How Deeply Does Britain’s Euroscepticism Run? British Social Attitudes 33; Prosser, Mellon and Green (2016) What Mattered Most to You When Deciding How to Vote in the EU Referendum? British Elections Study Blog. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press The Brexit Case These communities had little to lose and were more likely to support independence from the EU. This explanation is fundamentally a sociotropic one, rooted in the localized experiences of communities who saw that their community might do better. We contend that individual wealth, through its role in reducing risk aversion, should be a use- ful addition to these accounts. We propose that people in more economically deprived areas of the UK are more likely to prefer Brexit, which is consistent with prospect theory. But, within those areas, wealthier individuals are more likely to sustain their preferences in support of a change in the status quo. By providing insurance and reducing individuals’ tolerance to risk, wealth ‘de-risks’ the vote for a change in the status quo. We complement existing work by show- ing that individuals’ economic circumstances are just as important as sociotropic or local eco- nomic experiences. In other words, we expect wealthier individuals in ‘left-behind communities’ to be more likely to vote for Brexit than less wealthy individuals in those same areas and, similarly, wealthy voters to be more likely to support Brexit than less wealthy indivi- duals in prosperous areas. Indeed, this is consistent with considerable heterogeneity in the Brexit vote. The majority of Leave voters were not economically deprived or insecure. Brexit voters may have been more likely to live in parts of the country that had not benefited from rapid growth through globalization, immigration, and the expansion of high-skilled labour. The vote to remain in the EU was higher https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press British Journal of Political Science British Journal of Political Science 7 in cities, which are also composed of greater numbers of younger voters, graduates, and ethnic minorities (the demographic groups most likely to support Remain). Those living outside these metropolitan hubs may have felt resentful of more prosperous areas and groups (Green, Hellwig, and Fieldhouse 2022). However, at the individual level, more homeowners supported Brexit (73 per cent) than those who voted Remain (71 per cent), 60 per cent of those reporting to be very unlikely or unlikely to become unemployed supported Brexit compared to 53 per cent for Remain, 23 per cent of Leave voters reported being very unlikely or unlikely to encounter issues covering day-to-day living costs compared to 48 per cent of Remain.7 In short, Leave voters were, on average, more economically secure than Remain voters. 7Authors’ own calculations based on the raw post-EU referendum British Election Study data (Fieldhouse et al. 2016), weighted for national representativeness. 8See Fieldhouse et al. (2018) for further information about BES Wave 14 and Anderson et al. (2016) about the Bank of England survey. BES data collection, in which our experiment is also conducted, is subject to ethical approval at the University of Manchester. Data are collected by YouGov, who compensate respondents with points redeemed in payments according to YouGov processes on numbers (and lengths) of surveys completed. Note that no wealth data existed prior to the EU referendum, where EU referendum preferences were included. The Brexit Case Furthermore, economic left- right values exhibited no statistical association with Leave support, which was most strongly cor- related with liberal-authoritarian and immigration attitudes, which correlate with education and age rather than income (Fieldhouse et al. 2021). Such indicators of economic security are pre- dominant among older generations, who favour Leave, with greater social conservatism and more negative attitudes about immigration (Green and de Geus 2022). g g Applying this logic to the Brexit vote, we derive three hypotheses. The first is that, controlling for predictors of Brexit preference (the cost-benefit part of the Brexit calculation), wealth will have a positive relationship with support for leaving the EU. H1: At the individual level, wealth is positively associated with support for leaving t We also expect wealthier individuals to perceive themselves to have greater economic insurance, to be insulated against the economic consequences of Brexit. We can assess this through percep- tions about the personal and national economic consequences of Brexit. Consistent with our insurance argument, wealth should not predict expected national-level economic circumstances, but wealth should increase perceptions that personal economic circumstances should be unaffected by leaving the EU. H2: At the individual level, wealth is positively associated with expectations of no Brexit effects on personal economic circumstances. Finally, to further corroborate the theorized causal argument, we expect wealth, consistent with the existing literature, to lead to lower risk aversion. H3: An increase in wealth leads to a decrease in risk aversion. Data and Methods https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Data and Methods We test our expectations with observational data from the British Election Study (BES) internet panel and data from the Bank of England’s 2016–2018 panel survey of income and expenditure. These data sources offer a rare opportunity since national election surveys rarely include individual wealth measures (Nadeau, Foucault, and Lewis-Beck 2011) and federal banking datasets rarely include questions about political preferences, but include plenty of measures of individual wealth. We also designed an additional survey experiment administered as part of the BES by YouGov in 2019.8 https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press 8 Jane Green and Raluca L. Pahontu Most studies rely on household income to proxy wealth. However, as discussed earlier, the cor- relation between income and wealth is weak (Ansell 2019). Therefore, we try to better approxi- mate an individual’s economic circumstances and insurance leverage by relying on respondents’ reported financial and property wealth and adjusting all these measures for household size (Browning, Chiappori, and Lewbel 2013). We used the BES panel study to field a battery of questions on wealth. These wealth measures are available in 2018 (Wave 14). Usefully, the BES panel allows us to link individuals to their loca- tions, thereby enabling us to examine our findings across aggregate patterns. However, the BES data only provides a snapshot into citizens’ wealth in one wave of the BES panel and is limited in making broader claims about wealth accumulation and potential changes in political support. The Bank of England (BoE) data provides us with a way to tackle this shortcoming. It includes three waves of wealth measurement and Brexit support, asked to the same respondents, covering the period 2016–2018.9 Relying on both datasets further allows us to distinguish and account for respondents’ inten- tions and their current support for Brexit. The dependent variable in the BES asks respondents their prospective vote intention: ‘If there was a referendum on Britain’s membership of the European Union, how do you think you would vote?’ This divides respondents into two groups: Leavers and Remainers. BoE respondents are asked, ‘Taking everything into account, how do you [currently – as of 2016/2017/2018] view the UK voting to leave the EU (European Union) in the recent referendum – which has become known as “Brexit”?’ Possible responses vary from ‘very positive’ to ‘very negative’. 9Although the data was collected after the referendum, we were reassured of the validity of our analysis as individuals’ support for Brexit remained stable over time (Grynberg, Walter, and Wasserfallen 2020). In fact, in BES data, about 97 per cent of people and in BoE about 90 per cent of others maintained their preference across time. 10We exclude the undecided from the analysis and note that Brexit preferences are relatively stable over time (Grynberg, Walter, and Wasserfallen 2020). 11In our analysis, we control for whether a respondent owns their home, with or without a mortgage, and the results remain robust. 12Leavers appear less likely to report their home value. In order to address this, we report the robustness of our results in Fig. D.1 by including those who answered ‘do not know’ in the regression. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Data and Methods These attitudes are then mapped onto a binary Leave/Remain support scale.10 Our main explanatory variable, wealth, is similarly defined in the two datasets. To define financial wealth, we rely on individuals’ reported savings and debt accumulations and adjust this measure by the number of household members. Property wealth is defined as the respon- dent’s home value adjusted for the number of household members and as null if she does not own a house.11 We document all of these measurements in Table 1. We validate the BES and BoE wealth measures against official statistics in Appendix A (in the supplementary material) and document remarkable similarities between the central tendencies reported in the two surveys and those reported among the UK population. In Appendix B, we further explore the distribution of wealth in the BES and BoE samples and find few discrepancies between the two surveys. In Figure A.1, we report the relationship between an individual’s voting intention in the EU referendum and her likelihood of not reporting various items pertaining to her economic circumstances. Leave status does not appear to be correlated with the likelihood of reporting these items.12 Finally, in section C in the appendix, we document the geographical dis- tribution of wealth (at the LSOA level) and its distribution across several individual characteris- tics. As Figure C.1 shows, property wealth is not confined to London; it displays greater variance across the country. Additionally, Figures C.3 and C.4 provide supportive evidence that wealth is not confined to the economically active but is positively correlated with age and education. Further suggestive of the idea that there is no predefined beneficiary of Brexit, in Figure C.2 we report the fact that there are no differences in personal wealth across Remain or Leave voters. ppear less likely to report their home value. In order to address this, we report the robustness of our results in cluding those who answered ‘do not know’ in the regression. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press British Journal of Political Science 9 Table 1. Wealth items Wealth type Question text BES Financial Debt Do you and/or your partner (as applicable) have any debts, not including mortgage/student loans? If ‘yes’: Please pick the approximate value of your household debt. Savings Total amount of deposits and savings (continuous measure) If ‘yes’: Please pick the approximate value of your household savings. Property Homeowner Which of these applies to your home? Question text Do you and/or your partner (as applicable) have any debts, not including mortgage/student loans? If ‘yes’: Please pick the approximate value of your household debt. Total amount of deposits and savings (continuous measure) If ‘yes’: Please pick the approximate value of your household savings. Which of these applies to your home? [Owner, Owner with a mortgage, Rent, Housing Association] If ‘owner’: Please pick the approximate value of your home. Housing tenure [Owner, Owner with mortgage, Rent, Housing Association] If ‘owner’: House value (continuous measure) Based on these measures, we model the relationship between an individual’s wealth and her support for Brexit in the BES sample as follows: (1) Leave = a + b1Financial Wealth + b2Property Wealth + b3X + e (1) where X is a vector of covariates that includes respondents’ disposable income, age, gender, edu- cation, working and marital status, authoritarian values and location based on Office for National Statistics area classifications (to control for the possible effects of area on wealth and Brexit support).13 where X is a vector of covariates that includes respondents’ disposable income, age, gender, edu- cation, working and marital status, authoritarian values and location based on Office for National Statistics area classifications (to control for the possible effects of area on wealth and Brexit support).13 Estimates from Equation 1 may, however, be biased if there are systematic differences in wealthy voters’ unobserved characteristics that are correlated with higher support for Leave. We address this concern by modelling unobserved, time-invariant individual-level heterogeneity in the BoE panel dataset. We are able to estimate Leave support for each individual i at time t as follows: eit = g1Financial Wealthit + g2Property Wealthit + g3Xit + ji + lt + yit (2) (2) where, in addition to equation 1, we account for individual ξi and time λt specific e 13Established area classifications are produced using the 2011 Census and define areas by their economic activity, density, and ethnic diversity. 14This specification not only allows us to have a better claim at identifying a causal effect of wealth on the Leave vote, but allows us to account for the polarization in attitudes that happened as a result of the EU referendum. Data and Methods [Owner, Owner with a mortgage, Rent, Housing Association] If ‘owner’: Please pick the approximate value of your home. BoE Financial Debt Unsecured debt (continuous measure) Savings Total amount of deposits and savings (continuous measure) Property Homeowner Housing tenure [Owner, Owner with mortgage, Rent, Housing Association] If ‘owner’: House value (continuous measure) https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Main Effects Starting with the cross-sectional estimates in the BES sample, Table 2 reveals the importance of accounting for respondents’ wealth to understand their support for Brexit. The wealth measures are standardized to mean 0 and one standard deviation (because respondents may experience financial shortages once accounting for outgoing payments or debts). Hence, the coefficients are interpretable as a standard deviation increase in the intention to vote Leave. We also report the wealth effects visually in Fig. 2. 13Established area classifications are produced using the 2011 Census and define areas by their economic activity, density, and ethnic diversity. 14This specification not only allows us to have a better claim at identifying a causal effect of wealth on the Leave vote, but allows us to account for the polarization in attitudes that happened as a result of the EU referendum. 14This specification not only allows us to have a better claim at identifying a causal effect of wealth on the L allows us to account for the polarization in attitudes that happened as a result of the EU referendum. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Jane Green and Raluca L. Pahontu 10 Table 2. Wealth effect on leave support (BES) 1 2 HH Adj Financial wealth 0.011* −0.004 (0.006) (0.006) HH Adj Property wealth 0.027* 0.013 (0.006) (0.006) HH Adj Disposable income −0.076* −0.014 (0.006) (0.006) Gender −0.028* (0.010) Age 0.003* (0.001) Education: Enrolled in HE −0.078* (0.047) Education: have not completed HE −0.040* (0.021) Education: Graduated from HE −0.081* (0.012) Married −0.006 (0.011) Unemployed 0.058 (0.046) Student 0.041 (0.056) Retired 0.033 (0.016) Not in paid work 0.092* (0.019) Authoritarian-libertarian scale 0.089* (0.002) Controls X ✓ Observations 7,627 7,627 R2 0.018 0.244 Note: The dependent variable and leave vote intention, is binary (1 = Leave). Compared to model (1), model (2) includes controls for disposable income, gender, age, education, marital status, employment status, authoritarian values, and respondents’ location based on the ONS Super Area Group classification. The reference categories are as follows: Gender: male, Education: not enrolled in HE, ONS Area: Affluent England. Robust standard errors are reported in parentheses.*p < 0.01, p < 0.05, p < 0.1. Table 2. Table 2. Wealth effect on leave support (BES) Table 2. Wealth effect on leave support (BES) Note: The dependent variable and leave vote intention, is binary (1 = Leave). Compared to model (1), model (2) includes controls for disposable income, gender, age, education, marital status, employment status, authoritarian values, and respondents’ location based on the ONS Super Area Group classification. The reference categories are as follows: Gender: male, Education: not enrolled in HE, ONS Area: Affluent England. Robust standard errors are reported in parentheses.p < 0.01, p < 0.05, p < 0.1. Financial wealth does not seem to account for Remain or Leave support, whereas a standard deviation increase in property wealth increases Leave support by 1.3–2.7 percentage points. Since property wealth is significantly higher in the UK than financial wealth, these differences are expected, as our foregoing discussion showed.15 Overall, the effects are substantively important; they run in the opposite direction to those assumed in ‘left-behind’ accounts, including running in a counter direction to results at the aggregate level (Adler and Ansell 2020; Ansell 2019). In effect size terms, the effect can be benchmarked against the closeness of the referendum, tilted in favour of Leave by less than a 4 per cent difference, and by the effect sizes for other commonly cited variables, household income (as shown in Table 2), and also against the real-world size of increases in property wealth, which – in the UK case – has risen by an average of 8 percentage points over the five year period that preceded Brexit (Office for National Statistics, 2021). 15We explore the robustness of our results in Fig. D.2 to two alternative explanations correlated with support for Leave, the risk of unemployment and Euroscepticism. Main Effects Wealth effect on leave support (BES) 1 2 HH Adj Financial wealth 0.011 −0.004 (0.006) (0.006) HH Adj Property wealth 0.027* 0.013 (0.006) (0.006) HH Adj Disposable income −0.076* −0.014 (0.006) (0.006) Gender −0.028* (0.010) Age 0.003* (0.001) Education: Enrolled in HE −0.078* (0.047) Education: have not completed HE −0.040* (0.021) Education: Graduated from HE −0.081* (0.012) Married −0.006 (0.011) Unemployed 0.058 (0.046) Student 0.041 (0.056) Retired 0.033 (0.016) Not in paid work 0.092* (0.019) Authoritarian-libertarian scale 0.089* (0.002) Controls X ✓ Observations 7,627 7,627 R2 0.018 0.244 Note: The dependent variable and leave vote intention, is binary (1 = Leave). Compared to model (1), model (2) includes controls for disposable income, gender, age, education, marital status, employment status, authoritarian values, and respondents’ location based on the ONS Super Area Group classification. The reference categories are as follows: Gender: male, Education: not enrolled in HE, ONS Area: Affluent England. Robust standard errors are reported in parentheses.*p < 0.01, p < 0.05, p < 0.1. 16There may be greater heterogeneity in preferences among the wealthy such that, for example, they expect personal eco- nomic circumstances to improve after Brexit. We explore this possibility in Fig. D.3 and found no evidence that wealthier individuals expected circumstances to improve. However, as Fig. 3 shows, we find evidence that they expected no change in circumstances, consistent with an insurance mechanism. Mechanism The property wealth effect could be due to a couple of factors that relate to wealthy respondents’ expectations of the economic impact of Brexit. Consistent with our insurance argument, we 15We explore the robustness of our results in Fig. D.2 to two alternative explanations correlated with support for Leave, the risk of unemployment and Euroscepticism. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press British Journal of Political Science 11 Figure 2. Wealth increases leave support. Note: The dependent variable, leave vote intention, is binary (1 = Leave). Controls include household-adjusted disposable income, gen- der, age, education, marital status, employment status, authoritarian values, and respondents’ location based on the ONS Super Area Group classification (95% confidence intervals). Figure 2. Wealth increases leave support. Note: The dependent variable, leave vote intention, is binary (1 = Leave). Controls include household-adjusted disposable income, gen- der, age, education, marital status, employment status, authoritarian values, and respondents’ location based on the ONS Super Area Group classification (95% confidence intervals). expect that wealthier respondents are more likely to consider that they will be unaffected econom- ically by Britain’s departure from the EU. Figure 3 explores how national and personal economic situation evaluations vary by property wealth in predicting ‘no change’ in economic circum- stances.16 Consistent with our expectations, wealthier respondents are more likely to think that Brexit will bring no change to their own economic circumstances. However, that is not true about their beliefs about the national outcomes – denoted by a flat line across the wealth distribution. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Aggregate and Individual-Level Expectations of Brexit effect on national vs personal finances (BES). Note: The dependent variable is binary and takes the value 1 if the respondent believes leaving the EU will have no effect on her national or personal circumstances. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). p ( ) ndent variable is binary and takes the value 1 if the respondent believes leaving the EU will have no effect on her onal circumstances. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard % confidence intervals). we find a positive association between property wealth and Leave support when we explore the indi- vidual wealth effect, as in the top panel. Therefore, we explore heterogeneity in individual wealth by low- and high-priced LSOA areas in appendix Figure E.2. Consistent with Figure E.1 and prospect theory, we find evidence that poorer areas were more likely to support Brexit. However, within areas, wealthier individuals were more likely to support Brexit. We therefore contribute to the literature by providing evidence that, despite local conditions, personal wealth informs and increases Brexit support. These results suggest that higher sociotropic wealth is associated with higher Remain support, but individual-level wealth is associated with higher Leave support. Main Effects We proceed by exploring changes in wealth and related preferences over time. This allows us to account for all time-invariant unobserved individual-level characteristics, such as Britishness, xenophobia, education, etc. Table 3 reports significant wealth effects on Leave support, account- ing for individual-specific, unobserved characteristics. A standard deviation increase in property wealth increases Leave support by as much as 7.1 percentage points. We also report the wealth effects visually in Fig. 4. Contrary to the cross-sectional results in Fig. 2, a standard deviation change in financial wealth also increases Leave support, though its effect is half that of property wealth. This is in line with expectations pertaining to the higher absolute value of property rather than financial wealth and also shows how different types of wealth could be more or less insuring, given the economic and political context. Aggregate and Individual-Level Our results are, as discussed, different from aggregate-level patterns that identify the geographic relationship between wealth and higher Remain support. In what follows, we report the extent to which aggregate-level results may be unable to detect greater individual-level variations in wealth. We exploit BES data linkage to contextual data on median home prices at the Lower Super Output Areas (LSOA) level. We identify respondents who live in low-priced LSOA areas (below average median home prices) versus those in a high-priced LSOA area. The bottom panel of Figure E.1 in the online supplementary material replicates the results from the existing lit- erature, showing that individuals living in wealthier areas are more supportive of Remain. However, 16There may be greater heterogeneity in preferences among the wealthy such that, for example, they expect personal eco- nomic circumstances to improve after Brexit. We explore this possibility in Fig. D.3 and found no evidence that wealthier individuals expected circumstances to improve. However, as Fig. 3 shows, we find evidence that they expected no change in circumstances, consistent with an insurance mechanism. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Figure 3. Expectations of Brexit effect on national vs personal finances (BES). Note: The dependent variable is binary and takes the value 1 if the respondent believes leaving the EU will have no effect on her national or personal circumstances. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). 12 Jane Green and Raluca L. Pahontu Jane Green and Raluca L. Pahontu Figure 3. Expectations of Brexit effect on national vs personal finances (BES). Note: The dependent variable is binary and takes the value 1 if the respondent believes leaving the EU will have no effect on her national or personal circumstances. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). 12 Jane Green and Raluca L. Pahontu 12 Figure 3. Expectations of Brexit effect on national vs personal finances (BES). Figure 3. Expectations of Brexit effect on national vs personal finances (BES). Note: The dependent variable is binary and takes the value 1 if the respondent believes leaving the EU will have no effect on her national or personal circumstances. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). Figure 3. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Mechanism Similar to the BES data, in Fig. 5, we explore how evaluations of national and personal economic situations vary by property wealth by predicting ‘no change’ in economic circumstances. The g/10.1017/S0007123423000728 Published online by Cambridge University Press British Journal of Political Science 13 figure paints a similar picture to that displayed using the BES data in Fig. 3; higher wealth posi- tively correlates with an increase in expectations of no personal economic change.17 Results: Survey Experiment We complement this observational evidence by using a survey experiment that provides a hypo- 18 Table 3. Wealth effect on leave support (BoE) 1 2 HH Adj Financial wealth 0.036* 0.038 (0.016) (0.016) HH Adj Property wealth 0.071 0.062 (0.032) (0.030) HH Adj Disposable income 0.006 0.006 (0.019) (0.019) Age −0.031 (0.023) Age squared 0.000 (0.000) Education: high school −0.079 (0.077) Education: higher education 0.055 (0.111) Unemployed −0.053 (0.073) Student 0.074 (0.152) Retired −0.160 (0.120) Not in paid work −0.068 (0.095) Region: East Midlands 0.235 0.245 (0.153) (0.159) Region: Greater London 0.225 0.243 (0.254) (0.258) Region: North 0.015 −0.032 (0.218) (0.232) Region: North West −0.117 −0.179 (0.238) (0.262) Region: South East 0.134 0.112 (0.185) (0.188) Region: South West 0.124 0.104 (0.188) (0.195) Region: Wales −0.076 −0.152 (0.229) (0.256) Region: West Midlands 0.104 0.097 (0.188) (0.194) Region: Yorkshire & Humberside 0.129 0.088 (0.175) (0.184) Controls X ✓ Observations 6,242 6,242 R2 0.033 0.046 Number of ids 5,230 5,230 Note: The dependent variable is binary (1 = Leaver). Compared with model (1), model (2) includes controls for age, age squared, education, employment status, and respondent’s location. The reference categories are as follows: Education: not in higher education, Region: East Anglia. Clustered robust standard errors in parentheses. p < 0.01, p < 0.05, p < 0.1. Table 3. Wealth effect on leave support (BoE) (0.095) Region: East Midlands 0.235 0.245 (0.153) (0.159) Region: Greater London 0.225 0.243 (0.254) (0.258) Region: North 0.015 −0.032 (0.218) (0.232) Region: North West −0.117 −0.179 (0.238) (0.262) Region: South East 0.134 0.112 (0.185) (0.188) Region: South West 0.124 0.104 (0.188) (0.195) Region: Wales −0.076 −0.152 (0.229) (0.256) Region: West Midlands 0.104 0.097 (0.188) (0.194) Region: Yorkshire & Humberside 0.129 0.088 (0.175) (0.184) Controls X ✓ Observations 6,242 6,242 R2 0.033 0.046 Number of ids 5,230 5,230 Note: The dependent variable is binary (1 = Leaver). 17An alternative narrative could suggest that wealthier voters expect their properties to appreciate in value following Britain’s exit from the European Union. We entertained this possibility in Fig. D.5 across the property wealth distribution, but we do not find supportive evidence of this mechanism. 18 Mechanism Compared with model (1), model (2) includes controls for age, age squared, education, employment status, and respondent’s location. The reference categories are as follows: Education: not in higher education, Region: East Anglia. Clustered robust standard errors in parentheses. *p < 0.01, p < 0.05, p < 0.1. Note: The dependent variable is binary (1 = Leaver). Compared with model (1), model (2) includes controls for age, age squared, education, employment status, and respondent’s location. The reference categories are as follows: Education: not in higher education, Region: East Anglia. Clustered robust standard errors in parentheses. p < 0.01, p < 0.05, *p < 0.1. figure paints a similar picture to that displayed using the BES data in Fig. 3; higher wealth posi- tively correlates with an increase in expectations of no personal economic change.17 https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Results: Survey Experiment Results: Survey Experiment Results: Survey Experiment We complement this observational evidence by using a survey experiment that provides a hypo- thetical wealth treatment.18 In addition to bolstering our confidence in the relationship between 17An alternative narrative could suggest that wealthier voters expect their properties to appreciate in value following Britain’s exit from the European Union. We entertained this possibility in Fig. D.5 across the property wealth distribution, but we do not find supportive evidence of this mechanism. pp 18The hypothetical nature of this treatment ensures that the respondents are not deceived. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Jane Green and Raluca L. Pahontu 14 14 Figure 4. Wealth increases leave support. Note: The dependent variable is binary (1 = Leaver). All models include controls for age, age squared, education, employment status, and respondent’s location. Models include time and individual fixed effects (95% confidence intervals). Figure 4. Wealth increases leave support. Note: The dependent variable is binary (1 = Leaver). All models include controls for age, age squared, education, employment status, and respondent’s location. Models include time and individual fixed effects (95% confidence intervals). Figure 4. Wealth increases leave support. Note: The dependent variable is binary (1 = Leaver). All models include controls for age, age squared, education, employment status, and respondent’s location. Models include time and individual fixed effects (95% confidence intervals). Figure 5. Expectations of Brexit effect on national vs personal finances (BoE). Note: The dependent variable is binary and takes the value 1 if the respondent believes that the next 12 months will have no effect on national or her personal circumstances. Prospective evaluations are available for 2016–2018, and results are pooled across all respon- dents. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). Figure 5. Expectations of Brexit effect on national vs personal finances (BoE). Figure 5. Expectations of Brexit effect on national vs personal finances (BoE). Note: The dependent variable is binary and takes the value 1 if the respondent believes that the next 12 months will have no effect on national or her personal circumstances. Prospective evaluations are available for 2016–2018, and results are pooled across all respon- dents. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). Figure 5. Expectations of Brexit effect on national vs personal finances (BoE). Results: Survey Experiment Note: The dependent variable is binary and takes the value 1 if the respondent believes that the next 12 months will have no effect on national or her personal circumstances. Prospective evaluations are available for 2016–2018, and results are pooled across all respon- dents. Property wealth is denoted by the respondents’ home value, standardized to mean 0 and standard deviation 1 (95% confidence intervals). 15 British Journal of Political Science wealth and Brexit support, we use this experimental approach to test whether a wealth win decreases risk aversion, as specified in hypothesis 3. The experiment was fielded in BES Wave 19 in the post-election survey in December 2019. y First, the experiment randomizes respondents into a treatment group with a hypothetical wealth win and a control group. Each treatment condition then receives a question about their Brexit preference and willingness to take risks. In total, there are four groups: a control group who were only asked about their Brexit preference, a treatment group who received a hypothetical wealth win and asked about their Brexit preference (the Brexit Support Treatment), a control group who were only asked their risk aversion, and a treatment group who received a hypothetical wealth win and then requested their risk aversion (the Willingness to Take Risk Treatment). The protocol is described in Table 4. To measure risk aversion, we rely on a two-question battery of questions proposed by Barsky et al. (1997) to capture financial risk-taking. Similar questions have since been used in the Cooperative Congressional Election Study and the 1996 Panel Study of Income Dynamics, which have been validated in various contexts (Eckles et al. 2014; Hryshko, Luengo-Prado, and Sørensen 2011; Pahontu 2020). Around 15,000 people participated in the experiment, about half of them receiving the Brexit Support Treatment and the other half receiv- ing the Willingness to Take Risk Treatment. Figure D4 in the supplementary material gives us confidence that randomization was successful across all relevant observables. Main Effect The BES and Bank of England results have accounted for the effect of levels and changes in wealth on Brexit support. To complement this evidence, in Fig. 6 we report the treatment effect of a hypothetical £1 million home win on the respondent’s satisfaction with the UK’s vote to leave the EU, as noted in Table 4. Consistent with the observational data, property wealth increases satisfaction with Brexit by 0.2 to 0.25 on the scale, equivalent to a 4 per cent increase compared to the control. A ‘hypothetical’ wealth increase would not be sufficient evidence of wealth’s effect on voting for Brexit. However, in combination with evidence from two separate data sets, this evidence gives us greater confi- dence in our conclusions. Mechanism: Wealth and Risk Aversion Wealth increases leave support. Note: The dependent variable is measured on a scale from 0 to 10 (10 = Leave). This question is one of two randomized outcomes that were asked of the treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.1 (95% confidence intervals). Figure 6. Wealth increases leave support. Note: The dependent variable is measured on a scale from 0 to 10 (10 = Leave). This question is one of two randomized outcomes that were asked of the treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.1 (95% confidence intervals). Figure 7. Wealth treatment decreases risk aversion. Note: The dependent variable is measured on a scale from 0 to 3 (3 = risk averse). This question is one of two randomized outcomes that were asked of the treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.2 (95% confidence intervals). Figure 7. Wealth treatment decreases risk aversion. Note: The dependent variable is measured on a scale from 0 to 3 (3 = risk averse). This question is one of two randomized outcomes that were asked of the treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.2 (95% confidence intervals). Figure 7. Wealth treatment decreases risk aversion. Note: The dependent variable is measured on a scale from 0 to 3 (3 = risk averse). This question is one of two randomized outcomes that were asked of the treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.2 (95% confidence intervals). Figure 7. Wealth treatment decreases risk aversion. Note: The dependent variable is measured on a scale from 0 to 3 (3 = risk averse). This question is one of two randomized outcomes that were asked of the treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.2 (95% confidence intervals). t t eat e t dec eases s a e s o . dent variable is measured on a scale from 0 to 3 (3 = risk averse). This question is one of two randomized outcomes that e treatment and control group. The treatment group received a hypothetical wealth win. Full results in Table D.2 (95% vals). Mechanism: Wealth and Risk Aversion Mechanism: Wealth and Risk Aversion The observational evidence is consistent with the proposed insurance based mechanism that wealthier respondents enjoy in their support for Brexit. We also argued that the mechanism Table 4. Experimental conditions and outcome wording Conditions and outcomes Text Treatment Imagine you took part in a lottery, and you are now the lucky winner of a £1 million house! 1/2 of sample Brexit support Outcome 1 How satisfied or dissatisfied are you that the UK voted to leave the EU? 1/2 of the treated sample Willingness to take risks Outcome 2 Suppose you are the only income earner in the family, and you have a good job guaranteed to give you income every year for life. You are given the opportunity to take a new and equally good job, with a 50–50 chance it will double your income and a 50–50 chance that it will cut your income by a third. Would you take the new job? If ‘yes’: Suppose the chances were 50–50 that it would double your income, and 50–50 that it would cut it in half. Would you still take the new job? If ‘no’: Suppose the chances were 50–50 that it would double your income and 50–50 that it would cut it by 20 per cent. Would you then take the new job? 1/2 of the treated sample Table 4. Experimental conditions and outcome wording How satisfied or dissatisfied are you that the UK voted to leave the EU? 1/2 of the treated sample Suppose you are the only income earner in the family, and you have a good job guaranteed to give you income every year for life. You are given the opportunity to take a new and equally good job, with a 50–50 chance it will double your income and a 50–50 chance that it will cut your income by a third. Would you take the new job? If ‘yes’: Suppose the chances were 50–50 that it would double your income, and 50–50 that it would cut it in half. Would you still take the new job? If ‘no’: Suppose the chances were 50–50 that it would double your income and 50–50 that it would cut it by 20 per cent. Would you then take the new job? 1/2 of the treated sample https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Jane Green and Raluca L. Pahontu 16 Figure 6. Conclusion It is well-established that wealth provides self-insurance against economic risks, cushioning indi- viduals from possible income shocks and other risks (Ansell 2014; Ehrlich and Becker 1972; Tertytchnaya and De Vries 2019). Consequently, wealth leads to lower risk aversion (Pahontu 2020; Zanetti 2014). Despite these observations, the role of wealth has gone unstudied as a mech- anism that enables voters to opt for more risky political propositions, such as large-scale changes to the status quo where voters offset costs, benefits, and risks. q Studying the effect of wealth in the Brexit case, we demonstrate that variation in personal wealth – especially property wealth – enables wealthier individuals to support Brexit and less wealthy individuals to support Remain. We provide evidence that the mechanism linking wealth and higher Leave support comes via wealthy voters’ expectations that Brexit would not impact their personal finances; we also show that an increase in wealth lowers risk aversion. We explore these relationships using a rare combination of observational and experimental data across three separate contexts that include individual-level measures of wealth: a large cross-sectional electoral study, a separate panel study accounting for unobserved individual-level heterogeneity, and the use of a survey experiment. We also provide evidence that these results run contrary to aggregate-level relationships, although these findings could be complementary. We propose that wealth enables individuals to support Brexit and that sociotropic concerns also matter. This explains why our con- clusions differ from those of studies of contextual economic effects on support for Brexit (Carreras 2019; Colantone and Stanig 2018; Fetzer 2019). People living in left-behind areas were more likely to support Brexit than those living in prosperous areas. The gains of Brexit were perceived to be greater in areas of the country that had experienced economic decline (Carreras 2019). But within those areas, given people’s preferences, we show that wealthier individuals were more likely to vote for Brexit, and poorer individuals were more likely to vote for Remain. This individual-level find- ing is new and important for the Brexit case, which has otherwise focused on the economic deter- minants of preferences for Brexit rather than the role of wealth in economic risks. Our research design, relying on observational cross-sectional panel data and experimental data, allows us to validate our results across three separate datasets and increases confidence in the validity and magnitude of the causal estimate of wealth on Brexit support. Mechanism: Wealth and Risk Aversion through which wealth increases Brexit support is via wealth’s effect on reducing risk aversion. Like Pahontu (2020), we test whether the insurance provided by the respondents’ wealth reduces their risk aversion, thereby allowing those with underlying preferences to support a change in the status quo. Looking at the results in Fig. 7, we notice a substantial and significant increase in risk- through which wealth increases Brexit support is via wealth’s effect on reducing risk aversion. Like Pahontu (2020), we test whether the insurance provided by the respondents’ wealth reduces their risk aversion, thereby allowing those with underlying preferences to support a change in the status quo. Looking at the results in Fig. 7, we notice a substantial and significant increase in risk- https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press British Journal of Political Science British Journal of Political Science 17 taking among the treated, equivalent to a 5% increase in risk-taking behaviour. This is a very similar effect magnitude as in the case of Brexit support from Fig. 6. Taken together, the observational and experimental evidence increases our confidence that the mechanism linking wealth to Brexit support works through insurance and risk aversion; this insurance and lower risk aversion allows individuals to support a change to the status quo – in this case, Brexit. https://doi.org/10.1017/S0007123423000728 Published online by Cambridge University Press Conclusion While studies on Brexit rely almost exclusively on cross-sectional comparisons, leaving room for endogeneity concerns, we are the first to identify (to the best of our knowledge), at the individual level, a cau- sal estimate of wealth on Brexit support. We do so by exploiting the time dimension in Bank of England panel data and relying on a random variation in hypothetical wealth. We also offer a novel test of the individual-level mechanisms in the observational and experimental data. Our findings imply that, while many poorer individuals may have held a preference for Leave, they were less likely to vote for Brexit given their lack of economic insurance. Walter (2021) ques- tions why the British were willing to risk potentially imposing enormous economic self-harm on themselves. Our analysis shows that poorer individuals likely recognized a risk of economic self- harm due to their lack of economic insurance. These findings underline the importance of exam- ining causal effects at the individual level alongside broader trends at the aggregate level. They are also important for understanding Brexit’s long-term political and electoral implications. Support for Brexit might have been higher still had fewer poorer voters not perceived the economic risks associated with their lack of insurance. If the outcomes of Brexit are damaging to those who lack Jane Green and Raluca L. Pahontu 18 the insurance to withstand personal economic shocks, support for this political project may be significantly weakened. How might our findings generalize from the Brexit case and the context of Great Britain? One variation point may be the relative importance of financial and property wealth and the ability of different types of wealth to be more or less insuring. In Britain, owning a home has become an espe- cially reliable form of wealth. Brexit was the type of shock that would most likely disturb the econ- omy over the long term. In future applications of a ‘wealth-as-insurance’ lens in political behaviour, it would be useful to consider the possibility that different forms of economic insurance might be more important in different contexts. Our findings may apply more broadly where political choices over changes substantially affect the economy. This applies to other referenda on national secession. It may also apply if nations confront periods of economic instability due to a different kind of demo- cratic choice. Conclusion For example, it may apply to initiatives that change existing economic models moti- vated by decarbonization, which entails substantial economic instability. Wealthier voters may be more able to vote with their preferences over climate change reduction and support a party and its policies because they are insured against the immediate economic consequences. The general implications of our findings are normative, theoretical, and empirical. While there is a considerable amount of research on the relationship between inequality and redistributive preferences, we show that poorer voters do not just have different political preferences; they also lack the insurance to act on some of those preferences. Economic inequalities create inequal- ities in experience, preferences, and political risk-taking. Finally, supporting other research advo- cating for the unique role of wealth, in addition to standard predictors of economic voting (Lewis-Beck and Nadeau 2011; Nadeau, Foucault, and Lewis-Beck 2011; Nadeau, Lewis-Beck, and Foucault 2019), we have shown how it is important to measure economic positions in dif- ferent ways, how income and wealth are weakly correlated, and, as a result, how wealth will lead to different substantive conclusions than a focus only on income. Our findings show the importance of wealth for understanding political behaviour in a new and potentially important way. By providing insurance, wealth cushions individuals from the economic risks associated with a vote for a major change to the status quo. Supplementary Material. The supplementary material for this article can be found at https://doi.org/10.1017/ S0007123423000728. Data availability statement. Replication data for this article can be found in Harvard Dataverse at: https://doi.org/10.7910/ DVN/LOGVPZ. Acknowledgements. We thank the editor and the anonymous reviewers for their valuable comments. We are grateful for the helpful feedback provided by Pablo Beramendi, Jean-François Daoust, Andy Eggers, Tim Hicks, Rob Johns, David Rueda, and seminar participants at the Universities of Montreal, Essex, the Early Career Workshop hosted by the University of Southampton, and discussions at the Bank of England. Jane Green thanks colleagues in the British Election Study team, with whom survey measures were devised. Financial support. This work was supported by the Economic and Social Research Council funding for the British Election Study (Award ES/K005294/1) and from support provided by Nuffield College. Competing interests. None. Ethics standards. 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https://openalex.org/W3170225138	http://qspace.qu.edu.qa/bitstream/10576/22315/1/nutrients-13-02002%20%281%29.pdf	English	null	Validity and Reproducibility of a Food Frequency Questionnaire to Assess Macro and Micro-Nutrient Intake among a Convenience Cohort of Healthy Adult Qataris	Nutrients	2,021	cc-by	7,155	All content following this page was uploaded by Reema Tayyem on 12 June 2021. The user has requested enhancement of the downloaded file. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/352302743 The user has requested enhancement of the downloaded file. Validity and Reproducibility of a Food Frequency Questionnaire to Assess Macro and Micro-Nutrient Intake among a Convenience Cohort of Healthy Adult Qataris Hiba Bawadi 1,* , Rand T. Akasheh 2, Abdelhamid Kerkadi 1, Salma Haydar 1, Reema Tayyem 1 and Zumin Sh 1 Department of Nutrition, College of Health Sciences, QU-Health, Qatar University, Doha P.O. Box 2713, Qatar; abdel.hamid@qu.edu.qa (A.K.); salmahayder1@gmail.com (S.H.); reema.tayyem@qu.edu.qa (R.T.); zumin@qu.edu.qa (Z.S.) 2 Department of Nutrition and Dietetics, American University of Madaba, Madaba 11821, Jordan; r.akasheh@aum.edu.jo 1 Department of Nutrition, College of Health Sciences, QU-Health, Qatar University, Doha P.O. Box 2713, Qatar; abdel.hamid@qu.edu.qa (A.K.); salmahayder1@gmail.com (S.H.); reema.tayyem@qu.edu.qa (R.T.); zumin@qu.edu.qa (Z.S.) p g Q Q y Q abdel.hamid@qu.edu.qa (A.K.); salmahayder1@gmail.com (S.H.); reema.tayyem@qu.edu.qa (R.T.); zumin@qu.edu.qa (Z.S.) 2 Department of Nutrition and Dietetics, American University of Madaba, Madaba 11821, Jordan; r.akasheh@aum.edu.jo * Correspondence: hbawadi@qu.edu.qa 2 Department of Nutrition and Dietetics, American University of Madaba, Madaba 11821, Jordan; r akasheh@aum edu jo 2 Department of Nutrition and Dietetics, American University of Madaba, Madaba 11821, Jordan; r.akasheh@aum.edu.jo 2 Department of Nutrition and Dietetics, American University of Madaba, Madaba 11821, Jordan; r.akasheh@aum.edu.jo * Correspondence: hbawadi@qu.edu.qa 2 Department of Nutrition and Dietetics, American University of Madaba, Madaba 11821, Jordan; r.akasheh@aum.edu.jo * Correspondence: hbawadi@qu.edu.qa Abstract: This study aimed at developing a valid culture-sensitive quantitative food frequency questionnaire (FFQ) for Qatari adults. A convenient sample of healthy Qataris (n = 107) were recruited from family members of Qatar University students. The Diet History Questionnaire II of the US National Cancer Institute was translated to Arabic language, back-translated to English, pilot tested, and then modiﬁed accordingly to be used in Qatari setting. Participants were asked to complete the translated version of the FFQ. This FFQ was then validated against three 24 h diet recall (24 hDR) including a weekend day. Participants were asked to complete the FFQ again after one-month period to measure its repeatability. Dietary data were analyzed using the dietary analysis software ESHA. The validity and reliability of FFQ were assessed by comparing the median intake of nutrients and foods and by calculating the Pearson correlation coefﬁcients. The median nutrient intakes assessed by the second FFQ were higher than that reported in the baseline FFQ1 except for fat. The percentage of increase varies between 1.5% and 96%. Results of the second FFQ indicated an overestimation of intake for most nutrients (macro and micro). Macronutrient intakes assessed by the two FFQ and 24 hDR were strongly correlated. The correlation coefﬁcients for micronutrient intakes between FFQ2 and 24hDR were lower than that of the two FFQs except for calcium (r = 0.55) and sodium (r = 0.643). They ranged from (−0.17) for ﬂuorine to (0.643) for sodium. Citation: Bawadi, H.; Akasheh, R.T.; Kerkadi, A.; Haydar, S.; Tayyem, R.; Shi, Z. Validity and Reproducibility of a Food Frequency Questionnaire to Assess Macro and Micro-Nutrient Intake among a Convenience Cohort of Healthy Adult Qataris. Nutrients 2021, 13, 2002. https://doi.org/ 10.3390/nu13062002 Academic Editor: Laura Di Renzo Received: 11 April 2021 Accepted: 3 June 2021 Published: 10 June 2021 Keywords: Qatar; FFQ; validity Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Validity and Reproducibility of a Food Frequency Questionnaire to Assess Macro and Micro-Nutrient Intake among a Convenience Cohort of Healthy Adult Qataris Article in Nutrients · June 2021 Article in Nutrients June 2021 DOI: 10.3390/nu13062002 CITATIONS 0 READS 22 6 authors, including: Some of the authors of this publication are also working on these related projects: Dietary and Lifestyle Risk Factors for Developing Osteoporosis in Jordanian Postmenopausal Women. View project COMPARISON OF NUTRITIONAL STATUS BETWEEN LACTO-OVO VEGETARIANS AND NON-VEGETARIANS OF SELECTED SAMPLE AMONG JORDANIAN ADULTS View project Hiba Bawadi Qatar University 109 PUBLICATIONS 1,739 CITATIONS SEE PROFILE Abdelhamid Kerkadi Qatar University 44 PUBLICATIONS 250 CITATIONS SEE PROFILE Reema Tayyem Qatar University 168 PUBLICATIONS 1,772 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Some of the authors of this publication are also working on these related projects: Dietary and Lifestyle Risk Factors for Developing Osteoporosis in Jordanian Postmenopausal Women. View project COMPARISON OF NUTRITIONAL STATUS BETWEEN LACTO-OVO VEGETARIANS AND NON-VEGETARIANS OF SELECTED SAMPLE AMONG JORDANIAN ADULTS View project COMPARISON OF NUTRITIONAL STATUS BETWEEN LACTO-OVO VEGETARIANS AND NON-VEGETARIANS OF SELECTED SAMPLE AMONG JORDANIAN ADULTS View project COMPARISON OF NUTRITIONAL STATUS BETWEEN LACTO-OVO VEGETARIANS AND NON-VEGETARIANS OF SELECTED SAMPLE AMONG JORDANIAN ADULTS View project nutrients nutrients nutrients nutrients Validity and Reproducibility of a Food Frequency Questionnaire to Assess Macro and Micro-Nutrient Intake among a Convenience Cohort of Healthy Adult Qataris Hiba Bawadi 1,* , Rand T. Akasheh 2, Abdelhamid Kerkadi 1, Salma Haydar 1, Reema Tayyem 1 and Zumin Sh The agreement rates for classifying macronutrient intakes into same or adjacent quartile were between 79.4% and 100% for the two FFQs and between 71% and 100% for the second FFQ and 24hDR. The reported consumption of food groups estimated by FFQ2 was signiﬁcantly higher than that reported by FFQ1. In conclusion, the developed FFQ was sufﬁciently valid to assess energy and macronutrients but not micronutrients. The reliability was adequate for most nutrients. Citation: Bawadi, H.; Akasheh, R.T.; Kerkadi, A.; Haydar, S.; Tayyem, R.; Shi, Z. Validity and Reproducibility of a Food Frequency Questionnaire to Assess Macro and Micro-Nutrient Intake among a Convenience Cohort of Healthy Adult Qataris. Nutrients 2021, 13, 2002. https://doi.org/ 10.3390/nu13062002 2.1. Study Design and Participants 2.1. Study Design and Participants This study was approved by the IRB committee of Qatar University with an approval number of (QUSG-CAS-DHS-14\15-2). In this cross-sectional survey, a convenience sample of 500 Qatari adults from both genders were invited to participate in the study. Exclu- sion criteria entailed those who were suffering from chronic diseases that require dietary modiﬁcations such as diabetes, renal, and liver diseases and many others. Participants were asked to sign a consent form before completing the survey. One hundred and seven completed the study (40 males and 67 females), with an average age of 33 years 1. Introduction Different dietary assessment methods had been created to evaluate food and nutri- ents intake among individuals and populations. Food frequency questionnaire (FFQ) is considered an essential tool in epidemiologic research, and it is usually used to estimate the long-term relationship between diet and chronic diseases [1]. Food records and 24hDR may provide accurate ﬁgures on diet although they are costly to be used in epidemiolog- ical studies. The 24 h diet recalls (24hDR) is a retrospective method of diet assessment, where individuals are interviewed about their food and beverage consumption during the preceding 24 h [2]. However, a single 24hDR could not be considered representative of habitual diet at an individual level. At least 3-day recalls were recommended as the most appropriate method of dietary assessment [2]. Food records necessitate a high level Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/nutrients Nutrients 2021, 13, 2002. https://doi.org/10.3390/nu13062002 Nutrients 2021, 13, 2002 2 of 11 of literacy and cooperation and at least three days would be required to assess the current intake of nutrients and foods. This makes them less practical for epidemiological studies [2]. At present, FFQ is the preferred dietary assessment method in most epidemiological studies primarily because they are somehow inexpensive and easy to administer. Hence, FFQs have been continuously modiﬁed and validated to reﬂect each population’s traditional foods and true food intake [3–6]. Health challenges in Qatar, including the high prevalence of obesity and diabetes, urge the need for scientiﬁc research that expose risk factors—including diet—for these chronic diseases. Despite the research boom being witnessed in Qatar and despite having obesity and diabetes control as a national priority, there is no validated dietary tool available for use in Qatari settings. Therefore, developing a FFQ speciﬁc for the Qatari population is essential. The aim of the present study was to create a culture-sensitive quantitative FFQ for Qatari adults and to validate it against three-day 24hDR collected from a sample of Qatari population. 2.2. Dietary Assessment The Diet History Questionnaire II of the US National Cancer Institute provides struc- tured questions about the frequency of intake of 153 food items over a 12-month period and provides choices of three portion sizes for food quantiﬁcation. This DHQ was modiﬁed ac- cording to Qatari setting to create an FFQ, which was then tested for validity and reliability. The DHQ II was ﬁrst translated back by two bilingual experts. The ﬁrst expert translated the ﬁrst half of the DHQ into Arabic while the second translator translated the second half of the questionnaire. Thereafter, the questionnaire was back-translated to English where the ﬁrst translator took the second half of the translated questionnaire and the second translator took its ﬁrst half. An expert panel composed from researchers and translators were held to ﬁnalize the Arabic version of the questionnaire. A pilot screening was conducted on 50 individuals from different geographical areas in Qatar. The original FFQ was provided and participants were asked to identify uncommon and rarely consumed foods by Qataris. Participants were also asked to provide information about food items commonly consumed by Qataris but not included in the FFQ. Based on the data collected from the pilot test, food items common in the Qatari cuisine were added to the questionnaire, while items not relevant to the Qatari’s culture were removed. In the initial visit, participants were asked to complete a paper version of the translated FFQ and were oriented how to complete the dietary recall. Participants were told to start the recall from the most recent point and recall 24 h. They were also instructed to go on 2 rounds of recalls for each day. In the ﬁrst round, they will recall the major food items and in the second round, they recall the details related to amounts, preparation, toppings, etc. FFQ data collection was done via face-to-face interviews conducted by trained nutritionist to help participants complete the questionnaire. Three nutritionists were involved in data collection. To avoid variations between interviews, all nutritionists were oriented and trained of how to avoid leading questions, bias, and how to ask for additional details when needed. Food models and standard measuring tools were used to help participants in estimat- ing the consumed portion size. Participants’ responses were converted into average daily Nutrients 2021, 13, 2002 3 of 11 intakes. 2.3. Statistical Analyses The estimated intakes of energy, macronutrients, and micronutrients were used to assess the validity of the FFQ. Means (±SDs) as well as medians were calculated for energy and total nutrient intakes from the average of three-day 24hDRs and of each FFQ. In the study, we assessed the validity and reliability of the FFQ by (1) comparing the median intake of nutrient and food; and (2) calculating the correlations (Pearson correlation coefﬁcients) between nutrient and food intakes derived from different dietary survey methods (FFQ vs. 24hDR) and different surveys (ﬁrst and second FFQ). Wilcoxon signed rank test was also conducted to compare the nutrient intakes measured by different methods. In addition, agreement rates by quartile distribution of nutrient and food intake were also calculated. 2.2. Dietary Assessment On the other hand, three non-consecutive 24hDR (including a weekend day) were collected from participants. The 24hDRs were gathered a week after FFQ completion in order to reduce possible intra-individual day-to-day variation. Participants were asked to complete the FFQ two times separated by a one-month period to measure its repeatability. Food lists in the modiﬁed FFQ questions were classiﬁed based on types of foods: 21 items of vegetables; 16 items of meat such as red meat (lamb and beef), chicken, ﬁsh, cold meat, and others; 21 items of fruits and juices; nine items of milk and dairy products; eight items of cereals; four items of beans; four items of soups and sauces; ﬁve items of drinks; nine items of snacks and sweets; and 14 items of herbs and spices. The calculation of the amount of the food consumed was performed as follows: the average of the frequency category will be multiplied by the portion size then divide it by the number of days to provide the food amount/day. Dietary data were analyzed to estimate nutrient content using the dietary analysis software (ESHA Food Processor SQL version 10.1.1; ESHA, Salem, OR, USA) with addi- tional data on foods consumed in Qatar. The nutrients’ content of the mixed recipes and traditional foods was calculated from the Gulf Countries food composition table [7]. 3. Results Table 1 presents the comparison of median intakes of nutrients between the baseline FFQ, second FFQ and the average of 24hDRs. The median nutrient intakes assessed by the second FFQ were higher than that reported in the baseline FFQ1, except for fat. The percentage of increase varied between 15% and 96%. Results of second FFQ indicated an overestimation of intake for most nutrients, both macros and micros. The median nutrient intakes derived from FFQ2 were higher for trans-fat, vitamin A, selenium, omega 3, and omega 6 fatty acids as compared to FFQ1. To get more details on level of validity of the FFQ, Bland-Altman plots were obtained for energy and macronutrients intake. Based on the Bland-Altman plots, the FFQ had a good agreement with food record. The Bland- Altman plots are available online as supplementary Figure S1. Tables 2 and 3 shows the correlations of nutrient intakes assessed by two FFQs and 24hDR along with the agreement rates by quartile distributions. Results indicated that energy and macronutrient intakes assessed by the two FFQ were strongly correlated and all correlation coefﬁcients were above 0.5 (average r = 0.799). The same results were reported for energy and macronutrient intakes assessed by FFQ2 and 24hDR where correlation coefﬁcients were between 0.545 (ﬁber) and 0.974 (energy), except for trans-fat (0.076), omega 3 (0.263), and omega 6 (0.352). These ﬁndings parallel the agreement rates between the two FFQs and 24hDR in classifying energy and macronutrients. For energy, 100% of subjects were classiﬁed in the same or adjacent quartile of intake derived from the two FFQs or FFQ2 and 24hDR. Moreover, the agreement rates for classifying macronutrient intakes into the same or adjacent quartiles were between 79.5% and 100% for the two FFQs and between 71% and 100% for second FFQ and 24hDR; the lowest being again for trans-fat. Extreme misclassiﬁcation into the opposite quartile was low and ranged between 0% and 8.4% for the two FFQs and the two methods. Taken together, these ﬁndings indicated that the FFQ is valid and reliable for the estimation of energy and macronutrients. Nutrients 2021, 13, 2002 4 of 11 Table 1. Median comparison of nutrients intake between two FFQ surveys and average of the 24hDR recalls among adults. Median (25–75th Percentile) Wilcoxon Signed Racked Test (p-Value) Percentage of Median Difference Daily Intake 1st FFQ (25–75th) 2nd FFQ (25–75th) 24 h Recalls (25–75th) 1st FFQ vs. 3. Results 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ (%) 2nd FFQ vs. 3. Results 24 h Recalls (%) Energy intake (kcal) 2092 (1663, 2277) 2123 (1669, 2576) 2132 (1544, 2714) 0.00 0.30 −1.4 −0.5 Protein (g) 79.3 (61.8, 102.1) 78.8 (66.0, 108.6) 76.1 (54.9, 100.8) <0.01 <0.01 0.6 3.6 Carbohydrate (g) 243.1 (192.2, 299.3) 283.3 (226.0, 333.1) 264.0 (194.9, 343.0) 0.00 <0.01 −14.2 7.3 Fat (g) 81.9 (62.1, 109.2) 71.3 (55.5, 92.0) 77.6 (54.9, 113.8) 0.00 <0.01 15.0 −8.1 Saturated fat (g) 26.4 (17.3, 37.5) 24.4 (18.0, 35.0) 19.6 (13.2, 29.6) <0.01 0.00 8.4 24.5 Monosaturated fat (g) 9.1 (4.4, 14.7) 14.2 (10.7, 20.3) 24.1 (14.1, 37.9) 0.00 0.00 −36.0 −41.3 Polysaturated fat (g) 5.5 (2.0, 7.1) 7.9 (5.8, 11.3) 17.7 (9.5, 28.1) 0.00 0.00 −30.7 −55.7 Trans fat (g) 0.33 (0.03, 0.68) 0.86 (0.38, 1.88) 0.29 (0.02, 1.04) 0.00 0.00 −61.6 196.6 Fiber (g) 17.6 (13.5, 22.5) 21.7 (16.7, 29.3) 16.9 (12.3, 25.1) 0.00 0.00 −18.6 28.3 Vitamin A (µg) 1391 (846, 2963) 6800 (3789, 10,376) 2361 (1251, 4878) 0.00 0.00 −79.5 188.0 Vitamin B1 (mg) 0.67 (0.51, 0.89) 1.09 (0.85, 1.52) 1.5 (1.2, 2.0) 0.00 0.00 −38.2 −26.2 Vitamin B2 (mg) 0.53 (0.34, 0.76) 1.11 (0.81, 1.52) 1.5 (1.1, 2.1) 0.00 0.00 −52.3 −26.7 Vitamin B3 (mg) 9.6 (5.8, 15.0) 14.5 (11.5, 20.4) 18.4 (12.9, 24.0) 0.00 <0.01 −33.7 −21.4 Vitamin B6 (mg) 0.64 (0.37, 0.84) 1.10 (0.82, 1.68) 1.35 (0.90, 2.18) 0.00 0.02 −41.6 −18.6 Vitamin C (mg) 64.5 (27.9, 116.9) 105.1 (71.1, 147.0) 86.1 (38.4, 149.5) 0.00 0.01 −38.6 22.1 Vitamin D (µg) 2.4 (0.0, 28.8) 11.3 (6.9, 23.5) 8.5 (1.1, 24.8) 0.01 0.13 −78.3 32.3 Folate 152.5 (125.5, 203.2) 269.0 (193.3, 326.2) 366.6 (269.0, 465.7) 0.00 0.00 −43.3 −26.6 Vitamin K (mcg) 10.6 (3.0, 26.3) 98.9 (40.2, 181.9) 177.3 (94.3, 323.9) 0.00 0.00 −89.3 −44.2 B5 2.0 (1.1, 2.9) 3.7 (2.8, 4.7) 4.6 (3.7, 5.7) 0.00 0.00 −45.2 −20.1 Calcium (mg) 490.6 (379.9, 732.8) 644.4 (486.3, 833.2) 583.0 (351.1, 758.0) 0.00 <0.01 −23.9 10.5 Chromium 0.89 (0.22, 1.57) 2.0 (1.4, 2.9) 2.8 (1.6, 3.8) 0.00 <0.01 −55.1 −28.8 Copper 0.44 (0.34, 0.62) 0.73 (0.57, 0.98) 0.91 (0.71, 1.22) 0.00 0.00 −39.3 −20.3 Fluorine 0.01 (0.00, 0.05) 0.18 (0.12, 0.27) 0.25 (0.18, 0.46) 0.00 <0.01 −94.3 −30.0 Iodine 6.3 (2.6, 11.8) 28.2 (18.3, 36.9) 37.0 (24.4, 53.1) 0.00 0.00 −77.6 −23.8 Iron 10.9 (8.7, 13.0) 13.0 (10.7, 16.6) 14.7 (11.6, 20.1) 0.00 <0.01 −16.5 −11.4 Phosphate 431.2 (264.4, 685.6) 668.7 (530.3, 852.0) 808.9 (578.4, 992.0) 0.00 <0.01 −35.5 −17.3 Selenium (mcg) 39.0 (21.4, 66.2) 67.4 (46.4, 85.3) 29.3 (16.2, 52.9) 0.00 0.00 −42.2 130.2 Sodium (mg) 2824 (2229, 4106) 3377 (2601, 4738) 3732 (2342, 5055) 0.00 0.19 −16.4 −9.5 Zinc (mg) 3.2 (2.3, 4.4) 5.3 (4.0, 6.9) 6.1 (4.4, 8.5) 0.00 <0.01 −39.5 −13.8 Omega 3 (g) 0.32 (0.10, 0.91) 0.56 (0.33, 0.79) 0.33 (0.18, 0.65) 0.00 <0.01 −42.3 68.2 Omega 6 (g) 2.9 (1.1, 6.2) 5.8 (4.3, 9.5) 4.5 (2.0, 6.8) 0.00 0.00 −49.9 28.9 n of nutrients intake between two FFQ surveys and average of the 24hDR recalls among adults. 3. Results Percentage of Median Difference Nutrients 2021, 13, 2002 5 of 11 Table 2. Pearson’s correlation of nutrients intake between two FFQ, 24 h recall. Nutrients 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls Energy 0.991 0.974 Protein 0.903 0.610 Carbohydrates 0.931 0.803 Fat 0.929 0.799 Saturated fat 0.856 0.670 Monosaturated fat 0.651 0.617 Polysaturated fat 0.735 0.610 Trans fat 0.553 0.076 Fiber 0.652 0.545 Vitamin A 0.370 −0.014 Vitamin B1 0.710 −0.080 Vitamin B2 0.551 −0.106 Vitamin B3 0.722 −0.080 Vitamin B6 0.011 −0.034 Vitamin C 0.677 0.176 Vitamin D 0.535 0.098 Folate 0.563 −0.135 Vitamin K 0.276 −0.054 B5 0.624 −0.105 Calcium 0.764 0.550 Chromium 0.165 −0.008 Copper 0.609 0.034 Fluorine 0.257 −0.170 Iodine 0.339 −0.138 Iron 0.822 −0.095 Phosphate 0.643 0.031 Selenium 0.760 0.116 Sodium 0.853 0.643 Zinc 0.351 0.094 Omega 3 0.840 0.263 Omega 6 0.746 0.352 Table 2. Pearson’s correlation of nutrients intake between two FFQ, 24 h recall. As for micronutrient intakes, the correlation coefﬁcients between the two FFQs were in general higher than 0.5 for all except for vitamin A (r = 0.37), vitamin B6 (r = 0.011), vitamin K (r = 0.276), chromium (r = 0.165), ﬂuorine (r = 0.257), and iodine (r = 0.339). Furthermore, 69.2% to 98% of subjects were classiﬁed in the same or adjacent quartile of FFQs. On the other hand, the correlation coefﬁcients for micronutrient intakes between FFQ2 and 24hDR were lower than that of two FFQs, except for calcium (r = 0.55) and sodium (r = 0.643), and ranged from (r = −0.17) for ﬂuorine to (r = 0.643) for sodium. Moreover, 52% to 87% of participants were classiﬁed in the same or adjacent quartile intake based on FFQ and 24hDR. Extreme misclassiﬁcation into the opposite quartile was between 0% to 6.5% for reliability and 1.9% to 18.5% for validity. Overall, these data indicated that the FFQ is reliable with relatively weak validity for estimating most micronutrient intakes. y y g Median of food intakes are presented in Table 4. The reported consumption of food groups estimated by FFQ2 was signiﬁcantly higher than that reported by FFQ1, and parallels the ﬁndings on nutrient intakes. The median intakes of fruit, vegetables, grains, and milk derived from FFQ2 were signiﬁcantly lower than those reported by 24hDR. No signiﬁcant differences in median meat intakes between the two methods were detected. 3. Results Nutrients 2021, 13, 2002 6 of 11 Table 3. Agreement in quartile distribution of nutrients intake between two FFQ surveys and 24 h recall. Percent of Agreement Same Quartile Adjacent Quartile Skip One Quartile Opposite Quartile Daily Intake 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls Energy intake (kcal) 100 98.13 0 1.87 0 0 0 0 Protein (g) 71.03 46.73 28.04 42.99 0.93 6.54 0 3.74 Carbohydrate (g) 72.9 56.07 26.17 40.19 0.93 3.74 0 0 Fat (g) 75.7 62.62 24.3 30.84 0 6.54 0 0 Saturated fat (g) 68.22 39.25 29.91 50.47 1.87 9.35 0 0.93 Monosaturated fat (g) 49.53 42.06 39.25 38.32 10.28 18.69 0.93 0.93 Polysaturated fat (g) 54.21 38.32 32.71 41.12 10.28 16.82 2.8 3.74 Trans fat (g) 40.19 27.1 39.25 43.93 14.95 24.3 5.61 4.67 Fiber (g) 57.01 38.32 30.84 38.32 11.21 17.76 0.93 5.61 Vitamin A (µg) 34.58 28.97 34.58 39.25 28.04 19.63 2.8 12.15 Vitamin B1 (mg) 50.47 20.19 42.06 39.42 6.54 26.92 0.93 13.46 Vitamin B2 (mg) 37.38 26.92 46.73 31.73 14.02 25.96 1.87 15.38 Vitamin B3 (mg) 51.4 20.19 43.93 37.5 3.74 27.88 0.93 14.42 Vitamin B6 (mg) 39.25 19.23 41.12 41.35 13.08 24.04 6.54 15.38 Vitamin C (mg) 44.86 28.97 45.79 38.32 7.48 28.97 1.87 3.74 Vitamin D (µg) 54.21 22.43 36.45 44.86 8.41 23.36 0.93 9.35 Folate 42.06 25 42.99 28.85 12.15 28.85 2.8 17.31 Vitamin K (mcg) 29.91 25 42.99 35.58 20.56 24.04 6.54 15.38 B5 43.93 23.3 46.73 43.95 9.35 23.3 0 18.45 Calcium (mg) 44.86 40.19 50.47 42.99 4.67 14.02 0 2.8 Table 3. Agreement in quartile distribution of nutrients intake between two FFQ surveys and 24 h recall 7 of 11 Nutrients 2021, 13, 2002 Table 3. Cont. 3. Results Percent of Agreement Same Quartile Adjacent Quartile Skip One Quartile Opposite Quartile Chromium 34.58 29.13 47.66 26.21 14.95 33.01 2.8 11.65 Copper 47.66 25 43.93 42.31 7.48 23.08 0.93 9.62 Fluorine 31.78 22.33 40.19 33.01 21.5 26.21 6.54 18.45 Iodine 33.98 21.15 45.63 30.77 16.5 31.73 3.88 16.35 Iron 57.01 23.81 41.12 42.86 1.87 19.05 0 14.29 Phosphate 50.47 28.85 40.19 35.58 9.35 19.23 0 16.35 Selenium (mcg) 50.47 22.43 42.99 42.06 6.54 24.3 0 11.21 Sodium (mg) 64.49 49.53 31.78 37.38 3.74 11.21 0 1.87 Zinc (mg) 48.6 25.96 42.99 39.42 6.54 22.12 1.87 12.5 Omega 3 (g) 60.75 20.56 33.64 41.12 4.67 29.91 0.93 8.41 Omega 6 (g) 48.6 34.58 42.06 42.99 7.48 15.89 1.87 6.54 Table 3. Cont. Percent of Agreement Nutrients 2021, 13, 2002 8 of 11 Table 4. Median nutrients intake from the two FFQ surveys and average of the 24 h dietary recalls among adults in Qatar. Table 4. Median nutrients intake from the two FFQ surveys and average of the 24 h dietary recalls among adults in Qatar. FFQ1 FFQ2 24 h Recalls Median P25 P75 Median P25 P75 Median P25 P75 Fruit 0.46 0.00 1.25 1.38 1.01 1.88 2.70 1.94 4.15 Veg 1.16 0.52 1.94 1.64 1.15 2.19 2.04 1.32 2.96 Grain 4.73 3.66 6.17 6.19 4.92 7.47 8.39 6.07 10.82 Milk 0.56 0.33 1.08 0.77 0.51 1.06 0.99 0.47 1.60 Meat 4.72 2.98 6.69 4.43 3.03 6.20 4.01 2.87 5.58 Pearson correlation coefﬁcients for food groups are presented in Tables 5 and 6. Results indicate a strong correlation for all food groups between the two FFQs with an average r of 0.749, but a weak correlation for vegetable (0.028) and milk (0.06) between FFQ2 and 24hDR, and a negative correlation for fruit (−0.13), grain (−0.057), and meat (−0.192) between FFQ2 and 24hDR. When the median of food group intakes were categorized into quartiles, the ranges of agreement rates for the same or adjacent quartile were 84–95% for intakes reported by two FFQs, and 57–67% for those of 2nd FFQ and 24hDR. Classiﬁcation into the opposite quartile was lower than 3% for the two FFQs and 8–16% for second FFQ and 24hDR (Table 5). In summary, we observed that the FFQ is of strong reliability but relatively weak validity for estimating food group intakes. Table 5. Agreement in quartile distribution of food groups intake between two FFQ surveys and 24 h recall. 3. Results Percent of Agreement Same Quartile Adjacent Quartile Skip one Quartile Opposite Quartile Daily Intake 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls 1st FFQ vs. 2nd FFQ 2nd FFQ vs. 24 h Recalls Fruit 50.5 23.1 33.6 38.5 13.1 22.1 2.8 16.4 Vegetable 49.5 33.7 38.3 33.7 11.2 24.0 0.9 8.7 Grain 45.8 25.2 38.3 36.9 15.0 23.3 0.9 14.6 Milk 48.6 19.2 46.7 38.5 2.8 33.7 1.9 8.7 Meat 52.3 14.1 41.1 43.5 6.5 25.9 0 16.5 Table 5. Agreement in quartile distribution of food groups intake between two FFQ surveys and 24 h recall. Table 6. Pearson correlation coefﬁcients of food groups intake between two FFQ, 24 h recall. Table 6. Pearson correlation coefﬁcients of food groups intake between two FFQ, 24 h recall. FFQ1 vs. FFQ2 FFQ2 vs. FR Fruit 0.911 −0.130 Veg 0.749 0.028 Grain 0.614 −0.057 Milk 0.669 0.060 Meat 0.804 −0.192 4. Discussion Evaluating dietary habits in Qatar may help in spotting dietary risk factors that are likely contributing to the high obesity and diabetes rates in this country. Food frequency questionnaires are practical tools to assess the dietary intakes of large populations. How- ever, FFQs have to be validated against the time consuming—yet more accurate—24hDR. The aim of this study was to develop a FFQ tailored to the people in Qatar. To our knowl- edge, our study is the ﬁrst to attempt to create and validate a FFQ for this population. We translated the US National Cancer Institute DHQII and modiﬁed it to contain foods that are commonly consumed in Qatar. The FFQ contained questions on a total of 72 food items, administered to all participants twice, each one month apart, to assess the reliabil- ity. The average of three 24hDRs of the same participants was used to validate the FFQ. We used correlations, percentages of median difference, and agreement rates of distribution into quartiles to evaluate the validity and reliability of this FFQ. These are the standard statistical methods that have been typically and frequently used for this purpose [8–19] 9 of 11 Nutrients 2021, 13, 2002 Our results indicate that there was high agreement between intake of energy and macronutrient estimates from FFQ1 and FFQ2. This implies that the FFQ is reliable when estimating energy and macronutrients. When median intakes of energy, carbohydrates, protein, and fat estimated from FFQ2 and 24 h recall, results were also acceptable and reﬂect close estimates (Percentage of median difference ranged from 0.5% to 8%). Less valid data was obtained for micronutrients and food groups. This implies that the FFQ we developed was successful in predicting nutrient intakes, and indicates that the Qatari FFQ is valid for this purpose. Interestingly, the FFQ was remarkably successful in estimating energy intake in the study population, with a high correlation coefﬁcient of 0.974 and a 98.13% agreement rate with the energy intake reported through the 24hDR. This correlation is much stronger than what was previously reported on FFQs created for other populations. For example, a correlation coefﬁcient of 0.64 was reported between FFQ and 24hDR in the Shanghai’s Women Health Study, and 0.37 in a Canadian study [20,21]. Accurate estimation of energy intake through FFQ is important for drawing true conclusions on the connections between dietary habits and diseases. 4. Discussion Similar results have also been reported in the Belgium and the Dutch studies [22,23]. On the other hand, while the reliability of our FFQ was high in estimating most nutrients, its validity—especially for micronutrients—was relatively low as evidenced by low agreement rates (i.e., 19.23% for vitamin B6 between the two methods). Correlation coefﬁcients of nutrient intakes ranged between −0.17 (ﬂuorine) and 0.803 (carbohydrates) for validity, and 0.011 (vitamin B6) and 0.913 (carbohydrates) for reliability. In com- parison, El Kinany et al. used an adapted FFQ from the European Global Asthma and Allergy Network (GA2LEN) including traditional Moroccan foods, and found that the de-attenuated correlations for all nutrients were statistically signiﬁcant and positive, rang- ing from 0.24 (ﬁber) to 0.93 (total MUFA). For reproducibility, the intra-class correlation coefﬁcient were statistically signiﬁcant and ranged between 0.69 for fat and 0.84 for Vitamin A [24]. While, in the Shanghai study, Zang et al. reported that the adjusted Spearman’s correlations were 0.33–0.77 for validity and 0.46–0.79 for reliability [8]. Moreover, in a Peruvian study, Rodriguez et al. reported high validity, with an average Pearson’s corre- lation coefﬁcient of 0.70, and high reproducibility, with an average Pearson’s correlation coefﬁcient of 0.67 [25]. In a meta-analysis of the reproducibility of food frequency questionnaires, Cui and colleagues suggested that FFQs with a minimum correlation coefﬁcient of 0.5 for most nutrients may be considered reliable [26]. According to this criterion, we regard our FFQ to be reliable in general, with an average reliability coefﬁcient of 0.625 for all nutrients, and higher than 0.5 for most nutrients except for vitamins A and K, zinc, iodine, chromium, and ﬂuorine. This also paralleled good FFQ reliability of 0.749 for food group estimates. Importantly, the aforementioned meta-analysis also found that FFQ reproducibility may be improved with more food items, a 12-month interval for 24hDR—as opposed to less than 12 months—and shorter intervals between FFQs [25]. Therefore, future studies using our Qatari FFQ may improve reproducibility by modifying the dietary assessment protocol we used in our study. Previous studies collected FFQs with a one-year interval. Since limited reproducibility of FFQ is a known limitation of this method [26], we administered the two FFQs at a one-month interval to reduce intra-individual and seasonal variations in dietary choices. However, FFQ2 tended to overestimate nutrient intakes compared to FFQ1 and 24hDR for both macronutrients and micronutrients in our study. 4. Discussion While this may imply limited reproducibility of our FFQ, it could also reﬂect true changes in dietary intake or over- reporting in the FFQ2. Furthermore, FFQ2 also overestimated the intakes of some of the food groups including fruits, vegetables, grains, and dairy. Correlation coefﬁcients of food groups ranged between 0.614 (grains) and 0.911 (fruit) for reliability, and −0.192 (meat) to 0.028 (vegetables) for validity. Likewise, the estimations of micronutrients were poorly correlated between the two methods in our study, with some estimates even being nega- tively correlated between FFQ and 24hDR. However, since FFQ2 estimations for energy Nutrients 2021, 13, 2002 10 of 11 10 of 11 and macronutrients remained valid and reliable, this again likely means that our FFQ2 data are presenting true changes in food group and micronutrient intake patterns. Nonetheless, the food groups’ results should be interpreted cautiously when using the FFQ. Another possible explanation for the discrepancy in estimating micronutrients be- tween FFQ and 24hDR is that using energy adjustment in our analysis may have led to overestimation by FFQ2. Thus, using absolute rather than relative estimates of nutrient in- takes may circumvent this issue. Furthermore, while collecting additional 24hDRs around the time we conducted the FFQ2 may have provided a better explanation of this discrep- ancy, this may sensitize study participants to their food intake, making them answer the FFQ more accurately. The resulting training effect could then overinﬂate the true validity of FFQ. This issue has been previously described [21]. Additionally, it is well established that FFQ validity is greatly inﬂuenced by sex and to a lesser extent by age, BMI, and supplement use. Better validity in estimating many of the nutrients could be obtained if we adjust to remove the confounding effects of these variables [27,28]. This study has several strengths and limitations. A strength of this study was that it used primary data and used three 24 h recall. However, the sample size, as well as un ability to use biomarkers for FFQ validation. Another limitation of the study is the use of 24hDRs as the gold standard to compare with in the validation. It is known that 24hDRs does not reﬂect the long term intake. The use of FFQ can be reasonably reliable to rank individuals in epidemiological studies. 4. Discussion In conclusion, we developed a Qatari FFQ that provides a novel, comprehensive, and culturally-sensitive method for dietary evaluation that is suitable for this population. The performance of this FFQ fared better than many previously validated FFQs cited in epi- demiological studies, at least for the estimation of energy and macronutrient intakes. It also performed reliably in evaluating food groups and most nutrient intakes. Future studies may focus on improving the validity of this FFQ for estimating micronutrient intakes. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu13062002/s1, Figure S1: Bland-Altman plot for energy, carbohydrate, protein, fat intake. Author Contributions: Conceptualization, H.B. and R.T.; methodology, H.B., R.T., and Z.S.; formal analysis, Z.S.; investigation; H.B. and S.H.; writing—original draft preparation, A.K., R.T.A., and R.T.; writing—review and editing, H.B. supervision, H.B.; funding acquisition, H.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Qatar University (QUSG-CAS-DHS-14\15-2). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Qatar University (QUSG-CAS-DHS-14\15-2); 2016). 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. Data Availability Statement: Data is available from the PI upon request. Acknowledgments: Acknowledgement is due to Qatar University for funding the project. Conﬂicts of Interest: The authors declare no conﬂict of interest. , , [ ] Willett, W. Nutritional Epidemiology; Oxford University Press: New York, NY, USA, 2012; Volume 40. 1. Cade, J.E.; Burley, V.; Warm, D.; Thompson, R.; Margetts, B. Food-frequency questionnaires: A review of their design, validation and utilisation. Nutr. Res. Rev. 2004, 17, 5–22. [CrossRef] 2 Will W N i i l E id i l O f d U i i P N Y k NY USA 2012 V l 40 4. Gosadi, I.M.; Alatar, A.A.; Otayf, M.M.; AlJahani, D.M.; Ghabbani, H.M.; AlRajban, W.A.; Alrsheed, A.M.; Al-Nasser, K.A. Development of a Saudi Food Frequency Questionnaire and testing its reliability and validity. Saudi Med. J. 2017, 38, 636. [CrossRef] p gy y 3. Aoun, C.; Daher, R.B.; El Osta, N.; Papazian, T.; Khabbaz, L.R. Reproducibility and relative valid questionnaire to assess dietary intake of adults living in a Mediterranean country. PLoS ONE 2019, 14, p gy y 3. 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https://openalex.org/W2980316201	https://cris.unibo.it/bitstream/11585/734454/1/fonc-09-01004.pdf	English	null	Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia	Frontiers in oncology	2,019	cc-by	9,315	Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia Darina Ocadlikova 1, Mariangela Lecciso 1, Alessandro Isidori 2, Federica Loscocco 2, Giuseppe Visani 2, Sergio Amadori 3, Michele Cavo 1 and Antonio Curti 1 Darina Ocadlikova 1, Mariangela Lecciso 1, Alessandro Isidori 2, Federica Loscocco 2, Giuseppe Visani 2, Sergio Amadori 3, Michele Cavo 1 and Antonio Curti 1 1 Department of Hematology and Oncology, University Hospital S.Orsola-Malpighi, Institute of Hematology “L. and A. Seràgnoli”, Bologna, Italy, 2 Hematology and Stem Cell Transplant Center, AORMN Hospital, Pesaro, Italy, 3 Department of Medicine, Institute of Hematology, University Hospital Tor Vergata, Rome, Italy In solid tumors and hematological malignancies, including acute myeloid leukemia, some chemotherapeutic agents, such as anthracyclines, have proven to activate an immune response via dendritic cell-based cross-priming of anti-tumor T lymphocytes. This process, known as immunogenic cell death, is characterized by a variety of tumor cell modiﬁcations, i.e., cell surface translocation of calreticulin, extracellular release of adenosine triphosphate and pro-inﬂammatory factors, such as high mobility group box 1 proteins. However, in addition to with immunogenic cell death, chemotherapy is known to induce inﬂammatory modiﬁcations within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, such as the overexpression of indoleamine 2,3-dioxygensase 1, which may ultimately hamper anti-tumor T-cells via the induction of T regulatory cells. The aim of this review is to summarize the current knowledge about the mechanisms and effects by which chemotherapy results in both activation and suppression of anti-tumor immune response. Indeed, a better understanding of the whole process underlying chemotherapy-induced alterations of the immunological tumor microenvironment has important clinical implications to fully exploit the immunogenic potential of anti-leukemia agents and tune their application. Edited by: Edited by: Marcos De Lima, Case Western Reserve University, United States Reviewed by: Benjamin Bonavida, University of California, Los Angeles, United States Yona Keisari, Tel Aviv University, Israel *Correspondence: Darina Ocadlikova jennynka@seznam.cz Specialty section: This article was submitted to Hematologic Malignancies, a section of the journal Frontiers in Oncology Keywords: acute myeloid leukemia, immunogenic cell death, dendritic cells, T regulatory cells, immunosuppression Received: 02 July 2019 Accepted: 18 September 2019 Published: 09 October 2019 REVIEW REVIEW published: 09 October 2019 doi: 10.3389/fonc.2019.01004 published: 09 October 2019 doi: 10.3389/fonc.2019.01004 Keywords: acute myeloid leukemia, immunogenic cell death, dendritic cells, T regulatory cells, immunosuppression Citation: Ocadlikova D, Lecciso M, Isidori A, Loscocco F, Visani G, Amadori S, Cavo M and Curti A (2019) Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia. Front. Oncol. 9:1004. doi: 10.3389/fonc.2019.01004 Ocadlikova D, Lecciso M, Isidori A, Loscocco F, Visani G, Amadori S, Cavo M and Curti A (2019) Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia. Front. Oncol. 9:1004. doi: 10.3389/fonc.2019.01004 Acute myeloid leukemia (AML) is a clonal disorder sprouting from a rare population of leukemic stem cells with impaired diﬀerentiation capacity into fully mature myelocytic cells. Although new and potent drugs have recently entered the clinical stage, the induction therapy of AML is still principally based on cytotoxic drugs which are able to achieve complete remission (CR) in up to 70% of adult patients (1, 2). However, the probability of relapse remains elevated, in particular in elderly or prognostically “high risk” patients, unless transplantation of autologous or, more importantly, allogeneic hematopoietic stem cells is performed as post-CR consolidation strategy (3). October 2019 \| Volume 9 \| Article 1004 Frontiers in Oncology \| www.frontiersin.org Tregs During Immunogenic Cell Death in AML Ocadlikova et al. in morphology, but caspase-dependent and highly eﬃcient in immune response induction without any adjuvant (14, 15). Such a death process, called immunogenic cell death (ICD), was introduced for the ﬁrst time 10 years ago (16–20). During this process, the TAAs are released from dying tumor cells together with some factors, known as damage-associated molecular pattern molecules (DAMPs), generated in cell-stress conditions, hypoxia or nutrient depletion, which bind receptors expressed on immune cell surfaces, thus stimulating innate immune responses. In this context, specialized antigen-presenting cells, i.e., dendritic cells (DCs), play a crucial role in eﬃciently priming TAA-speciﬁc T cells (21). Subsequent studies have identiﬁed various mechanisms of the ICD process, and also highlighted the importance of the host capacity to detect the ICD events and induce a therapeutically relevant immune response against dying cells (16, 17, 20, 22, 23). In the last few years, cancer immunotherapy, which is based on the ability of the immune system to recognize tumor- associated antigens (TAAs) and mediate a highly speciﬁc cytolytic response against tumor cells, is gaining much interest due to its unique characteristics, such as the absence of conventional drug resistance mechanisms and low grade of toxicity. ICD EVENTS AND IMMUNE SYSTEM ACTIVATION ICD biology has been actively studied over the last 10 years. Very schematically, ICD is represented by the coordinated emission of a series of DAMPs (24–29), including the translocation of the endoplasmic reticulum (ER) chaperones such as calreticulin (CRT) and heat shock proteins 70 and 90 (HSP70 and 90) on cell surface, the adenosine triphosphate (ATP) active secretion, the non-histone chromatin-binding protein high mobility group box 1 (HMGB1) release from nucleus in extracellular milieu (30–37) and ﬁnally, the release of immunostimulatory cytokines, such as type I IFN (38). AML is a neoplasm with characteristics which make it suitable to elicit eﬀective speciﬁc immune responses. Indeed, 50–90% of cases reveal chromosomal anomalies, above all translocations, which give rise to rejected tumor antigens, namely neo-antigens, not expressed by normal cells. Moreover, leukemia cells express elevated levels of TAAs, which can be recognized by the immune system and can induce a T cell-targeted response. These TAAs are proteinase 3, the receptor for hyaluronan mediated motility and Wilms tumor protein (6). Moreover, various cell types such as αβ and γδ T cells, and NKT and NK cells have proven to be functional against AML cells together with a series of eﬀector molecules such as perforin and tumor necrosis factor-related apoptosis-inducing ligand, but also IFN-γ, IFN type I, and IL- 12 (7–9). Based on these premises, since the immune system is activated against leukemia cells, the possibility to harness immunity against AML to obtain a durable leukemia-speciﬁc immune response should not be underestimated in the clinical management of AML patients. In the early phase, CRT translocates from the ER to the outer leaﬂet of the plasma membrane, thus initiating the apoptotic caspase-dependent process. Simultaneously, the HSP70 and HSP90 bind TAAs and contribute to stimulate DC maturation. During the tardive post-apoptotic phase, pro-inﬂammatory factor HMGB1, which binds toll-like receptor 4 (TLR 4) on DCs, is released from the nucleus in the extracellular space. Finally, autophagy-dependent active secretion of ATP, which binds purinergic receptors (P2Rs) on DCs, promotes DC recruitment, survival and diﬀerentiation (39, 40). When emitted in the correct spatiotemporal context, these DAMPs recruit DCs in the proximity of ICD and activate them to engulf TAAs. Citation: In AML, the immunotherapy ﬁeld is evolving and expanding. In this scenario, recent promising clinical results support the full development of immune-based strategies for the management of AML patients. IMMUNOGENICITY OF AML AND IMMUNOTHERAPY The data demonstrating increased incidence of solid tumors in immune-compromised patients, spontaneous immune- based regression of some tumors and favorable prognostic impact of tumor-inﬁltrating cytotoxic T lymphocytes (CTLs) or serum tumor-speciﬁc antibodies support the hypothesis that the immune system plays a very important role in tumor development, growth, and progression (4). The most clear demonstration of tumor eradication by the immune system comes from the setting of hematopoietic stem cell transplantation (SCT), where the existence of the Graft vs. Leukemia eﬀect accounts for the prominent therapeutic activity of transplantation (5). ICD EVENTS AND IMMUNE SYSTEM ACTIVATION As a consequence, DCs become fully matured and competent in skewing cytokine production toward immunostimulatory cytokines, like IL-1β, IL-12p70, and IL-6, in spite of immunosuppressive cytokines, such as IL-10 (30, 34, 36), this process being strictly required for the adequate polarization of IFN-γ producing CD8+ T cells. The activation of APCs generally proceeds in two sequential phases, i.e., recruitment of T cells followed by their activation into IL-17- secreting γδ T cells, αβ Th1 T cells (IFN-γ secreting CD4+ T cells), and αβ cytotoxic T cells (IFN-γ secreting CD8+ T cells) (27, 31, 34). The latter are not only capable of mediating direct anti-tumor eﬀects, but also underlie the establishment of host-protective immunological memory. Importantly, CRT exposure, HMGB1 release, and ATP secretion are indispensable for ICD. Indeed, the absence of just Frontiers in Oncology \| www.frontiersin.org IMMUNOGENIC CANCER CELL DEATH In recent years, the concept of two principal forms of cell death which can promote tolerance (e.g., apoptosis) or immunity (e.g., necrosis) was ﬁrst challenged and then surpassed, and a number of factors determining whether a death is tolerogenic or immunogenic was identiﬁed (10). An example is represented by the type of cell death, induced by therapeutics such as ionizing radiations or cytotoxic chemotherapy. Selected antineoplastic agents, in particular ionizing irradiation, anthracyclines, oxaliplatin, cyclophosphamide, mitoxantrone, and others (11– 13), are able to induce a type of cell death which is apoptotic October 2019 \| Volume 9 \| Article 1004 Frontiers in Oncology \| www.frontiersin.org 2 Tregs During Immunogenic Cell Death in AML Ocadlikova et al. one of these ICD hallmarks cancels out the eﬃcacy of ICD in mouse model (41). be released into the extracellular space (HSPs 70 and 90). HSP exposure/release from cells that underwent ER stress represents one of the distinctive factors of chemotherapy-induced ICD (48). HSPs can potentiate immunogenicity in diﬀerent ways. On one hand, when present on the cell surface of tumor cells they can improve the recognition and up-take of dying cells by DCs. On the other hand, TAAs derived from dying tumor cells can bind HSPs, thus enhancing eﬃcient antigen presentation. HSP-antigen complex recognition is mediated by TLR4, which facilities intracellular processing and presentation of TAAs (48). be released into the extracellular space (HSPs 70 and 90). HSP exposure/release from cells that underwent ER stress represents one of the distinctive factors of chemotherapy-induced ICD (48). HSP Exposure HSPs play an important role as chaperones ensuring the correct folding of newly synthesized proteins or damaged proteins as a consequence of cellular stress and preventing their aggregation. However, HSPs can have a double role based on cellular localization. In the case of HSP 90, the intracellular localization determines a cytoprotective function responsible for addressing of damaged proteins toward proteasome degradation, thus maintaining protein homeostasis (48). On the contrary, when located inside the cells, HSP70 interacts with various components of apoptotic machinery at both pre- and post-mitochondrial level, thus preventing an inappropriate ICD induction caused by stress-induced cell damage. Importantly, the HSPs can translocate to the outer plasma membrane leaﬂet (HSP 70) or can Release of HMGB1 From the Nucleus HMGB1 is a nuclear protein, which participates in the folding of DNA in the chromatin structure, thus inﬂuencing transcription and other nuclear functions. In contrast to histones, which are part of nucleosomes, the interaction of HMGB1 with chromatin is rather loose, which means that HMGB1 can exit from the nucleus to the cytoplasm. Importantly, HMGB1 also acts as an extracellular signal molecule, DAMP, and can be released from cells by non-canonical secretion pathways or passively released through the permeabilized plasma membrane of dead cells (49). Indeed, after cellular stress, HMGB1 translocates to cytosol and is then released to the extracellular space. When it binds to speciﬁc receptors, together with other cytokines, HMGB1 can induce myeloid DC maturation by CD40, CD54, CD80, CD83, and MHC II upregulation (20). CRT exposure represents an “eat me signal” for DCs, the crucial component of immune system activation by chemotherapy (45). CRT initiates phagocytosis of apoptotic cells binding to the CD91 receptor (known as LDL-correlate receptor protein; LRP) on phagocytic cells. The presence of a CRT speciﬁc receptor on DCs and its activation are essential for immunogenicity of tumor cell death. Interestingly, CRT translocation also occurs in viable malignant cells (46), suggesting that apoptosis may not be necessarily required for CRT translocation, and that “ER stress” induction can be suﬃcient to promote its cell surface expression (47). Under certain circumstances, cells dying by apoptosis or autophagy can release HMGB1, as observed in the case of DNA damage induced by UV radiation or platinization, where HMGB1 is sequestrated in the nucleus and the ICD inducers, such as anthracyclines, and stimulate HMGB1 release in the late phase of apoptosis (20). HMGB1 released in the extracellular space binds mainly the TLR4 present on DCs, thus facilitating TAA processing and presentation through the inhibition of phagosome and lysosome fusion, and the prevention of early degradation and by allowing their transport to eﬀector immune cells (48). Moreover, it has been demonstrated that HMGB1 released during tumor cell necrosis induces not only DC maturation, but also secretion of IL-12 by DCs and IFN-γ by T cells acting as a potent stimulus for polarization of Th1 response (20). Early ICD Events CRT Translocation HSPs can potentiate immunogenicity in diﬀerent ways. On one hand, when present on the cell surface of tumor cells they can improve the recognition and up-take of dying cells by DCs. On the other hand, TAAs derived from dying tumor cells can bind HSPs, thus enhancing eﬃcient antigen presentation. HSP-antigen complex recognition is mediated by TLR4, which facilities intracellular processing and presentation of TAAs (48). CRT can be localized in the cytoplasm, on the cell membrane or in the extracellular matrix, operating in both extra and intracellular space. Inside the ER, CRT plays an essential role in the regulation of intracellular Ca2+ homeostasis and storage, thus participating in a large variety of Ca2+-dependent signal transduction mechanisms. Moreover, CRT is involved in CRT/calnexin cycles where, interacting with calnexin and 57- kDa protein ER (ERp57), it ensures the correct folding of newly synthesized proteins and glycoproteins. In this context, CRT is fundamental also for assembly of major histocompatibility complex (MHC) molecules, which are essential for class I antigens presentation (42). Exposure to ICD inducers like anthracyclines, oxaliplatin, or ionizing radiation is able to induce translocation of CRT/ERp57 complex to the cell surface. Although the whole process underlying CRT protein exposure is far from being fully elucidated, three steps have been clearly identiﬁed: ER stress induction, apoptosis and translocation. Initially, stress response induction causes activation of reticulum PERK serin/treonin kinase which phosphorylates the eukaryotic translation initiation factor 2α (eIF2α) following the partial caspase-8 activation, the caspase-8-mediated cleavage of BAP31 and structural activation of pro-apoptotic proteins BAX and BAK. Finally, the translocation process predicts exocytosis through a SNARE-dependent mechanism in which CRT and ERp57 are transported inside vesicles to the outer plasma membrane leaﬂet of Golgi apparatus (43, 44). Collectively, these ﬁndings indicate that the presence of HSPs on dying tumor cells is critical for tumor cell recognition by DCs, full DC maturation and, thus, for the induction of a tumor- speciﬁc immune response. Late ICD Events Release of HMGB1 From the Nucleus Release of HMGB1 From the Nucleus Frontiers in Oncology \| www.frontiersin.org ATP Extracellular Release One of the most distinctive features of ICD is represented by the active extracellular release of ATP from dying cells during the tardive phase of apoptosis. Normally located inside the cells, ATP is considered the most important factor for bioenergetics, connecting anabolism and catabolism, with a well-established crucial role in some important processes, such as cellular motility, phosphorylation, and active transport. By speciﬁcally testing which of the P2Rs is involved, as well as the type or the optimal concentration of released nucleotides within the extracellular October 2019 \| Volume 9 \| Article 1004 3 Tregs During Immunogenic Cell Death in AML Ocadlikova et al. the antigenicity of cancer cells (increased TAA expression or presentation) (i); increasing the immunogenicity of cancer cells (DAMP production and release) (ii) and increasing the susceptibility of cancer cells (better recognition and killing of cancer cells by immune eﬀectors) (iii) (68). As for the enhancement of antigenicity, cyclophosphamide, oxaliplatin and γ irradiation have been shown to increase MHC I molecule expression by cancer cells (69, 70), whereas γ irradiation, 5- ﬂuorouracil or vemurafenib increased TAA expression (69, 71, 72). Regarding immunogenicity, anthracyclines, oxaliplatin, mafosfamide, bortezomib, and some other types of chemotherapy agents are eﬀective in inducing CRT and HSP exposure (11, 12, 32, 47, 59, 61), as well as ATP secretion (31, 52, 55) and HMGB1 release (12, 73) from various tumor cells including leukemias. Finally, to increase the susceptibility of cancer cells, diﬀerent anticancer agents including anthracyclines have been shown to sensitize murine tumor cells to the cytotoxic function of CTLs (74). Moreover, other pieces of evidence indicate that chemotherapy favors breast cancer cell inﬁltration by myeloid and granzyme B-expressing cells, while increasing the intra-tumoral CD8+ and CD4+ T cell ratio (75). Taken together, accumulating evidence suggests that, in some settings, tumor-speciﬁc immune responses induced during chemotherapy drive the destiny of cancer patients (76, 77). environment, various and seminal studies demonstrated the role of extracellular nucleotides in the regulation of cell proliferation, migration, and death (50, 51). g While diﬀerent mechanisms of ATP release are known, the elevated release of ATP during ICD induced by chemotherapy principally depends on the induction of the autophagy process. Autophagy is a multistep process that involves cytoplasmic material sequestration within double-membraned organelles, autophagosomes, and their fusion with lysosomes (52). ATP Extracellular Release Importantly, besides its role as a DAMP molecule, extracellular ATP represents a strong “ﬁnd me” signal which facilitates DC recruitment in sites of massive apoptosis (53). DC recruitment in tumor sites is mediated by P2Y2 receptors (54), whereas activation of P2Y11 receptors on monocytes and DCs induces their maturation (55). Once recruited, naive immune cells need activation signals to increase their anti-tumoral activity. It has recently been demonstrated that P2XRs are essential for the immune response induced by chemotherapy. ATP released from dying cells binds to the P2X7 receptor present on DCs, thus determining assembly and activation of inﬂammasome NOD-like Receptor protein 3 (NLRP3)/ASC/caspasi-1 driving the IL-1β secretion. The IL-1β is fundamental for adequate recruitment of γδ T lymphocytes secreting IL-17 and cytotoxic CD8+IFN-γ+ tumor-speciﬁc T lymphocyte generation (56, 57). For hematological malignancies, recent studies have demonstrated that anthracyclines trigger ICD in vitro and in murine models (78) including AML (34, 47). In particular, in AML patients, following anthracycline administration, CRT translocates from the nucleus to the leukemia cell surface. Indeed, Fredly et al. has demonstrated that CRT is exposed by apoptotic primary human AML cells in 65% of tested patients and that, in vitro, cultured AML cells showed spontaneous release of HSP70 and 90 (62). Of note, similarly to solid tumors, including neuroblastoma, non-small cell lung carcinoma, ovarian cancer, and colorectal carcinoma, where CRT exposure has been shown to be an important prognostic factor (79–81), CRT exposure by AML cells has been recently correlated by Fucikova et al. with a strong anticancer immune response, improving the clinical outcome of AML patients (59, 60). Surprisingly, these authors have found that DAMP emission from AML may also be chemotherapy-independent. In particular, 82% of AML patients exhibited positivity for CRT expression prior to treatment and a similar pattern was observed also for HSP exposure and HMGB1 release, thus suggesting that DAMP production may represent an intrinsic feature of some types of AML, which make them more prone to interact with the immune system. Indeed, CRT exposure was associated with enhanced anti-leukemia immune response and better prognosis. Transcriptional and phenotyping signature analysis in patients with AML has revealed robust vs. weak CRT exposure on blasts. Moreover, AML patients are prognostically divided into two groups based on the median percentage of circulating ecto-CRT, HSP70, or HSP90 positive cells, thus revealing that ICD-associated DAMPs correlate with improved disease outcome (60). ATP Extracellular Release CRT exposure on malignant blasts predicts a cellular anticancer immune response in patients with AML (61). Frontiers in Oncology \| www.frontiersin.org Clinical trials in AML Blocking of CTLA-4 pathway—increase of anti-leukemia T-cell immune response translated in prolonged tumor regression (91, 92). 1. Anti-CTLA-4 Ipilimumab: NCT00039091, NCT02890329, NCT02397720 CD200R/CD200 CD200R—inhibitory receptor. Ligand: CD200. CD200-CD200R signaling—down-regulation of immune responses preventing inﬂammation and immune pathology (83). CD200R/CD200—immunosuppressive signal transmission, macrophages inhibition, Tregs induction and tumor-speciﬁc T cells inhibition (93). Expression of CD200 on human AML cells (94)—worse overall survival of some AML subsets (83). Blocking of CD200—enhanced cytotoxicity of NK cells, restored proliferative capacity of T cells, dampens tumor-reactive immune responses (95), but also favors tumor progression due to enhanced pro-tumorigenic inﬂammation (96). 1. Anti-CD200 Samalizumab: NCT03013998 Lag-3 Lag-3 receptor of negative co-stimulation. Ligand: MHC II. Lag-3/MHCII signaling - tolerance maintenance (83, 97). Lag-3 signaling-suppression of CTL activity in tumors (97, 98). Blocking of PD-L1, CTLA-4 and Lag-3—effective and enduring immunotherapy for disseminated leukemia in murine model (98). To date—no clinical trials available Tim-3 Tim-3—receptor of negative co-stimulation. Ligands: gal-9/HMGB1/phosphatidyl serin. Tim-3/gal-9 signaling—regulation of T-cell tolerance (83). Tim-3 released by AML—reduce ability of T cells to secrete IL-2 required for NK and CTLs activation (99). TIM-3 and PD-1 co-expression on T cells was associated with AML progression in mouse and human (7) and with relapse in AML patients after allo-SCT (100). TIM-3—overexpression on AML (stem) cells (101) and T cells of newly diagnosed AML -(102). Blocking of TIM-3 and PD-1—reduced tumor burden and improved survival in AML murine model (7). 1. AntiPD-1 + TIM-3 PDR001+MBG453+ Decitabine: NCT03066648 IDO and Tregs IDO –immunosuppressive and tolerogenic enzyme responsible for tryptophan degradation in kynurenines with subsequent T cell inhibition and Tregs expansion. Tregs—role in maternal tolerance, autoimmune disease regulation, suppression of transplant rejection (85). IDO signaling—Tregs induced by IDO-expressing leukemic DCs impair leukemia-speciﬁc CTL (103). Increased IDO activity—lower CR rates and shorter OS in AML (103–105). Blocking of IDO—effective immune response in AML in vitro (103–106). 1. Anti-IDO Epacadostat: NCT03444649 Different inhibitory pathways and their role in both physiological and AML contexts are correlated with clinical trials ongoing for speciﬁc pathways. CD200R/CD200 CD200R—inhibitory receptor. Ligand: CD200. CD200-CD200R signaling—down-regulation of immune responses preventing inﬂammation and immune pathology (83). 1. AntiPD-1 + TIM-3 PDR001+MBG453+ Decitabine: NCT03066648 Tim-3 released by AML—reduce ability of T cells to secrete IL-2 required for NK and CTLs activation (99). TIM-3 and PD-1 co-expression on T cells was associated with AML progression in mouse and human (7) and with relapse in AML patients after allo-SCT (100). ICD IN SOLID TUMORS AND LEUKEMIAS Recent data support the role of chemotherapy in activating the immune response both in solid tumors (9, 11, 12, 44, 58) and, recently, in leukemias (34, 47, 59–62), with important therapeutical implications. For a long time, the immune system was considered as a passive bystander of cancers, until the antineoplastic potential of new drugs in immunodeﬁcient murine models was tested. Accumulating preclinical evidence has indicated that murine tumors respond more eﬃciently to therapies in immunocompetent individuals than in immunodeﬁcient hosts (63, 64), suggesting an important role of the immune system mediating the chemotherapy eﬀects. Several anticancer agents, for example anthracyclines in colorectal cancer (9), ﬁbrosarcomas (57), and methylcholanthrene-induced tumors (65), cyclophosphamide in mesotheliomas (66), oxaliplatin in colorectal carcinomas and ﬁbrosarcomas (12, 31), and cisplatin in combination with digoxin in ﬁbrosarcomas (67) were tested and proven to activate the immune system, which ultimately and crucially contributes to the clinical response of cancers to chemotherapy treatment. Regarding chemotherapeutic drugs, it is very important to diﬀerentiate between the direct immunogenic eﬀects that such therapeutic regimens exert on tumor cells, and the capacity of chemotherapy-treated tumor cells to interact with the host immune system, resulting in reactivation of immune eﬀectors, or in relief of immune-suppressive mechanisms. Three principal ways in which antineoplastic agents may stimulate the immune system were deﬁned by Zitvogel et al.: increasing October 2019 \| Volume 9 \| Article 1004 4 Tregs During Immunogenic Cell Death in AML Ocadlikova et al. TABLE 1 \| Inhibitory pathways in AML. Inhibitory pathway/check point Physiological role Role in AML Clinical trials in AML PD-1/PD-L1 axes PD-1—receptor of negative co-stimulation. Ligands: PD-L1 and PD-L2. PD-1/PD-L1 axes—control of normal immune responses, involved in periphery tolerance, autoimmunity regulation, allergy, infections, and antitumor immunity (87). PD-1/PD-L signaling—dampening of anti-leukemic immunity in AML. PD-L1 and PD-L2 expression on human AML cells at diagnosis and relapse (88). Blocking of PD-1/PD-L1 axis—increase of anti-leukemia immune response and prevention of AML progression in murine model (83, 84, 89). 1. Anti-PD-1 Nivolumab: NCT02275533, NCT02397720, NCT02532231, NCT03092674, NCT02464657, NCT02275533, NCT03066648 Pembrolizumab: NCT02708641, NCT02845297, NCT02996474, Clinical trials in AML Clinical trials in AML PD-1/PD-L signaling—dampening of anti-leukemic immunity in AML. PD-L1 and PD-L2 expression on human AML cells at diagnosis and relapse (88). Blocking of PD-1/PD-L1 axis—increase of anti-leukemia immune response and prevention of AML progression in murine model (83, 84, 89). PD-1/PD-L signaling—dampening of anti-leukemic immunity in AML. PD-L1 and PD-L2 expression on human AML cells at diagnosis and relapse (88). Blocking of PD-1/PD-L1 axis—increase of anti-leukemia immune response and prevention of AML progression in murine model (83, 84, 89). 1. Anti-PD-1 Nivolumab: NCT02275533, NCT02397720, NCT02532231, NCT03092674, NCT02464657, NCT02275533, NCT03066648 Pembrolizumab: NCT02708641, NCT02845297, NCT02996474, NCT02771197, NCT02768792 Avelumab: NCT02953561 2. Anti-PD-L1 Durvalumab: NCT02775903 Atezolizumab: NCT02892318, NCT03154827 CTLA-4/CD80/CD86—hampering T cell immunity against hematological malignancies (83) and modulating immune responses in AML (90). Blocking of CTLA-4 pathway—increase of anti-leukemia T-cell immune response translated in prolonged tumor regression (91, 92). 1. Anti-CTLA-4 Ipilimumab: NCT00039091, NCT02890329, NCT02397720 CD200R/CD200—immunosuppressive signal transmission, macrophages inhibition, Tregs induction and tumor-speciﬁc T cells inhibition (93). Expression of CD200 on human AML cells (94)—worse overall survival of some AML subsets (83). Blocking of CD200—enhanced cytotoxicity of NK cells, restored proliferative capacity of T cells, dampens tumor-reactive immune responses (95), but also favors tumor progression due to enhanced pro-tumorigenic inﬂammation (96). 1. Anti-CD200 Samalizumab: NCT03013998 Lag-3 signaling-suppression of CTL activity in tumors (97, 98). Blocking of PD-L1, CTLA-4 and Lag-3—effective and enduring immunotherapy for disseminated leukemia in murine model (98). To date—no clinical trials available Tim-3 released by AML—reduce ability of T cells to secrete IL-2 required for NK and CTLs activation (99). TIM-3 and PD-1 co-expression on T cells was associated with AML progression in mouse and human (7) and with relapse in AML patients after allo-SCT (100). TIM-3—overexpression on AML (stem) cells (101) and T cells of newly diagnosed AML -(102). Blocking of TIM-3 and PD-1—reduced tumor burden and improved survival in AML murine model (7). 1. AntiPD-1 + TIM-3 PDR001+MBG453+ Decitabine: NCT03066648 IDO signaling—Tregs induced by IDO-expressing leukemic DCs impair leukemia-speciﬁc CTL (103). Increased IDO activity—lower CR rates and shorter OS in AML (103–105). Blocking of IDO—effective immune response in AML in vitro (103–106). 1. Anti-IDO Epacadostat: NCT03444649 ts are correlated with clinical trials ongoing for speciﬁc pathways. NCT02892318, NCT03154827 CTLA-4 CTLA-4—receptor of negative co-stimulation. Ligands: CD80 and CD86. CTLA-4/CD80/CD86 pathway—regulation of T cell response (83). CTLA-4/CD80/CD86—hampering T cell immunity against hematological malignancies (83) and modulating immune responses in AML (90). Frontiers in Oncology \| www.frontiersin.org Clinical trials in AML TIM-3—overexpression on AML (stem) cells (101) and T cells of newly diagnosed AML -(102). Blocking of TIM-3 and PD-1—reduced tumor burden and improved survival in AML murine model (7). IDO and Tregs IDO signaling—Tregs induced by IDO-expressing leukemic DCs impair leukemia-speciﬁc CTL (103). Increased IDO activity—lower CR rates and shorter OS in AML (103–105). Blocking of IDO—effective immune response in AML in vitro (103–106). Blocking of IDO—effective immune response in AML in vitro (103–106). October 2019 \| Volume 9 \| Article 1004 Frontiers in Oncology \| www.frontiersin.org Frontiers in Oncology \| www.frontiersin.org Tregs During Immunogenic Cell Death in AML Ocadlikova et al. FIGURE 1 \| Balance between immune activation and tolerance during ICD in AML. Immunogenic chemotherapy causes the release of DAMPs (CRT, HSPs, ATP, and HMGB1) which bind to receptors on DCs as CD91, TLR4, and P2X7. DCs up-regulate maturation markers (CD80, CD86, and CD83) and produce IL-1β resulting in activation of T cells producing IFN-γ At the same time, DCs up-regulate IDO1 which is responsible for the production of kynurenines which in turn stimulate induction of Tregs producing IL-10 and inhibit effector T cells. IDO1 is expressed also on AML cells and Treg cells, thus participating to the suppressive local milleu. Immune check points receptors (ICRs) as PD-1, Tim-3, Lag-3, CD200R, and CTLA-4 can contribute to the cell composition of tumor microenvironment. In this context, IDO1 seems to play a key role in the balance between immune system activation and tolerance in AML during ICD. MECHANISMS OF IMMUNOLOGIC TOLERANCE IN AML induction of immunoregulatory populations expansion as Tregs (83–85). The most known suppressive mechanism in AML is the up-regulation of IDO1 expression on leukemia cells. IDO1 is responsible for catalyzing the initial rate-limiting step of tryptophan degradation resulting in increased ﬁnal product kynurenines. The kynurenines have suppressive properties and increase the conversion of CD4+25−T cells into Tregs. In addition, their suppressive eﬀect relies on the fact that they can reduce the activity of NK cells, DCs or proliferating T cells, in response to inﬂammation or infection (85, 86). The most known inhibitory pathways in AML are reported in Table 1. Along with the well-known cell-intrinsic mechanisms by which leukemic cells can develop drug resistance, which leads to enhanced proliferation and survival, the role of cell- extrinsic factors, partly derived from AML bone marrow, the immunosuppressive microenvironment has recently been investigated (82). It is known that both the innate and adaptive immune systems are deeply aﬀected and profoundly deregulated by the interaction with leukemia cells. This happens as a result of several diﬀerent immunosuppressive mechanisms, which, in turn, may lead to the escape of leukemia cells from the natural immunological control (82). Many of these regulatory mechanisms seem to be shared by solid tumors and hematology neoplasms including over-expression of inhibitory check-point receptors on T cells and their ligands on AML cells or DCs such as PD-1/PD-L1, CTLA-4/CD80/CD86, Tim- 3/galectine-9 (gal-9), and Lag-3/MHCII, enzymes as IDO and Frontiers in Oncology \| www.frontiersin.org CONCLUDING REMARKS Some antineoplastic agents are capable of activating the immune system through the release of inﬂammatory signals from dying tumor cells. However, recent evidence indicates that chemotherapy may also provide the tumor microenvironment with a number of tolerogenic signals, mainly resulting in Tregs induction, which negatively inﬂuence immune response activation. Interestingly, the same mechanisms leading to immune activation are suggested to be also responsible for tolerance induction. Then, to fully exploit the immunogenic potential of chemotherapy, it is necessary to concomitantly act by inhibiting tolerance induction. Indeed, early clinical studies are testing the safety and early eﬃcacy of new immunological agents contrasting tolerogenic mechanisms, such as IDO1 and immune checkpoint inhibitors, in combination with immunogenic chemotherapy. Similarly to solid tumors, also in AML it is well-known that inducing a suppressive microenvironment by expanding Tregs may hamper the anti-leukemia immune response (103, 109). Interestingly, early lymphocyte recovery in 20 patients undergoing induction chemotherapy for newly diagnosed AML indicated that recovering T cells were predominantly activated Tregs with suppressive activity. Despite an initial burst of thymopoiesis, most recovering Tregs were of peripheral origin and showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven (108). Wang et al. too demonstrated a rapid turnover of Tregs in AML patients after chemotherapy compared to healthy controls (110). Together, these ﬁndings suggest an important role of Tregs induction after chemotherapy in AML. Although this dual process is relevant in many tumors, it is particularly important in the setting of AML, where chemotherapy still constitutes the most powerful and curative therapeutical tool for most patients. For these reasons, in the AML ﬁeld these studies will help in better understanding the biology of ICD, including the critical balance between activation and tolerance, thus providing the rationale for moving another step forward for an integrated immunological approach to AML therapy. We recently investigated the mechanisms underlying the eﬀects of chemotherapy on Tregs induction. In particular, we focused on the tolerogenic role of leukemia-inﬁltrating DCs after chemotherapy. Our in vitro and in vivo data demonstrate that during ICD a population of DCs expressing IDO1 is responsible for the induction of Tregs (106). In particular, we demonstrated that ATP released from chemotherapy-treated AML cells is responsible for IDO1 up-regulation on DCs through the P2X7 receptor and consequent Tregs enrichment, resulting in the establishment of an immune suppressive microenvironment. FUNDING This study was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) 2017 IG20654, FATRO/Foundation Corrado and Bruno Maria Zaini-Bologna, Fabbri1905, Bologna AIL (Associazione Italiana contro le Leucemie)/Section of Bologna and AIL Pesaro Onlus. Taken together, these ﬁndings suggest that IDO and related downstream pathways resulting in Tregs induction may play an important regulatory role in the choice between tolerance or immunity in response to dying tumor cells (Figure 1) and are in line with other recent studies which use preclinical CONCLUDING REMARKS Moreover, the analysis of the T-cell composition emerging in AML patients after induction chemotherapy revealed an enrichment and activation of the most suppressive Tregs- subpopulation expressing FOXP-3, CTLA-4, CD39, PD-1, and Ki-67 (106). These results demonstrated that ATP released from chemotherapy-treated dying leukemic cells during ICD has a role in the induction of the immune suppressive microenvironment, which comprises Tregs and IDO1-expressing DCs (106). TOLEROGENIC MECHANISMS DURING ICD In the ICD scenario, some recent reports indicate that, along with the activation of the immune system, a wide variety of tolerogenic mechanisms is also induced, mostly resulting in Tregs induction (53, 107, 108). In particular, Tregs induction after immunogenic October 2019 \| Volume 9 \| Article 1004 Frontiers in Oncology \| www.frontiersin.org 6 Tregs During Immunogenic Cell Death in AML Ocadlikova et al. chemotherapy was observed in some solid tumors. Bugaut et al. demonstrated that bleomycin, an anti-tumor antibiotic glycopeptide produced by bacterium Streptomyces and used for the treatment of cancer testis and Hodgkin disease, induces both ICD resulting in anti-tumor CD8+ T cell response and Tregs accumulation in vivo. Speciﬁcally, bleomycin induces expansion of Foxp3+ Tregs via its capacity to induce transforming growth factor beta (TGF-β) secretion by tumor cells. Accordingly, Tregs or TGF-β depletion dramatically potentiates the antitumor eﬀect of bleomycin. Based on these premises, it is conceivable that in order to fully exploit the activatory capacity of immune response by immunogenic chemotherapy, it may be fundamental to concomitantly block chemotherapy-driven Tregs induction. models of self-tolerance and autoimmunity (85). In this scenario, chemotherapy-induced ICD can prompt both immune tolerance and activation through the same mechanisms, and the balance between these phenomena can be fundamental for the ﬁnal immune system response. 1. Döhner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Büchner T, et al. Diagnosis and management of AML in adults: 2017 ELN AUTHOR CONTRIBUTIONS DO wrote and revised the manuscript and was the major contributor. ML wrote and revised sections of the manuscript. FL collected the related papers. SA, GV, MC, and AI participated in the design of the review and helped to draft and revise the manuscript. 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(2012) 26:2148–51. doi: 10.1038/leu.2012.77 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. 96. Rygiel TP, Meyaard L. CD200R signaling in tumor tolerance and inﬂammation: a tricky balance. Curr Opin Immunol. (2012) 24:233–8. doi: 10.1016/j.coi.2012.01.002 97. Lichtenegger FS, Rothe M, Schnorfeil FM, Deiser K, Krupka C, Augsberger C, et al. Targeting LAG-3 and PD-1 to enhance T cell activation by antigen- presenting cells. Front Immunol 9:385. doi: 10.3389/ﬁmmu.2018.00385 Copyright © 2019 Ocadlikova, Lecciso, Isidori, Loscocco, Visani, Amadori, Cavo and Curti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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https://openalex.org/W3123784072	https://molecularbrain.biomedcentral.com/track/pdf/10.1186/s13041-020-00712-3	English	null	Fibroblasts from idiopathic Parkinson’s disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2	Molecular brain	2,021	cc-by	11,704	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fibroblasts from idiopathic Parkinson’s disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2 Ria Thomas†, Elizabeth B. Moloney†, Zachary K. Macbain, Penelope J. Hallett* and Ole Isacso Abstract Lysosomal dysfunction is a central pathway associated with Parkinson’s disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism. Keywords: GBA, LIMP2, Idiopathic PD fibroblasts, Lysosomal dysfunction Thomas et al. Mol Brain (2021) 14:16 https://doi.org/10.1186/s13041-020-00712-3 Open Access Introduction vesicular transport deficits, immune response activation, and protein aggregation pathways [1–3]. Recent studies highlight a critical role for lysosomal dysfunction in the etiology of genetic and sporadic forms of PD [4, 5]. Glu- cocerebrosidase (GCase), a lysosomal hydrolase encoded by the GBA gene, is responsible for the metabolism of the glycosphingolipid substrates glucosylceramide (Glc- Cer) and glucosylsphingosine (GlcSph). While homozy- gous mutations in this gene lead to the most prevalent lysosomal storage disorder Gaucher’s disease, heterozy- gous loss-of-function mutations in GBA are one of the Parkinson’s disease (PD) is a multifactorial neurodegen- erative disorder, and several cell biological pathways that contribute to PD etiology have been described, includ- ing mitochondrial dysfunction, oxidative stress, lysoso- mal dysfunction, lipid and lipid transport abnormalities, Correspondence: phallett@mclean.harvard.edu; ole_isacson@hms.harvard. edu †Ria Thomas and Elizabeth B. Moloney contributed equally to this work Neuroregeneration Research Institute, Harvard Medical School/McLean Hospital, Belmont, MA 02478, USA Correspondence: phallett@mclean.harvard.edu; ole_isacson@hms.harvard. edu Page 2 of 12 Page 2 of 12 Thomas et al. Mol Brain (2021) 14:16 most common genetic risk factors identified in PD [5–7]. Reduced GCase activity is observed in PD patient tis- sues and in in vitro cellular systems harboring muta- tions in GBA gene, as well as, in the brain, cerebrospinal fluid (CSF) and blood from sporadic PD patients with no GBA mutations [4, 8–17]. Aging is the most signifi- cant risk factor for developing PD and several pathologi- cal processes of the disease are phenocopied in normal aging. Interestingly, lysosomal GCase activity is reduced in normal aging in humans and rodents with parallel elevations of glycosphingolipids [4, 18]. Furthermore, overexpression of GBA in two rodent models of PD is protective against dopamine neuron degeneration and reduces alpha synucleinopathy [19]. Some studies have also postulated a potential interaction between GBA and alpha-synuclein whereby accumulation of the substrates GlcCer and GlcSph, dysfunction of autophagy and ubiq- uitin proteasome system observed in models with mutant GBA was associated with increased accumulation of alpha-synuclein, and increased level of alpha-synuclein result in reduced GCase activity [12, 20]h significant association with PD, clinical studies have also reported that LIMP2 transcript and protein levels, [34] and GCase activity [28] have not been shown to associate with these SNP genotypes. PGRN, encoded by the GRN gene, is a secreted gly- coprotein comprising seven and a half granulin motifs connected by short linker regions. Heterozygous muta- tions in GRN lead to the development of frontotempo- ral dementia (FTD) and homozygous mutations cause the lysosomal storage disorder, neuronal ceroid lipo- fuscinosis [35–37]. In addition to its role in regulating a multitude of functions including embryogenesis, tumo- rigenesis, inflammation and wound repair [38, 39], recent studies have identified its role in lysosomal function as a chaperone of several lysosomal enzymes such as GCase [40, 41], cathepsin D [42] and Hex A [43]. Two modes of regulation of GBA by PGRN have been identified so far. Within the cytosol, PGRN binds to GBA through the c-terminal granulin E domain and is required for the lysosomal localization of the GBA/LIMP2 complex [40]. Within the lysosomes, PGRN is required for the process- ing of prosaposin to saposinC, which in turn, acts a co- activator for GBA [44, 45]. Cortical neurons derived from FTD PGRN iPSCs display reduced processing of prosa- posin to saposinC and reduced lysosomal GCase activity compared to isogenic controls [45]. y The activity and function of GBA is regulated by sev- eral accessory proteins including lysosomal integral membrane protein 2 (LIMP2) and progranulin (PGRN). Unlike other lysosomal enzymes that utilizes mannose 6 phosphate receptor for trafficking to lysosomes, GBA is transported from the endoplasmic reticulum (ER) through Golgi to lysosomes as part of a complex with the specific receptor LIMP2 (encoded by SCARB2 gene) in association with adaptor proteins 1 and 3 [21–25]. Homozygous mice deficient for LIMP2 exhibit signifi- cantly reduced GCase activity in peripheral tissues with a concomitant increase in the level of its substrate Glc- Cer, and in several regions of the brain paralleled by increased alpha synuclein levels, neutral lipid load, and lysosomal dysfunction [24, 26]. Deficiency of LIMP2 in these animals caused mistargeting and extracellular secretion of the GBA protein leading to its increased expression and activity in serum [24]. Overexpression of functional LIMP2 in murine fibroblasts and human neuroglioma cells stably expressing alpha synuclein led to increased trafficking of the GBA protein from ER to post ER compartments, increased enzyme activity, and dose dependent reduction in alpha synuclein levels [26]. Genome-wide association studies (GWAS) studies in several cohorts have identified that two short nucleotide polymorphisms (SNPs), rs6825004 (located within the SCARB2 gene) [27] and rs6812193 (located immediately upstream of SCARB2 gene) [28–30], are significantly associated with sporadic PD. However, studies performed in populations with differing genetic backgrounds indi- cate that the PD risk generated by these polymorphisms may depend on such genetic contexts. [31–33]. Despite p g Even though primarily a symptomatic neurological disease, PD is widely systemic with multiple cellular sys- tems and organs affected [46, 47]. Even in the rare clas- sic mendelian families with evident genetic etiology, the associated mutations and cellular phenotypes are present in all cells of the organism, so potentially there are also systemic interactions that lead to the neurologi- cal manifestations and neurodegenerative changes. The degeneration of specific cellular systems upon expo- sure to PD-associated environmental, aging and genetic stressors depends on increased intrinsic vulnerability of certain cell types to the disease-causing mechanisms, and even within the brain there is regional and cellular differ- ences in susceptibility to PD pathology [48, 49]. Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controlsi Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controls y p y j Our previous findings demonstrated that GCase activ- ity progressively declines with age, and is decreased in PD patient substantia nigra compared to healthy patients [4]. To understand this further, in the current study a large cohort of fibroblasts derived from patients with idiopathic PD (PD), those harboring N370S mutation in the GBA gene (gPD-GBA N370S) and age-matched healthy subject controls (HS) were utilized to meas- ure basal levels of lysosomal GCase activity. In addition to the expected reduction of GCase activity in gPD- GBA N370S cells (60.71%, p = 0.0001), a significant decrease in the enzyme activity was also observed in cells derived from idiopathic PD (33.27%, p = 0.0012) patients (Fig. 1a). All cell lines from the PD group of fibroblasts were sequenced for the entire GBA gene and were con- firmed to be devoid of any nonsense and missense muta- tions in the coding region. To examine whether the reduction of GCase enzyme activity in PD cells was due to reduced transcript or protein level, qPCR and immu- noblot assays were performed. While no difference in GBA transcript expression was observed between the three groups of cells (Fig. 1b), GBA protein level was sig- nificantly upregulated in PD cells compared to HS con- trols (p = 0.0461) and gPD-GBA N370S cells (p = 0.0331) (Fig. 1c, d). To exclude the possibility of altered lysoso- mal load contributing to the reduced lysosomal GCase activity in PD fibroblasts, an immunoblot assay was per- formed for LAMP1, and no change in its expression was observed between the PD-derived cells compared to HS controls (Additional file 1: Fig. S1A, B, Additional file 2). Furthermore, regression analysis performed between GCase activity and age of disease onset (PD, r = − 0.1259, p = 0.5487; gPD-GBA N370S, r = 0.7724, p = 0.1258) (Additional file 1: Fig. S2A, Additional file 2), and GCase activity and disease duration (PD, r = 0.3403, p = 0961; gPD-GBA N370S, r = − 0.7546, p = 0.1404) (Additional file 1: Fig. S2B, Additional file 2) across the idiopathic PD and gPD-GBA N370S cells showed no significant correla- tion between the variables in both groups of PD cells. Neurons are relatively more vulnerable than other cellular systems and organs, such as the 100-fold higher vulnerability of iPSC-derived neurons with PINK1 and LRRK2 muta- tions to mitochondrial stress compared to fibroblasts from the same patients [50]. In order to study systemic and cell biological mechanisms associated with PD, eas- ily accessible, peripheral cell types such as fibroblasts and blood cells can be used as in vitro models [9, 51–55]. The current study utilized a large cohort of idiopathic and genetic (with GBA N370S mutation) PD patient-derived skin fibroblasts as in vitro cellular model to establish mechanisms leading to the deficiency of GCase in vari- ous forms of PD. Our findings identified that lysosomal Page 3 of 12 Thomas et al. Mol Brain (2021) 14:16 GCase activity was reduced in idiopathic PD patient cells and was associated with decreased LIMP2 levels. These data confirm that GCase activity, and more broadly, lys- osomal dysfunction is perturbed in idiopathic forms of PD, and suggest that reduced GBA function in idiopathic PD could be the result of a LIMP2-mediated trafficking deficit. GBA trafficking receptor LIMP2 is reduced and its levels correlate with GCase activity in idiopathic PD patient‑derived fibroblast cells GBA trafficking receptor LIMP2 is reduced and its levels correlate with GCase activity in idiopathic PD patient‑derived fibroblast cells i Activity of GBA is regulated by several accessory proteins and we analyzed two such proteins, PGRN and LIMP2, in detail. PGRN encoded by GRN gene has been reported to regulate GCase activity either directly by acting as a co-chaperone with Hsp70 [40], or indirectly by promot- ing the processing of prosaposin to saposin C [45]. While qPCR analysis for GRN transcripts showed a significant reduction in PD cells compared to controls (p = 0.01) (Additional file 1: Fig. S3A, Additional file 2), the protein levels were increased in this group of cells compared to HS (p = 0.0425) and gPD-GBA N370S cells (p = 0.0096) (Fig. 2A, B). Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controlsi g LIMP2 (also known as SCARB2) is the protein respon- sible for trafficking of GBA from ER to Golgi and finally to the lysosomes [24], and its expression was analyzed in this cohort of fibroblasts using qPCR and ELISA- based quantification assays. Interestingly, LIMP2 levels displayed a significant reduction at both the transcript (p = 0.0072) (Fig. 2c) and protein levels in PD (p < 0.0001) (Fig. 2d) cells compared to HS controls. Importantly, no change in this protein was observed in the gPD-GBA N370S cells (Fig. 2c, d). As the primary protein responsi- ble for trafficking of GBA, its reduction in PD cells could lead to alteration in localization of the GBA protein. To examine this, Endo-H and PNGaseF digestion was per- formed in fibroblast lysates. GBA is heavily glycosylated and undergoes several carbohydrate modifications as it passes through the secretory pathway from ribosomes through ER to Golgi. The N-linked glycans present on GBA can be cleaved by the Endo-H enzyme (Endo-H- sensitive ER fraction). However, as the proteins pass through Golgi, these N-linked glycans are converted to complex oligosaccharides that are no longer respon- sive to the action of Endo-H enzyme (Endo-H-insen- sitive post ER fraction). The ratio of Endo-H-sensitive to -insensitive fractions of the GBA protein provides an indication of the localization of the protein between the ER and post ER compartments [9, 26]. Interestingly, both PD (p = 0.0013) and gPD-GBA N370S (p = 0.0266) cells displayed a reduction in post-ER/ER ratio com- pared to HS (Fig. 2e, Additional file 1: Fig. S3B, Addi- tional file 2), suggesting a higher retention of the protein in the ER and thus an altered localization of the protein in both groups of cells. Importantly, regression analysis between LIMP2 and GCase activity levels across all cells showed that a significant correlation between the two variables was observed only in the case of idiopathic PD cells (r = 0.7288, p < 0.0001), and not in HS (r = 0.0768 p = 0.8028) or gPD-GBA N370S (r = 0.3864, p = 0.4493) cells (Fig. 2f). Thomas et al. Mol Brain (2021) 14:16 Page 4 of 12 Thomas et al. Mol Brain a b c d Fig. 1 Idiopathic PD fibroblasts exhibit reduced basal lysosomal GCase activity. Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controlsi a Basal level of lysosomal GCase activity was measured in fibroblasts derived from idiopathic PD patients (PD), those harboring GBA N370S mutation (gPD-GBA N370S) and age-matched healthy subject controls (HS) using 4-MU glucopyranoside substrate (n = 14, HS; n = 31, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,48) = 12.25, p < 0.0001). b GBA transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S) and c, d protein level (normalized to GAPDH) measured across HS, PD and gPD-GBA N370S group of cells (n = 13, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,45) = 5.203, p = 0.0093). Data represented as mean ± SEM. * = p < 0.05; = p < 0.01; * = p < 0.001 a b c d d Fig. 1 Idiopathic PD fibroblasts exhibit reduced basal lysosomal GCase activity. a Basal level of lysosomal GCase activity was measured in fibroblasts derived from idiopathic PD patients (PD), those harboring GBA N370S mutation (gPD-GBA N370S) and age-matched healthy subject controls (HS) using 4-MU glucopyranoside substrate (n = 14, HS; n = 31, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,48) = 12.25, p < 0.0001). b GBA transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S) and c, d protein level (normalized to GAPDH) measured across HS, PD and gPD-GBA N370S group of cells (n = 13, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,45) = 5.203, p = 0.0093). Data represented as mean ± SEM. * = p < 0.05; = p < 0.01; * = p < 0.001 Numerous GWAS have identified the presence of two SNPs, rs6825004 and rs6812193, to be associated with increased risk for PD in several study cohorts [27–30, 33]. Furthermore, both these SNPs are expres- sion quantitative trace loci (eQTL) for SCARB2 in sev- eral tissues including cultured fibroblast cells [56]. To examine whether these nucleotide changes have a role in the reduced LIMP2 levels observed in PD fibroblasts, we sequenced a partial cohort of idiopathic PD (11 for rs6812193 and 14 for rs6825004 out of the total 31 PD lines) and gPD-GBA N370S (1 out of 6) cell lines for the two SNPs. Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controlsi Details of the distribution of genotype variants for rs6812193 and rs68225004 across the vari- ous cell lines sequenced in PD and gPD-GBA N370S groups are provided in a tabular form (Additional file 1: Fig. S4A, Additional file 2). Further analysis performed across the various SNP genotypes in the idiopathic PD group of cells showed that there was no signifi- cant difference in LIMP2 protein level nor lysosomal GCase activity between the cell lines with the various rs6812193 and rs6825004 polymorphisms (Additional file 1: Fig. S4B–E). Thomas et al. Mol Brain (2021) 14:16 Page 5 of 12 Thomas et al. Mol Brain a b c d e f Fig. 2 Idiopathic PD fibroblasts exhibit altered levels of PGRN, LIMP2 and localization of the GBA protein. a Representative image and b quantification of PGRN protein (normalized to GAPDH) in HS, PD and gPD-GBA N370S group of cells (n = 10, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,42) = 6.577, p = 0.0033). c LIMP2 transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,45) = 6.147, p = 0.0044) and d ELISA-based LIMP2 protein expression across the three groups of cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,39) = 11.32, p = 0.0001). e Ratio of the post ER to ER fraction of GBA protein was measured in cell lysates using Endo-H and PNGase F digestion (n = 7, HS; n = 25, PD; n = 3, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,32) = 8.084, p = 0.0014). f Correlation analysis between LIMP2 protein and GCase activity was performed and Pearson’s correlation coefficient was determined between the two variables across HS, PD and gPD-GBA N370S cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S). Data represented as mean ± SEM. * = p < 0.05; = p < 0.01; ** = p < 0.0001 a b c d f e f Fig. 2 Idiopathic PD fibroblasts exhibit altered levels of PGRN, LIMP2 and localization of the GBA protein. Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controlsi e Ratio of the post ER to ER fraction of GBA protein was measured in cell lysates using Endo-H and PNGase F digestion (n = 7, HS; n = 25, PD; n = 3, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,32) = 8.084, p = 0.0014). f Correlation analysis between LIMP2 protein and GCase activity was performed and Pearson’s correlation coefficient was determined between the two variables across HS, PD and gPD-GBA N370S cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S). Data represented as mean ± SEM. * = p < 0.05; = p < 0.01; ** = p < 0.0001 N370S cells. Together, these results suggest that while reduced GCase activity is observed in fibroblasts derived from both idiopathic PD and a genetic form of PD with the GBA N370S mutation, reduced LIMP2 levels (both at transcript and protein level) are observed only in the case of idiopathic PD cells, indicative of different path- ways leading to this deficit in these two PD cohorts. While the presence of the N370S mutation in the GBA gene is responsible for improper folding, increased retention of the protein in the ER, and increased lyso- somal cholesterol accumulation, and thus reduced lyso- somal GCase activity in gPD-GBA N370S group of cells [9], the reduced trafficking of GBA to lysosomes due to Idiopathic PD patient fibroblasts display reduced basal GCase activity compared to healthy subject controlsi a Representative image and b quantification of PGRN protein (normalized to GAPDH) in HS, PD and gPD-GBA N370S group of cells (n = 10, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,42) = 6.577, p = 0.0033). c LIMP2 transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,45) = 6.147, p = 0.0044) and d ELISA-based LIMP2 protein expression across the three groups of cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,39) = 11.32, p = 0.0001). e Ratio of the post ER to ER fraction of GBA protein was measured in cell lysates using Endo-H and PNGase F digestion (n = 7, HS; n = 25, PD; n = 3, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,32) = 8.084, p = 0.0014). f Correlation analysis between LIMP2 protein and GCase activity was performed and Pearson’s correlation coefficient was determined between the two variables across HS, PD and gPD-GBA N370S cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S). Data represented as mean ± SEM. * = p < 0.05; = p < 0.01; ** = p < 0.0001 Fig. 2 Idiopathic PD fibroblasts exhibit altered levels of PGRN, LIMP2 and localization of the GBA protein. a Representative image and b quantification of PGRN protein (normalized to GAPDH) in HS, PD and gPD-GBA N370S group of cells (n = 10, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,42) = 6.577, p = 0.0033). c LIMP2 transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,45) = 6.147, p = 0.0044) and d ELISA-based LIMP2 protein expression across the three groups of cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,39) = 11.32, p = 0.0001). Elevation of PGRN in idiopathic PD fibroblast cells pi Investigation into one of the accessory proteins that reg- ulate GBA function, PGRN, showed that while GRN tran- script was downregulated, PGRN protein expression was significantly elevated in idiopathic PD cells. This discrep- ancy between transcript and protein level could suggest the presence of a post-translational modification leading to an increased half-life of the protein, and further exper- imentation would be required to confirm it. However, GCase activity is regulated by the PGRN protein (not the transcript) and its increase in PD cells, similar to that observed with GBA, could indicate a cellular compensa- tion for reduced GCase activity in these cells. Along with HSP70, PGRN acts as a co-chaperone for GBA/LIMP2 complex and is required for its proper localization to the lysosome in vivo in PGRN KO mice under ovalbumin- mediated inflammatory stress [40]. Overexpression of the complete PGRN protein or the c-terminal granulin E domain, which includes the binding site for GBA, abro- gated the accumulation of GlcCer and increased GCase activity in fibroblasts isolated from Gaucher’s disease (GD) patients [40]. Furthermore, treatment of various GD mouse models (PGRN KO mice treated with ovalbu- min and GBA D409V/- mice) with recombinant progran- ulin led to an increase in the appearance of lysosomal GCase, reduction of accumulated glycolipid substrates and the number and size of gaucher cells [62], indicating a potential therapeutic function for this protein in GD. However, despite the upregulation of PGRN in idiopathic PD fibroblasts in the current study, the lysosomal GCase activity was not increased. PGRN acts a co-chaperone of the GBA/LIMP2 complex and our results point to a reduction of LIMP2 in idiopathic PD cells. The decreased level of LIMP2 might serve as a limiting factor and could be the reason why lysosomal GCase activity was not res- cued by the upregulation of PGRN in the idiopathic PD group of cells. f The reduced lysosomal GCase activity observed in fibroblasts derived from idiopathic PD patients in the current study was not due to changes in the correspond- ing transcript and protein expression. There was no reduction of mRNA expression and in fact, idiopathic PD fibroblasts exhibited a significant elevation of GBA protein compared to controls and gPD-GBA N370S cells, indicative of a possible cellular compensatory response. Reduced GCase activity in anterior cingulate cortex [13], cerebellum and substantia nigra [10] of sporadic PD post-mortem brain tissue was accompanied by reduced level of GBA protein. Discussionh This study demonstrates that fibroblasts derived from idiopathic PD patients, devoid of mutations in the GBA gene, display reduced levels of lysosomal GCase activity, approaching that of cells with the GBA N370S mutation. In the fibroblasts from idiopathic PD patients, this GCase activity reduction was accompanied by a reduced pro- tein expression of the GBA trafficking receptor, LIMP2, and by altered localization and increased ER retention of the GBA protein in these cells. Furthermore, regression analysis showed that LIMP2 expression levels correlated significantly with GCase activity levels only in idiopathic PD fibroblasts and not in healthy subject or gPD-GBA Thomas et al. Mol Brain (2021) 14:16 Page 6 of 12 Thomas et al. Mol Brain (2021) 14:16 decreased LIMP2 levels could be responsible for the sig- nificantly reduced GCase enzymatic activity in the idi- opathic PD cohort. However, further experimental evidence using immu- nocytochemistry for lysosomal markers would serve to confirm this result. Furthermore, correlation analysis performed between lysosomal GCase activity levels and age of onset and disease duration excluded the influence of these factors in the reduced enzyme activity observed in idiopathic PD cells. decreased LIMP2 levels could be responsible for the sig- nificantly reduced GCase enzymatic activity in the idi- opathic PD cohort. Elevation of PGRN in idiopathic PD fibroblast cells However, unlike patient-derived fibroblasts cells, post-mortem brain tissue represents an advanced stage of the disease, and it is plausible that mechanistic differences exist between these two tissue systems and cells under study. The GCase activity assay used in the current study specifically measures the activ- ity of the lysosomal GBA enzyme and alterations in the overall lysosomal load within the cell can influence these results. No change was observed in the expression of the lysosomal marker LAMP1 between PD patient and con- trol fibroblasts, suggesting that the decreased lysosomal GCase activity in idiopathic PD cells in the current study was not due to a general reduction of the lysosomal load. Reduced expression of LIMP2 correlates significantly with reduced GCase activity in idiopathic PD cells The significant downregulation of LIMP2, observed both at the transcript and protein level in idiopathic PD cells, but not in gPD-GBA N370S, suggests that the reduced lysosomal GCase activity in the former group of cells could be due to a defect in trafficking of the GBA Fibroblasts from idiopathic Parkinson’s disease patients exhibit reduced GCase activity levelsi Numerous lines of research have identified lysosomal function to be a central pathway perturbed in PD [3, 57] and GWAS studies have identified heterozygous GBA variants and mutations to be present in 2–20% of PD patients from various cohorts [3, 5, 7, 58, 59]. In addi- tion to a significantly large reduction of lysosomal GCase activity observed in tissues derived from PD patients harboring GBA mutations [8, 10, 15], intermediate defi- cits in this enzyme activity have also been observed in blood, CSF, and post-mortem brain tissue from spo- radic PD patients [4, 8, 10, 13, 15]. Using a large cohort of fibroblasts (HS, n = 15; idiopathic PD, n = 31; gPD- GBA N370S, n = 6), this study demonstrates that, simi- lar to deficiencies observed in the human post-mortem PD brain tissue, basal lysosomal GCase activity is highly reduced in gPD-GBA N370S cells (60.71%) and to an intermediary level in idiopathic PD cells (33.27%). Con- trasting these results, three previous studies conducted in idiopathic PD fibroblasts concluded that these cells do not show differences in lysosomal GCase activity com- pared to controls [9, 60, 61]. However, the limited cohort of cell lines used in these studies (n = 2–5) might account for the observed differences between the results. Conclusionshi The current findings can help to define the cell biologi- cal mechanisms regulating GCase activity in idiopathic PD. Decreased levels of the GBA trafficking protein LIMP2, which was observed in idiopathic PD patient fibroblasts, would disrupt GBA trafficking from the ER/ Golgi to lysosomes resulting in the observed reduction in lysosomal GCase activity. Alterations in lysosomal enzyme activities and subsequent lysosomal dysfunction can lead to deficits in cellular degradation pathways. In vulnerable cell types such as neurons, increased cellu- lar glycosphingolipid load and lipid dyshomeostasis can precipitate abnormal protein, vesicular and neuroim- mune interactions, eventually leading to their degenera- tion [1, 2, 64]. The current data also illustrate that cells peripheral to the brain, such as fibroblasts, can serve as platforms for experimental and therapeutic paradigms aimed at manipulation of cellular GCase levels. GBA related cell biological pathways seem to be dysregulated in the majority of PD cases. In addition to PD, mutations in GBA and reduced GCase activity are also observed in the related disorder, dementia with Lewy bodies (DLB) [65]. Interestingly, one of the SCARB2 associated SNPs analyzed in this study (rs6812193) has also been identi- fied to be a risk loci for DLB [66], and further studies to examine a LIMP2-associated influence on GCase activ- ity in DLB would be of interest. Therapeutic modalities aimed at improving lysosomal function by increasing the activity of lysosomal enzymes, increasing lysosomal enzyme transport through LIMP2, or reducing glycolipid substrate accumulation, serve as attractive therapeutic targets and are currently under development. f Several GWAS studies identified that the two SNPs rs6825004 and rs6812193 exhibit a population-depend- ent association with PD [27–30] which prompted us to study the influence of these variances on LIMP2 level in our work. HS controls were not sequenced for these SNPs in our study, and hence, we cannot conclude on their association with PD based on our data. In line with the two previous reports [28, 34], our study provides further evidence that neither the LIMP2 level nor GCase activ- ity are affected by the genotype at rs681193 or rs6825004 locus in PD fibroblast cells, and hence, does not explain the reduction of LIMP2 observed at the transcript and protein level in idiopathic PD group of cells compared to age matched controls. Reduced expression of LIMP2 correlates significantly with reduced GCase activity in idiopathic PD cellshi We note that there is a trend for reduced LIMP2 protein level in gPD-GBA N370S cells, however, with the current sample size this did not reach statistical significance. Further- more, regression analysis showed that a significant cor- relation of LIMP2 protein levels with GCase activity was observed only in the idiopathic PD group of cells, and not in HS and gPD-GBA N370S cells, further supporting the involvement of LIMP2 in the regulation of GBA enzyme activity exclusively in fibroblasts derived from idiopathic PD cohort. Changes in LIMP2 level can affect the pool of functional GCase enzyme entering the lysosome. A mutation in LIMP2 (p.Glu471Gly) was identified to be a modifier of GD and displayed inefficient lysosomal local- ization of GBA followed by accumulation of downstream GCase targets such as GlcSph and GlcCer [63]. Further- more, LIMP2 deficient mice (LIMP2−/−) display dimin- ished GCase activity (and protein) in the brain compared to WT littermate controls [24, 26]. These studies support our finding that reduced level of LIMP2 can indeed result in reduced lysosomal GCase activity. In a previous, unre- lated study, staining for LIMP2 in PD human post-mor- tem brain tissue showed that the surviving dopaminergic neurons in the substantia nigra displayed elevated LIMP2 levels compared to controls [26]. However, differences between the two systems being studied here (fibroblasts and brain) and the stage of the disease they represent might account for the differences in the results. the mechanisms by which they could, potentially, confer increased susceptibility to the disease. Conclusionshi Closer analysis of these data show very marginal effects, in particular the eQTL inference is barely significant and does not indicate much change in expression levels. Further analysis, with increased sample size and consideration for the ethnicity of the study sub- jects would need to be performed to confirm any poten- tial association of these polymorphisms with PD and Reduced expression of LIMP2 correlates significantly with reduced GCase activity in idiopathic PD cellshi The significant downregulation of LIMP2, observed both at the transcript and protein level in idiopathic PD cells, but not in gPD-GBA N370S, suggests that the reduced lysosomal GCase activity in the former group of cells could be due to a defect in trafficking of the GBA Thomas et al. Mol Brain (2021) 14:16 Page 7 of 12 protein. In line with this, idiopathic PD fibroblasts dis- played an alteration in the localization of GBA protein, suggesting an increased retention of the protein in the ER compared to post-ER fraction. Additional experimenta- tion using immunocytochemistry with GBA and an ER marker would serve to confirm this finding. We note that there is a trend for reduced LIMP2 protein level in gPD-GBA N370S cells, however, with the current sample size this did not reach statistical significance. Further- more, regression analysis showed that a significant cor- relation of LIMP2 protein levels with GCase activity was observed only in the idiopathic PD group of cells, and not in HS and gPD-GBA N370S cells, further supporting the involvement of LIMP2 in the regulation of GBA enzyme activity exclusively in fibroblasts derived from idiopathic PD cohort. Changes in LIMP2 level can affect the pool of functional GCase enzyme entering the lysosome. A mutation in LIMP2 (p.Glu471Gly) was identified to be a modifier of GD and displayed inefficient lysosomal local- ization of GBA followed by accumulation of downstream GCase targets such as GlcSph and GlcCer [63]. Further- more, LIMP2 deficient mice (LIMP2−/−) display dimin- ished GCase activity (and protein) in the brain compared to WT littermate controls [24, 26]. These studies support our finding that reduced level of LIMP2 can indeed result in reduced lysosomal GCase activity. In a previous, unre- lated study, staining for LIMP2 in PD human post-mor- tem brain tissue showed that the surviving dopaminergic neurons in the substantia nigra displayed elevated LIMP2 levels compared to controls [26]. However, differences between the two systems being studied here (fibroblasts and brain) and the stage of the disease they represent might account for the differences in the results. protein. In line with this, idiopathic PD fibroblasts dis- played an alteration in the localization of GBA protein, suggesting an increased retention of the protein in the ER compared to post-ER fraction. Additional experimenta- tion using immunocytochemistry with GBA and an ER marker would serve to confirm this finding. Human dermal fibroblast linesi The amplicon sizes for GBA gene, rs6812193 and rs68504 were 7766 bp, 554 bp and 552 bp respectively. PCR amplified products were pooled at an equimolar concentration and normalized to 0.2 ng/ul. Library construction of each amplicon pool to produce sequence ready indexed libraries was carried out using the Illumina Nextera XT DNA Sam- ple Prep Kit as per manufacturer’s instructions. Quality and control fibroblast lines used in this study Catalogue ID Description Sex Age at biopsy ND34769 Control Female 68 ND34791 Control Female 60 ND35046 Control Male 60 ND36091 Control Female 63 AG11743 Control Female 76 AG06959 Control Male 67 AG04061 Control Male 66 AG13220 Control Male 66 AG04355 Control Male 67 AG11489 Control Male 66 AG07141 Control Male 66 AG05265 Control Female 61 AG06010 Control Female 62 AG06241 Control Male 61 AG06281 Control Male 67 AG20439 Idiopathic PD Male 55 AG20445 Idiopathic PD Male 60 ND30159* Idiopathic PD Female 76 ND35302* Idiopathic PD Male 69 ND35976* Idiopathic PD Male 63 ND39538* Idiopathic PD Female 72 ND39999* Idiopathic PD Male 63 ND34106* Idiopathic PD Male 65 ND29541* Idiopathic PD Male 65 ND39528* Idiopathic PD Female 67 ND39183 Idiopathic PD Male 70 ND32462* Idiopathic PD Male 75 ND39955* Idiopathic PD Male 55 ND31508* Idiopathic PD Male 71 ND32157* Idiopathic PD Female 52 ND32697* Idiopathic PD Male 58 ND34265* Idiopathic PD Male 62 ND38528* Idiopathic PD Female 65 ND34854* Idiopathic PD Female 68 ND37609* Idiopathic PD Male 68 AG08395 Idiopathic PD Female 85 ND29494 Idiopathic PD Male 80 ND33424 Idiopathic PD Male 57 ND38020 Idiopathic PD Male 86 ND38865 Idiopathic PD Male 51 ND38791 Idiopathic PD Female 69 ND39450 Idiopathic PD Female 72 ND39510 Idiopathic PD Male 69 ND39957 Idiopathic PD Female 70 ND41125 Idiopathic PD Male 70 ND40260 Idiopathic PD Male 78 ND29756 GBA N370S Het Female 55 ND34982 GBA N370S Het Female 82 ND34263 GBA N370S Hom Male 65 Human dermal fibroblast linesi i Healthy subject-derived fibroblasts (HS, n = 15), idi- opathic PD patient (PD, n = 31), and mutant GBA PD patient fibroblast (gPD-GBA N370S, n = 6) lines were obtained from Coriell, and NINDS repositories (see Table 1 for overview). The skin samples used for gener- ating the fibroblast cell lines were collected under Cori- ell and NINDS’s informed consent and deidentification of subjects. Biospecimens obtained from the NINDS Human Cell and Data Repository were not considered to be human subject research because conducting research with the samples does not involve an intervention or interaction with the individual and the samples do not contain identifiable private information. Idiopathic cells used in this study are defined as non-familial form of PD where cause of the disease is not known yet. Fibroblasts Thomas et al. Glucocerebrosidase activity Fibroblasts were plated onto tissue culture plates at 15,000 cells/cm2. They were supplemented with 0.1% DMSO in fibroblast culture medium the next day. Three days post-plating, the cells were harvested with lysis buffer (pH 7, 10 mM Tris, 0.1% Ipegal, 1 × Halt protease and phosphatase inhibitor cocktail with 0.5 M EDTA (Thermo fisher, #78440)) and mechanically homogenized with a sonicator (BioLogics Inc, Model 150 V). GCase activity was measured in cell lysates diluted three times in GCase-activity sample diluent (50 mM citric acid, 0.1 M sodium phosphate, and 2 mg/mL bovine serum albumin, pH 5). 10µL of diluted sample was added to 75µL of 5 mM 4-Mu-β-D-glucopyranoside (Sigma, #M3633) substrate prepared in GCase-activity substrate diluent (50 mM citric acid, 0.1 M sodium phosphate, 6 mg/mL sodium taurocholate (Sigma, #86339), 0.3% Tween20, pH 5). After incubation with the substrate for 60 min at 37 °C, the reaction was terminated using 200µL stop solution (333 mM glycine, 207 mM sodium carbon- ate, pH 10.7). Plates were read (Ex 360/Em 460) using a SPECTRAmax plate reader (Molecular Devices). Enzy- matic activity of triplicate measurements of each sample was assessed from a 4-Mu standard curve (100–0.391 µM standard range prepared from a 1 mM 4-methylumbellif- eryl sodium salt solution (Sigma, #M1508)) and normal- ized to total protein content in each sample. Human dermal fibroblast linesi Mol Brain (2021) 14:16 Page 8 of 12 Table 1 Case information on the Parkinson’s disease and control fibroblast lines used in this study Catalogue ID Description Sex Age at biopsy ND34769 Control Female 68 ND34791 Control Female 60 ND35046 Control Male 60 ND36091 Control Female 63 AG11743 Control Female 76 AG06959 Control Male 67 AG04061 Control Male 66 AG13220 Control Male 66 AG04355 Control Male 67 AG11489 Control Male 66 AG07141 Control Male 66 AG05265 Control Female 61 AG06010 Control Female 62 AG06241 Control Male 61 AG06281 Control Male 67 AG20439 Idiopathic PD Male 55 AG20445 Idiopathic PD Male 60 ND30159* Idiopathic PD Female 76 ND35302* Idiopathic PD Male 69 ND35976* Idiopathic PD Male 63 ND39538* Idiopathic PD Female 72 ND39999* Idiopathic PD Male 63 ND34106* Idiopathic PD Male 65 ND29541* Idiopathic PD Male 65 ND39528* Idiopathic PD Female 67 ND39183 Idiopathic PD Male 70 ND32462* Idiopathic PD Male 75 ND39955* Idiopathic PD Male 55 ND31508* Idiopathic PD Male 71 ND32157* Idiopathic PD Female 52 ND32697* Idiopathic PD Male 58 ND34265* Idiopathic PD Male 62 ND38528* Idiopathic PD Female 65 ND34854* Idiopathic PD Female 68 ND37609* Idiopathic PD Male 68 AG08395 Idiopathic PD Female 85 ND29494 Idiopathic PD Male 80 ND33424 Idiopathic PD Male 57 ND38020 Idiopathic PD Male 86 ND38865 Idiopathic PD Male 51 ND38791 Idiopathic PD Female 69 ND39450 Idiopathic PD Female 72 ND39510 Idiopathic PD Male 69 ND39957 Idiopathic PD Female 70 ND41125 Idiopathic PD Male 70 ND40260 Idiopathic PD Male 78 ND29756 GBA N370S Het Female 55 ND34982 GBA N370S Het Female 82 ND34263 GBA N370S Hom Male 65 Table 1 Case information on the Parkinson’s disease and control fibroblast lines used in this study were maintained in culture as described previously [53], and cells were not used for experiments beyond passage 20. HS, PD and gPD-GBA N370S cells were age-matched and the average ages were 65, 67.2 and 68.6 years respec- tively. Apart from being sequenced for the GBA gene, a subset of the fibroblasts (indicated with ) were also sequenced and confirmed to be devoid of any mutations in the LRRK2 gene as part of a previous study [53]. were maintained in culture as described previously [53], and cells were not used for experiments beyond passage 20. HS, PD and gPD-GBA N370S cells were age-matched and the average ages were 65, 67.2 and 68.6 years respec- tively. Human dermal fibroblast linesi Apart from being sequenced for the GBA gene, a subset of the fibroblasts (indicated with ) were also sequenced and confirmed to be devoid of any mutations in the LRRK2 gene as part of a previous study [53]. and cells were not used for experiments beyond passage 20. HS, PD and gPD-GBA N370S cells were age-matched and the average ages were 65, 67.2 and 68.6 years respec- tively. Apart from being sequenced for the GBA gene, a subset of the fibroblasts (indicated with ) were also sequenced and confirmed to be devoid of any mutations in the LRRK2 gene as part of a previous study [53]. Glucocerebrosidase activity Fibroblasts were plated onto tissue culture plates at 15,000 cells/cm2. They were supplemented with 0.1% DMSO in fibroblast culture medium the next day. Three days post-plating, the cells were harvested with lysis buffer (pH 7, 10 mM Tris, 0.1% Ipegal, 1 × Halt protease and phosphatase inhibitor cocktail with 0.5 M EDTA (Thermo fisher, #78440)) and mechanically homogenized with a sonicator (BioLogics Inc, Model 150 V). GCase activity was measured in cell lysates diluted three times in GCase-activity sample diluent (50 mM citric acid, 0.1 M sodium phosphate, and 2 mg/mL bovine serum albumin, pH 5). 10µL of diluted sample was added to 75µL of 5 mM 4-Mu-β-D-glucopyranoside (Sigma, #M3633) substrate prepared in GCase-activity substrate diluent (50 mM citric acid, 0.1 M sodium phosphate, 6 mg/mL sodium taurocholate (Sigma, #86339), 0.3% Tween20, pH 5). After incubation with the substrate for 60 min at 37 °C, the reaction was terminated using 200µL stop solution (333 mM glycine, 207 mM sodium carbon- ate, pH 10.7). Plates were read (Ex 360/Em 460) using a SPECTRAmax plate reader (Molecular Devices). Enzy- matic activity of triplicate measurements of each sample was assessed from a 4-Mu standard curve (100–0.391 µM standard range prepared from a 1 mM 4-methylumbellif- eryl sodium salt solution (Sigma, #M1508)) and normal- ized to total protein content in each sample. Sequencing of GBA gene and SNPs DNA was extracted from fibroblast pellets using DNeasy blood and tissue kit (Qiagen, #69504). Prim- ers between 18–22 bp and with a Tm of 52–62 °C were designed using the Primer3 software. DNA was ampli- fied in a 25 ul reaction containing, 0.3 uM each PCR primers, 0.3 mM dNTP mix, 1X Kapa HiFi Fidelity Buffer with MgCl2 and 1U Kapa HiFi DNA Polymerase (Kapa Biosystems, #KR0368). Immunoblotting Fib bl b control of the libraries were carried out by running them on a High Sensitivity DNA Tape on the Tapesta- tion 2200 Instrument (Agilent Technologies) to meas- ure library size (average size of libraries = 250 bp), and library concentrations were measured using the Quant- iT PicoGreen dsDNA BR Assay Kit (Life Technologies). Equimolar quantities of each uniquely indexed library were pooled and 10 pmol/L of the pooled libraries were subsequently run on the Illumina NextSeq 550 instru- ment to generate 150-bp paired-end sequencing reads. Generated sequencing reads were analyzed using a Burrows-Wheeler Aligner (BWA (mem), v0.7.17) for alignment, and the Genome Analysis Toolkit (GATK, (HaplotypeCaller) v4.0.3.0) unified Genotyper for variant calling (BWA and GATK are developed by the Broad Institute, Cambridge, MA). Genome ampli- fication and sequencing was performed at the Part- ners HealthCare Personalized Medicine Translational Genomics Core (Cambridge, MA). The GBA sequenc- ing carried out for this study were used in two previous publications [53, 67]. The primers used for the sequenc- ing library construction are the following; control of the libraries were carried out by running them on a High Sensitivity DNA Tape on the Tapesta- tion 2200 Instrument (Agilent Technologies) to meas- ure library size (average size of libraries = 250 bp), and library concentrations were measured using the Quant- iT PicoGreen dsDNA BR Assay Kit (Life Technologies). Equimolar quantities of each uniquely indexed library were pooled and 10 pmol/L of the pooled libraries were subsequently run on the Illumina NextSeq 550 instru- ment to generate 150-bp paired-end sequencing reads. Generated sequencing reads were analyzed using a Burrows-Wheeler Aligner (BWA (mem), v0.7.17) for alignment, and the Genome Analysis Toolkit (GATK, (HaplotypeCaller) v4.0.3.0) unified Genotyper for variant calling (BWA and GATK are developed by the Broad Institute, Cambridge, MA). Genome ampli- fication and sequencing was performed at the Part- ners HealthCare Personalized Medicine Translational Genomics Core (Cambridge, MA). The GBA sequenc- ing carried out for this study were used in two previous publications [53, 67]. The primers used for the sequenc- ing library construction are the following; GBA, Forward: 5′ CGACTTTACAAACCTCCCTG 3′. GBA, Forward: 5′ CGACTTTACAAACCTCCCTG 3′. GBA, Reverse: 5′ CCAGATCCTATCTGTGCTGG 3′. rs6812193, Forward: 5 CCCTAGGGGGAAATATGT GA 3′. rs6812193, Reverse: 5′ TGTTCCTGCAGCTCCTTT TT 3′. rs6825004, Forward: 5′ AAAGGACGTGTTTGTGTC CC 3′. rs6825004, Reverse: 5′ AAAGCCATTCATTTTCAG GG 3′. Sequencing of GBA gene and SNPsi DNA was extracted from fibroblast pellets using DNeasy blood and tissue kit (Qiagen, #69504). Prim- ers between 18–22 bp and with a Tm of 52–62 °C were designed using the Primer3 software. DNA was ampli- fied in a 25 ul reaction containing, 0.3 uM each PCR primers, 0.3 mM dNTP mix, 1X Kapa HiFi Fidelity Buffer with MgCl2 and 1U Kapa HiFi DNA Polymerase (Kapa Biosystems, #KR0368). The amplicon sizes for GBA gene, rs6812193 and rs68504 were 7766 bp, 554 bp and 552 bp respectively. PCR amplified products were pooled at an equimolar concentration and normalized to 0.2 ng/ul. Library construction of each amplicon pool to produce sequence ready indexed libraries was carried out using the Illumina Nextera XT DNA Sam- ple Prep Kit as per manufacturer’s instructions. Quality Page 9 of 12 Thomas et al. Mol Brain (2021) 14:16 Thomas et al. Mol Brain (2021) 14:16 Table 1 (continued) Catalogue ID Description Sex Age at biopsy ND35843 GBA N370S Hom Male 61 ND31630 GBA N370S Het Male 69 ND37180 GBA N370S Het Male 80 GAPDH, #QT00079247). qPCR reaction was run on a StepOnePlus real time PCR system (Applied Biosystems) and analysis was performed using the 2(-delta delta C(T)) method [68]. Immunoblotting Fib bl b Fibroblasts to be harvested for lysis were washed once with cold 1X PBS buffer followed by the addition of an appropriate amount of cold RIPA buffer (Thermo Fisher, #PI89900) supplemented with Halt protease along with phosphatase inhibitor cocktail and EDTA (Thermo fisher, #78440). The cells were scraped off the bottom of the culture dish and transferred to a microcentrifuge tube kept on ice. Cells were incubated on ice for 30 min after which they were sonicated (BioLogics Inc, Model 150 V) and spun down. Protein concentration of the supernatant was determined using BCA assay (Thermo fisher, #23225). Equal amount of protein was mixed with Pierce lane marker reducing sample buffer (Thermo fisher, #39000), boiled at 95 ◦C for 5 min, loaded onto precast 4–20% gradient Criterion Tris–HCl protein gels (Bio-rad, #3450033) and was electrophoresed at 120 V for 2 h. The proteins were transferred to a PVDF mem- brane (Bio-rad, #1704157) using the Trans-blot turbo system (Bio-rad) at 25 V and 1.3 Amps for 15 min, fol- lowed by blocking of the membranes in blocking buffer comprising 1 × Tris-buffered saline (Bio-rad, #170-6435) with 0.1% Tween 20 (American Bioanalytical, #AB02038- 01000) and 5% blotting grade blocker (Bio-rad, #170- 6404). Membranes were then incubated overnight at 4 ◦C (on a shaker) with the following primary antibodies diluted in blocking buffer: anti-GBA (Sigma, #G4171, 1:500), anti-PGRN (Sigma, #SAB4200310, 1:1000), anti LAMP1 (Abcam, #24170 1:1000) and anti-GAPDH (Sigma-Aldrich, #AB2302, 1:5000). The membranes were washed 4 times (10 min incubation on the shaker at room temperature) in TBST (1 × Tris-buffered saline (Bio-rad, #170-6435) with 0.1% Tween 20 (American Bioanalytical, #AB02038-01000) to remove excess unbound antibod- ies after which they were incubated in appropriate HRP conjugated secondary antibodies (Jackson Immunore- search, #103-035-155, 1:5000; Bio-rad, #1706515, 1:5000) diluted in blocking buffer, for 1 h at room temperature (on the shaker). Following another 4 washes with TBST (10 min incubations on the shaker at room temperature), the membranes were developed using Advansta West- ernBright Sirius chemiluminescent substrate (Advansta, K-12043-D20) or SuperSignal West Pico Plus chemilu- minescent substrate (Thermo fisher, #34579), and imaged using Chemidoc XRS with Image Lab software. Densi- tometry analysis was performed using ImageJ software and all protein bands were normalized to GAPDH. Ethics approval and consent to participate The skin samples used for generating the fibroblast cell lines were collected under Coriell and NINDS’s informed consent and deidentification of subjects. Biospecimens obtained from the NINDS Human Cell and Data Repository were not considered to be human subject research because conducting research with the samples does not involve an intervention or interaction with the individual and the samples do not contain identifiable private information. Availability of data and materials All results and methods utilized in this study are mentioned in the article and the supplementary information files. Individual data values for each of the analyses can be obtained from the corresponding authors upon reasonable request. Statistical analysis This research was supported by NIH/NIA R01AG060195, NIH/NINDS 1R01NS092667, DoD W81XWH2010368, DoD W81XWH2010371, the Orchard Foundation, the Harold and Ronna Cooper Family and the Consolidated Anti- Aging Foundation. Statistical data analysis was performed in GraphPad Prism software version 8.4.2. All data are expressed as arithmetic mean ± SEM. Unpaired two-tailed student’s t-test or One-way ANOVA followed by post hoc testing was used as appropriate and the test used for each analy- sis is mentioned in the figure legend. In all cases, outliers identified using the iterative Grubb’s function in Graph- Pad Prism, with alpha set at 0.05, were removed from subsequent analyses. P value < 0.05 was considered sig- nificant for all analyses. Consent for publication Not applicable. Additional file 1: Figure S1. Lysosomal load was not altered in PD patient-derived cells. (A) Representative image and (B) quantification of LAMP1 level (normalized to GAPDH) in whole cell lysates from HS, PD and gPD-GBA N370S cells (n = 13, HS; n = 28, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test). Data represented as mean ± SEM. Figure S2: GCase activity does not correlate with the age of onset or disease duration in PD and gPD-GBA N370S group of cells. Correlation analysis between (A) GCase activity and age of onset (years) and, (B) GCase activity and disease duration (years) was performed in PD and gPD-GBA N370S group of cells and Pearson’s correlation coefficient was determined between the two variables (n = 25, PD; n = 5, gPD-GBA N370S). Figure S3: GRN transcript was reduced in idiopathic PD cells. (A) GRN transcript levels were measured across HS, PD and gPD-GBA N370S cells (n = 13, HS; n = 24, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F(2,40) = 4.772, p = 0.0138) using qPCR. (B) Representative image of immunoblot performed using Endo-H and PNGaseF digested lysates from HS, PD and gPD-GBA N370S cells for GBA protein (Quantification in Fig. 2E). Data represented as mean ± SEM. = p < 0.05. Figure S4: LIMP2 and GCase activity levels do not correlate ELISA‑based measurement of LIMP2 protein levels ELISA‑based measurement of LIMP2 protein levels with rs6812193 or rs6825004 genotypes in idiopathic PD cells. (A) Table depicting the distribution of various genotypes at rs6812193 and rs6825004 locus across the cells from PD and gPD-GBA N370S group of cells. (B, C) LIMP2 levels and (D, E) GCase activity levels between PD cells with various genotypes for rs6812193 and rs6825004 SNPs. Data represented as mean ± SEM. Figure S5: Uncropped immunoblots used in the manuscript. Uncropped images of blots from (A) Fig. 1C, (B) Fig. 2A, (C) Supplementary Fig. 1A and (D) Supplementary Fig. 3B. Equal concentration of cell lysates (RIPA lysates collected as mentioned above) were used to perform ELISA assay (RayBiotech, #ELH-LIMP-II) according to the manufac- turer’s instructions. Samples and standards were run in triplicates and the final colorimetric readout was per- formed using a SPECTRAmax plate reader (Molecular Devices). Additional file 2. Information on processed data used to generate all figures in this study. Authors’ contributions RT, EBM, PJH and OI designed the study. RT, EBM and ZKM performed the experiments. RT, EBM, PJH and OI contributed to the data analysis and interpretation. RT, EBM, PJH and OI wrote the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 19 August 2020 Accepted: 7 December 2020 Received: 19 August 2020 Accepted: 7 December 2020 Abbreviations Endo H and PNGase F treatment of cell lysates Endo H (New England BioLabs, #P0702L) and PNGase F (New England BioLabs, #P0704L) was used accord- ing to manufacturer’s instructions. 20ug of cell lysates (RIPA lysates collected as mentioned above) was digested using 250 units of Endo H and 1000 units of PNGase F enzymes after which they were used for immunoblotting. A condition that included all steps of the digestion with- out the inclusion of the enzymes was included as a nega- tive control. PD: Parkinson’s disease; GCase: Glucocerebrosidase; GlcCer: Glucosylceramide; GlcSph: Glucosylsphingosine; LIMP2: Lysosomal integral protein 2; PGRN: Progranulin; ER: Endoplasmic reticulum; GWAS: Genome wide association studies; SNPs: Short nucleotide polymorphisms; CSF: Cerebrospinal fluid; GD: Gaucher’s disease; DLB: Dementia with lewy bodies. with rs6812193 or rs6825004 genotypes in idiopathic PD cells. (A) Table depicting the distribution of various genotypes at rs6812193 and rs6825004 locus across the cells from PD and gPD-GBA N370S group of cells. (B, C) LIMP2 levels and (D, E) GCase activity levels between PD cells with various genotypes for rs6812193 and rs6825004 SNPs. Data represented as mean ± SEM. Figure S5: Uncropped immunoblots used in the manuscript. Uncropped images of blots from (A) Fig. 1C, (B) Fig. 2A, (C) Supplementary Fig. 1A and (D) Supplementary Fig. 3B. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13041-020-00712-3. Quantitative RT‑PCR RNA extraction was performed with the RNeasy Mini kit (Qiagen, #74104) and cDNA was synthesized using QuantiTect Reverse Tranascription kit (Qiagen, #205311) according to manufacturer’s instructions. qPCR reactions were performed using Power SYBR® Green PCR master mix (Thermo Fisher, #4367659) with 2 ng of cDNA and commercial primers (all Qiagen QuantiTect Primer Assays: SCARB2, #QT00041566; GBA, #QT00047551; GRN, Qiagen #QT01001686; and Thomas et al. Mol Brain (2021) 14:16 Page 10 of 12 References Parnetti L, Paciotti S, Eusebi P, Dardis A, Zampieri S, Chiasserini D, et al. Cerebrospinal fluid β-glucocerebrosidase activity is reduced in parkin- son’s disease patients. Mov Disord. 2017;32:1423–31. 37. Smith KR, Damiano J, Franceschetti S, Carpenter S, Canafoglia L, Morbin M, et al. 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https://openalex.org/W2946344448	https://europepmc.org/articles/pmc6528248?pdf=render	English	null	Using Facebook to reduce smoking among Australian Aboriginal and Torres Strait Islander people: a participatory grounded action study	BMC public health	2,019	cc-by	21,016	Hefler et al. BMC Public Health (2019) 19:615 https://doi.org/10.1186/s12889-019-6918-7 Hefler et al. BMC Public Health (2019) 19:615 https://doi.org/10.1186/s12889-019-6918-7 Open Access Using Facebook to reduce smoking among Australian Aboriginal and Torres Strait Islander people: a participatory grounded action study a Hefler1* , Vicki Kerrigan1, Becky Freeman2, Gordon Robert Boot3 and David P. Thomas1 Marita Hefler1* , Vicki Kerrigan1, Becky Freeman2, Gordon Robert Boot3 and David P. Thom * Correspondence: marita.hefler@menzies.edu.au Correspondence: marita.hefler@menzies.edu.au 1Tobacco Control Research Program, Wellbeing & Preventable Chronic Diseases Division, Menzies School of Health Research, PO Box 41096, Casuarina, NT 0811, Australia Full list of author information is available at the end of the article Abstract Background: There is limited evidence for the effectiveness of social media to promote healthy behaviour among Indigenous Australians, including to reduce smoking. Social media has significant potential to stimulate interpersonal influence to quit, however an important knowledge gap is how and what content people choose to share with friends and family. This paper explores the decision making processes of community members for sharing tobacco control content with family and friends on Facebook. Methods: Community researchers were paid to choose and share at least one tobacco control post per week for a period of 6 months on their personal Facebook page. They documented reasons for their choices, which were coded and analysed to determine features of messages most likely to be shared, and salient considerations in the decision- making process. Results: Posts which are child-focused, feature Indigenous content, and are perceived as practical, relevant and Results: Posts which are child-focused, feature Indigenous content, and are perceived as practical, relevant and credible, with a direct and unambiguous message, were most likely to be shared. Posts which included disgusting imagery about health impacts, were focused on the environment, or were ambiguous or sarcastic were less likely to be shared. Decisions were also based on whether content was perceived to contain new information, to be helpful for their friends, and to be consistent with the participant’s online identity, as well as the perceived sensitivity of content. The potential impact on expensive mobile data for videos was also a factor. Conclusions: When designing tobacco control messages to be shared on social media, health promoters should take into account how information will align with positive self-image and can contribute to social capital among the intended audience, and generate interpersonal engagement. Content should complement, rather than attempt to replicate, some message features that are effective on traditional broadcast media. This study shows the potential for health services to incorporate a strategy of using paid local social media ‘champions’ or ‘ambassadors’ to disseminate tobacco control messages on Facebook through community networks. Keywords: Smoking, Social media, Health communication, Qualitative research, Indigenous health Background For Australian Aboriginal and Torres Strait Islander peo- ples, social media have the potential to encourage social support and enact an agenda of self-determination and em- powerment which aligns with Indigenous notions of health [1, 2]. Although limited current statistics are available, re- search suggests social media use is higher among Aborigi- nal and Torres Strait Islander people than non-Indigenous Australians [3]. A 2014 survey found that 60% of Indigen- ous people used Facebook, compared to 42% of the Austra- lian population at that time [4], and in most communities Facebook use has continued to increase since then. Abori- ginal and Torres Strait Islander organisations and leaders have been at the forefront of social media use to advance health [5], particularly for online activism, community development, advocacy, citizen journalism, and countering racism and negative stereotypes [2, 5–9]. Social media are used by Aboriginal and Torres Islander people in a range of ways which can impact health and wellbeing, including developing and expressing Indigenous identity [10–12], help seeking and support for suicide and self-harm [13], and communication and support connected to death and grieving [14]. However, there is limited evidence for the ef- fectiveness of social media to promote healthy behaviour among Indigenous Australians, [15, 16] including to reduce smoking [17]. This study forms part of a larger project to investigate how social media can be effectively used to enhance Indi- genous tobacco control in Australia. The first exploratory study for the project found that tobacco control content was rarely shared or seen by participants on Facebook, the most commonly used social media platform [36]. In this study we nested smoking prevention messages within exist- ing Facebook networks, by employing community-based peer researchers (hereafter called participants) to share to- bacco control content on their personal Facebook pages. The aim of this paper is to explore the decision-making processes of what content was shared. Although the interactive nature of social media is the key to their potential power to achieve health promotion goals [18–20], many health organisations are failing to harness these capabilities [21]. Research about health promotion using social media tends to focus on approaches that use so- cial media as a one-way tool for health education [1]. © 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. Hefler et al. BMC Public Health (2019) 19:615 Page 2 of 21 Hefler et al. BMC Public Health (2019) 19:615 Tobacco use is a leading cause of disease and prema- ture mortality, and reducing smoking prevalence among Aboriginal and Torres Strait Islander people is an im- portant Australian health priority. Smoking prevalence is around three times higher among Indigenous than non-Indigenous Australians (39 and 14% respectively, in 2014–5 for people aged 15 years and over), however there is limited Indigenous-specific evidence for most tobacco control approaches [17]. Internationally, there is a strong evidence base for mass media communication as part of a comprehensive tobacco control approach, with effectiveness evident across different population groups [33–35]. Messages about negative health effects are most effective at generating increased knowledge and promoting quitting, however impact depends on sufficient intensity and duration of exposure [33]. Achieving exposure at the level necessary to have an im- pact may be unlikely through social media, as users can control how and to what extent they engage with con- tent, and exposure is highly dependent on sharing from personal contacts. There is therefore a need to under- stand what tobacco control messages are most likely to be shared through social media, and how messages may need to be adapted to maximise impact. Background How to maximise engagement is a challenge not unique to health promotion; research focused on generating consumer en- gagement for commercial purposes has highlighted a similar tendency towards broadcasting content that involves passive consumption [22]. Both health and commercially-focused research highlights the importance of a user-centred ap- proach to engaging target audiences, taking into account the interpersonal aspects of social media use, its role in building social capital and relationships, and interaction with con- sumers’ self-image and identity [22–24]. Methods A ‘grounded action’ [37] approach based on a combin- ation of grounded theory [38] and participatory action research [39] was used. It combined a structured inter- vention with qualitative data about decision-making pro- cesses for sharing content. The study duration was 26 weeks, from December 2016 to June 2017. Consultation with participants about the study design occurred from September to December 2016, in order to ensure max- imum participation and minimal burden. There is an emerging body of research about char- acteristics of highly shared health content on social media [25–27], however there is little existing re- search which examines decision-making processes for sharing health-related content with friends and family on social media. Given the potential for peer-to-peer influence through social media [28, 29] and the po- tential to use social media networks to shape health behaviours [30], this is an important knowledge gap [20]. This is particularly so for tobacco control, given that anti-smoking messages can stimulate interpersonal influence to quit [31, 32]. Participants, recruitment and sampling Thirteen participants were employed on a casual basis at study commencement. Inclusion criteria were: (1) the par- ticipant identified as Aboriginal and/or Torres Strait Is- lander, (2) was a regular Facebook user (at least weekly), and (3) a significant proportion of their Facebook network Hefler et al. BMC Public Health (2019) 19:615 Page 3 of 21 are taken from both interviews and written information sent by text or email. was people with whom they had an existing offline con- nection and regular contact. Participants were recruited from Darwin and Alice Springs, Northern Territory, Australia. Eleven participants had been employed in the initial exploratory study of the project which investigated what types of health-related content is being shared by Indigenous people on social media [36]. Two additional participants were recruited for this study – one recom- mended by a participant in the previous study, and one responded to information distributed through local community networks. We sought to achieve maximum variation in terms of gender, age, residential location, socioeconomic and employment/study status, and smok- ing status. Two participants were male, all others were female. The age range was from early 20s through to approximately 60. One participant was a never- smoker, three were ex-smokers. All others were current smokers at study commencement. Four identified as not currently ready to quit, while all others were either contemplating or actively trying to quit. Study intervention Participants were asked to share at least one tobacco con- trol post per week for the study duration. Three options were provided each week, drawn from a content library cre- ated by the research team (MH and VK). It contained video and still images, international content, Australian content (general, as well as tailored for Aboriginal and Torres Strait Islander people), content produced for government cam- paigns, and content created by non-government organisa- tions and others. Selected content was not screened for evidence of impacting smoking attitudes and behaviour, however it was assessed to ensure it did not inadvertently promote smoking, contain factually incorrect information, or was produced by the tobacco industry or entities work- ing to promote its interests. The full list of content options for each week is provided as Table 1. Participants were free to post more than one option each week, not post anything if they did not feel any options were suitable, or to post al- ternative content they had sourced or created themselves (subject to screening by the project team). Ethical issues Two non-Indigenous researchers (MH & VK) worked in close collaboration with the partner Aboriginal Commu- nity Controlled Health Services, and the participants, to ensure an inclusive, respectful and culturally appropriate approach which adheres to Australia’s guidelines for eth- ical conduct in Aboriginal & Torres Strait Islander health research [40]. Written consent was obtained from participants, who were free to withdraw from the study at any time. Identifying information such as names and profile pictures (of participants and their Facebook friends) was removed from all data during analysis. Pseudonym codes have been used for direct quotes in this article. As several participants were smokers who were in the process of attempting to quit, there was significant potential for them to experience distress. Counselling and other support services were available as needed; several referrals were provided to both general counselling services and Quitline. Data analysis Data was inductively coded, adapted from a grounded the- ory approach, using Microsoft Word to create codes and record memos [38]. Initial coding was developed by the research team and was based on content characteristics, using both screenshots of the Facebook posts and the in- formation about decision-making provided by participants each week. Additional data collected through monthly in- terviews was also coded, and insights added to memos. An iterative approach was used; as categories were devel- oped, these were compared to earlier data from each par- ticipant, and interpretations were checked with participants to allow their feedback to be integrated. A final reflection interview was held with each participant to further refine and develop the categories developed through the 6 months of data collection. At the comple- tion of the data collection period, all posts were sorted by popularity based on the total number of times they were posted by participants, (provided as Table 2) and the per- centage of participants each week who shared the post, as well as broad themes of the content. The quantitative and qualitative analyses were then compared to further de- velop the key themes. The initial collated findings were presented to the participants and project partners at a project meeting held in September 2017, for further devel- opment, refinement and validation. In particular, discrep- ancies regarding posts that appeared to be popular based on quantitative analysis and participants’ perceptions reported in interviews were discussed and resolved. The themes presented here represent consensus agreement about themes that shaped participants’ choices. Data collection Participants were asked to: document the reasons for the content they did and did not select, and track the inter- actions with each post – both online (e.g. likes, com- ments, shares), and offline (e.g. discussions prompted by post content). This information, together with screen- shot(s) of the content posted was sent to the research team each week. The research team interviewed the participants each month to clarify or collect additional information about the decision-making process for selecting posts. Participant quotes in the results section Page 4 of 21 Hefler et al. BMC Public Health (2019) 19:615 Weekly content options provided to peer researchers Option A Option B Option C Video: Catmageddon (Truth Initiative, USA) https://www.youtube.com/watch?v=tLtschJxRy8 Video: 1200 die (American Legacy Foundation) https://www.youtube.com/ watch?v=4XmyGQJ2WWE Image: Life meter Description: shows a cigarette with numbers to represent lifespan, with the ash burning towards the number 60. URL not provided as original source not able to be determined. Video: Quit for you, quit for two (Australian Government campaign, pregnancy focus) http://www.quitnow.gov.au/internet/quitnow/ publishing.nsf/Content/quit-you-quit-two Video: Skinnyfish Yaka Ngarali (created by local record label and Aboriginal community controlled health service) https://www.youtube.com/watch?v= 9yPwNVaOPMY&list= PLhVW2l7sc6giYqfvL9qN6l0Q89kx0vAzd&index= 1 Image: Smoking burns money Cartoon-like image showing a hand holding a lit cigarette, with the ash replaced by coins. URL not provided as original source of image not able to be determined. Video: Break the Chain (Australian Government campaign, Indigenous focus) https://www.youtube.com/watch?v=0yvjBU- E0aw Video: 5 things the tobacco industry doesn’t want you to know (produced by Public Health Ottawa) https://www. youtube.com/watch?v=UB1biuoDkEQ Image: smokers teeth Description: photo of young attractive women holding a cigarette and smiling, showing extremely strained teeth. URL not provided as original source not able to be determined. Video: don’t make smokes your story (Australian Government campaign, Indigenous focus) https://www.facebook.com/healthgovau/videos/ 1089321171111579/?v=1089321171111579 Video: Allen Carr top tips on quitting https://www.youtube.com/watch?v= 0TL2Vh7goJc Image: Smoking good for environment Close up photo of lower half of face with cigarette and caption “Smoking is good for the environment because it kills humans”. URL not provided as original source not able to be determined. Video: Bryan Curtis story (YouTube user generated content) https://www.youtube.com/watch?v= dVLtNgAhPRg Video: Quit smoking timeline Video of how the body recovers from smoking. Produced by online quit smoking community www.quitsmoking. com. https://www.youtube.com/watch?v= fLbQfMmrISE Image: Dream boy Poster with a photo of a young Indigenous boy dressed for boxing, and the caption “Fill my head with dreams, not my lungs with smoke”. Data collection URL not provided as original source of image not able to be determined. Video: 5 things the tobacco industry doesn’t want you to know (produced by Public Health Ottawa) https://www. youtube.com/watch?v=UB1biuoDkEQ Image: smokers teeth Description: photo of young attractive women holding a cigarette and smiling, showing extremely strained teeth. URL provided as original source no able to be determined. Video: Allen Carr top tips on quitting https://www.youtube.com/watch?v= 0TL2Vh7goJc Image: Smoking good for environment Close up photo of lower half o face with cigarette and captio “Smoking is good for the environment because it kills humans”. URL not provided as original source not able to be determined. Video: Quit smoking timeline Video of how the body recovers from smoking. Produced by online quit smoking community www.quitsmoking. com. https://www.youtube.com/watch?v= fLbQfMmrISE Image: Dream boy Poster with a photo of a youn Indigenous boy dressed for boxing, and the caption “Fill m head with dreams, not my lun with smoke”. Current poster U not available. Website listed o poster: www. giveupsmokesforgood.org.au Video: Social farting/smoking Available from Middlesex London Health Unit YouTube channel. https://www.youtube.com/watch?v= Image: smokers’ funeral Photo of two people smoking a room, with the ceiling painte to look like they are looking u Table 1 Weekly content options provided to peer researchers Week Option A Option B 1. Video: Catmageddon (Truth Initiative, USA) https://www.youtube.com/watch?v=tLtschJxRy8 Video: 1200 die (American Legacy Foundation) https://www.youtube.com/ watch?v=4XmyGQJ2WWE 2. Video: Quit for you, quit for two (Australian Government campaign, pregnancy focus) http://www.quitnow.gov.au/internet/quitnow/ publishing.nsf/Content/quit-you-quit-two Video: Skinnyfish Yaka Ngarali (created by local record label and Aboriginal community controlled health service) https://www.youtube.com/watch?v= 9yPwNVaOPMY&list= PLhVW2l7sc6giYqfvL9qN6l0Q89kx0vAzd&index= 1 Image: Smoking burns money Cartoon-like image showing a hand holding a lit cigarette, with the ash replaced by coins. URL not provided as original source of image not able to be determined. 3. Video: Break the Chain (Australian Government campaign, Indigenous focus) https://www.youtube.com/watch?v=0yvjBU- E0aw Video: 5 things the tobacco industry doesn’t want you to know (produced by Public Health Ottawa) https://www. youtube.com/watch?v=UB1biuoDkEQ 4. Video: don’t make smokes your story (Australian Government campaign, Indigenous focus) https://www.facebook.com/healthgovau/videos/ 1089321171111579/?v=1089321171111579 Video: Allen Carr top tips on quitting https://www.youtube.com/watch?v= 0TL2Vh7goJc 5. Video: Bryan Curtis story (YouTube user generated content) https://www.youtube.com/watch?v= dVLtNgAhPRg Video: Quit smoking timeline Video of how the body recovers from smoking. Produced by online quit smoking community www.quitsmoking. com. https://www.youtube.com/watch?v= fLbQfMmrISE 6. Video: Thai smoking kids Advertisement produced by Ogilvy Asia for the Thai Health Promotion Foundation. Data collection Current poster URL not available. Website listed on poster: www. giveupsmokesforgood.org.au Video: Thai smoking kids Advertisement produced by Ogilvy Asia for the Thai Health Promotion Foundation. https://www.youtube.com/watch?v= Video: Social farting/smoking Available from Middlesex London Health Unit YouTube channel. https://www.youtube.com/watch?v= Image: smokers’ funeral Photo of two people smoking in a room, with the ceiling painted to look like they are looking up Image: smokers teeth Description: photo of young attractive women holding a cigarette and smiling, showing extremely strained teeth. URL not provided as original source not able to be determined. Image: Smoking good for environment Close up photo of lower half of face with cigarette and caption “Smoking is good for the environment because it kills humans”. URL not provided as original source not able to be determined. Image: Dream boy Poster with a photo of a young Indigenous boy dressed for boxing, and the caption “Fill my head with dreams, not my lungs with smoke”. Current poster URL not available. Website listed on poster: www. giveupsmokesforgood.org.au Image: smokers’ funeral Photo of two people smoking in a room, with the ceiling painted to look like they are looking up Image: smokers teeth Description: photo of young attractive women holding a cigarette and smiling, showing extremely strained teeth. URL not provided as original source not able to be determined. Image: Smoking good for environment Close up photo of lower half of face with cigarette and caption “Smoking is good for the environment because it kills humans”. URL not provided as original source not able to be determined. Image: Dream boy Poster with a photo of a young Indigenous boy dressed for boxing, and the caption “Fill my head with dreams, not my lungs with smoke”. Current poster URL not available. Website listed on poster: www. giveupsmokesforgood.org.au Image: smokers’ funeral Photo of two people smoking in a room, with the ceiling painted to look like they are looking up Option B Option C Video: 1200 die (American Legacy Foundation) https://www.youtube.com/ watch?v=4XmyGQJ2WWE Image: Life meter Description: shows a cigarette with numbers to represent lifespan, with the ash burning towards the number 60. URL n provided as original source no able to be determined. Video: Skinnyfish Yaka Ngarali (created by local record label and Aboriginal community controlled health service) https://www.youtube.com/watch?v= 9yPwNVaOPMY&list= Image: Smoking burns money Cartoon-like image showing a hand holding a lit cigarette, with the ash replaced by coins. Data collection https://www.youtube.com/watch?v= Video: Social farting/smoking Available from Middlesex London Health Unit YouTube channel. https://www.youtube.com/watch?v= Option B Video: 1200 die (American Legacy Foundation) https://www.youtube.com/ watch?v=4XmyGQJ2WWE Video: Skinnyfish Yaka Ngarali (created by local record label and Aboriginal community controlled health service) https://www.youtube.com/watch?v= 9yPwNVaOPMY&list= Image: Smoking burns money Cartoon-like image showing a hand holding a lit cigarette, with the ash replaced by coins. URL not provided as original source of image not able to be determined. Video: 5 things the tobacco industry doesn’t want you to know (produced by Public Health Ottawa) https://www. youtube.com/watch?v=UB1biuoDkEQ Video: Allen Carr top tips on quitting https://www.youtube.com/watch?v= 0TL2Vh7goJc Video: Quit smoking timeline Video of how the body recovers from smoking. Produced by online quit smoking community www.quitsmoking. com. https://www.youtube.com/watch?v= fLbQfMmrISE Video: Social farting/smoking Available from Middlesex London Health Unit YouTube channel. https://www.youtube.com/watch?v= Table 1 Weekly content options provided to peer researchers W k O A 5. Page 5 of 21 Hefler et al. BMC Public Health (2019) 19:615 Table 1 Weekly content options provided to peer researchers (Continued) Week Option A Option B Option C qHH2LsAHeHc&feature=youtu.be 9rIj2DWQRdw from a grave with people attending their funeral looking on. URL not provided as original source not able to be determined. 7. Video: Everybody knows Video created by Cancer Institute NSW, which shows clips from a range of well-known Austra- lian anti-smoking campaigns, set to Leonard Co- hen song “Everybody knows”. Link not provided, as original URL not able to be determined. Video: Cigarettes & child labour Information video produced by AJplus news media. https://www.youtube.com/watch?v= t6pVsA6hdYs Image: cup of butts Photo showing a teacup full of cigarette butts and a teaspoon with a butt above it. URL not provided as original source not able to be determined. 8. Video: Culture or killer Produced for Lakes Entrance Aboriginal Health Association in Victoria, Australia. https://vimeo.com/127003575 Video: smoking and drinking. Video showing animation of the impacts of smoking and drinking on the body. (Appears to be YouTube user- generated content) https://www.youtube.com/watch?v= XXwpTrI74BQ Image: Quit gains Poster with the question “What have you gained from quitting?” in the centre, surrounded by line drawings to suggest improved health and money saved. URL not provided as original source not able to be determined. 9. Video: exercise & quitting https://www.youtube.com/watch?v=- dHzc4xqHks Video: smoking damage. Motion graphic video on health impacts of smoking. Australian government, Indigenous focus. Data collection https://www.youtube.com/watch?v= IfIBckA0hAc Image: smoking increases stress Monotone mage of a cigarette pack shaped like a coffin, with the words “Smoking reduces stress. False” in red. Further down “Although smoking temporarily relieves the stress of withdrawal ovided to peer researchers (Continued) Option B Option C eHc&feature=youtu.be 9rIj2DWQRdw from a grave with people attending their funeral looking on. URL not provided as original source not able to be determined. ybody knows ted by Cancer Institute NSW, which s from a range of well-known Austra- moking campaigns, set to Leonard Co- Everybody knows”. Link not provided, URL not able to be determined. Video: Cigarettes & child labour Information video produced by AJplus news media. https://www.youtube.com/watch?v= t6pVsA6hdYs Image: cup of butts Photo showing a teacup full of cigarette butts and a teaspoon with a butt above it. URL not provided as original source not able to be determined. ure or killer or Lakes Entrance Aboriginal Health in Victoria, Australia. eo.com/127003575 Video: smoking and drinking. Video showing animation of the impacts of smoking and drinking on the body. (Appears to be YouTube user- generated content) https://www.youtube.com/watch?v= XXwpTrI74BQ Image: Quit gains Poster with the question “What have you gained from quitting?” in the centre, surrounded by line drawings to suggest improved health and money saved. URL not provided as original source not able to be determined. cise & quitting w.youtube.com/watch?v=- ks Video: smoking damage. Motion graphic video on health impacts of smoking. Australian government, Indigenous focus. https://www.youtube.com/watch?v= IfIBckA0hAc Image: smoking increases stress Monotone mage of a cigarette pack shaped like a coffin, with the words “Smoking reduces stress. False” in red. Further down “Although smoking temporarily relieves the stress of withdrawal symptoms, scientists have found that nicotine actually increases your level of stress hormones” is printed in black. URL not provided as original source not able to be determined. t steps poster with 7 steps, accompanied by o help with quitting smoking: set a h ashtrays, call the Quitline (with mber prominently included, exercise, ealthy foods, drink water and have less alcohol. URL not provided as urce not able to be determined. Image: start repairing Australian government poster with a photo of a person, pointing to different parts of the body, and examples of both short and longer-term (5 days up to 1 year) benefits of stopping smoking. Video: Real cost – your skin US Government Food & Drug Administration campaign advertisement. Data collection Can be viewed at https://www. ispot.tv/ad/7BnU/the-real-cost- your-skin smoking USA Truth campaign, image of the t: dogs and cats are twice as likely to if their owner smokes”. Video: Donna, Alice Springs Don’t make smokes your story. Australian Government campaign, Indigenous focus, with real local people. https://www.youtube.com/watch?v= rOy1vO-HlnA Image: Trigger Poster with the heading “if you smoke…” With four boxes containing animated graphics and the words: “In the morning, try a new morning routine”, “after meals, have a mint or a cup of Option C from a grave with people attending their funeral looking on. URL not provided as original source not able to be determined. Image: cup of butts Photo showing a teacup full of cigarette butts and a teaspoon with a butt above it. URL not provided as original source not able to be determined. Image: Quit gains Poster with the question “What have you gained from quitting?” in the centre, surrounded by line drawings to suggest improved health and money saved. URL not provided as original source not able to be determined. Image: smoking increases stress Monotone mage of a cigarette pack shaped like a coffin, with the words “Smoking reduces stress. False” in red. Further down “Although smoking temporarily relieves the stress of withdrawal symptoms, scientists have found that nicotine actually increases your level of stress hormones” is printed in black. URL not provided as original source not able to be determined. Video: Real cost – your skin US Government Food & Drug Administration campaign advertisement. Can be viewed at https://www. ispot.tv/ad/7BnU/the-real-cost- your-skin Image: Trigger Poster with the heading “if you smoke…” With four boxes containing animated graphics and the words: “In the morning, try a new morning routine”, “after meals, have a mint or a cup of Option C from a grave with people attending their funeral looking on. URL not provided as original source not able to be determined. Image: cup of butts Photo showing a teacup full of cigarette butts and a teaspoon with a butt above it. URL not provided as original source not able to be determined. Image: Quit gains Poster with the question “What have you gained from quitting?” in the centre, surrounded by line drawings to suggest improved health and money saved. URL not provided as original source not able to be determined. Data collection Image: smoking increases stress Monotone mage of a cigarette pack shaped like a coffin, with the words “Smoking reduces stress. False” in red. Further down “Although smoking temporarily relieves the stress of withdrawal symptoms, scientists have found that nicotine actually increases your level of stress hormones” is printed in black. URL not provided as original source not able to be determined. Video: Real cost – your skin US Government Food & Drug Administration campaign advertisement. Can be viewed at https://www. ispot.tv/ad/7BnU/the-real-cost- your-skin Image: Trigger Poster with the heading “if you smoke…” With four boxes containing animated graphics and the words: “In the morning, try a new morning routine”, “after meals, have a mint or a cup of ded to peer researchers (Continued) Option B Option C &feature=youtu.be 9rIj2DWQRdw from a grave w attending their on. URL not pro source not able determined. dy knows by Cancer Institute NSW, which m a range of well-known Austra- ng campaigns, set to Leonard Co- rybody knows”. Link not provided, not able to be determined. Video: Cigarettes & child labour Information video produced by AJplus news media. https://www.youtube.com/watch?v= t6pVsA6hdYs Image: cup of b Photo showing cigarette butts with a butt abo provided as ori able to be dete or killer akes Entrance Aboriginal Health Victoria, Australia. om/127003575 Video: smoking and drinking. Video showing animation of the impacts of smoking and drinking on the body. (Appears to be YouTube user- generated content) https://www.youtube.com/watch?v= XXwpTrI74BQ Image: Quit gai Poster with the have you gaine in the centre, s drawings to sug health and mo not provided a not able to be & quitting outube.com/watch?v=- Video: smoking damage. Motion graphic video on health impacts of smoking. Australian government, Indigenous focus. https://www.youtube.com/watch?v= IfIBckA0hAc Image: smoking Monotone mag pack shaped lik the words “Smo stress. False” in “Although smo relieves the stre Image: start repairing Australian government poster with a photo of a person, pointing to different parts of the body, and examples of both short and longer-term (5 days up to 1 year) benefits of stopping smoking. Video: Donna, Alice Springs Don’t make smokes your story. Australian Government campaign, Indigenous focus, with real local people. Data collection Department website: https://www.mass.gov/ massachusetts-tobacco-cessation- and-prevention-program-mtcp Image: smoking sarcasm Cartoon like image of a lit cigarette with arms ‘eating’ a person through the lit end of the cigarette. Accompanied by a caption “Share if you are against smoking”. URL not provided as original source not able to be determined. Image: Butts are litter Image of a cigarette being stubbed out with the words “Cigarettes butts are LITTER too…” URL not provided as original source not able to be determined. Video: Sugar sugar Produced for Make Smoking History campaign by Cancer Council WA, Western Australia. Campaign website: https:// makesmokinghistory.org.au/ https://www.youtube.com/ watch?v=0fXdLCTND1A Option C tea instead”, in your car, clean it out to get rid of the smell and keep healthy snacks with you” and “when you’re stressed, get enough sleep, stay active and talk about what is bothering you”. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. URL of poster not provided as original source not able to be determined. Department website: https://www.mass.gov/ massachusetts-tobacco-cessation- and-prevention-program-mtcp Image: smoking sarcasm Cartoon like image of a lit cigarette with arms ‘eating’ a person through the lit end of the cigarette. Accompanied by a caption “Share if you are against smoking”. URL not provided as original source not able to be determined. Image: Butts are litter Image of a cigarette being stubbed out with the words “Cigarettes butts are LITTER too…” URL not provided as original source not able to be determined. Video: Sugar sugar Produced for Make Smoking History campaign by Cancer Council WA, Western Australia. Campaign website: https:// makesmokinghistory.org.au/ https://www.youtube.com/ watch?v=0fXdLCTND1A Image: Quit tip Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitTip Ask friends who smoke to promise not to offer you any cigarettes”. Current poster URL not available. Campaign website: https://web.archive.org/web/ 20190304210745, https://campaigns. health.gov.au/smokes Image: timebomb Photo of a bundle of cigarettes tide to a clock to resemble a time bomb, with a hand with a lighter about to light the fuse. Accompanied by the caption “Every breath you take will eventually destroy your future”. URL not provided as original source not able to be determined. Image: Four D’s Image of a poster with the heading “Practice the Four D’s to help you get through a craving. Each D is listed (Delay, Drink water, Deep breathing, Distract) with explanatory text and a graphic. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. Data collection https://www.youtube.com/watch?v= rOy1vO-HlnA Image: Quit steps Australian poster with 7 steps, accompanied by graphics, to help with quitting smoking: set a date, banish ashtrays, call the Quitline (with Quitline number prominently included, exercise, snack on healthy foods, drink water and have drinks with less alcohol. URL not provided as original source not able to be determined. Image: pet smoking Form the USA Truth campaign, image of the words “Fact: dogs and cats are twice as likely to get cancer if their owner smokes”. Image: Quit steps Australian poster with 7 steps, accompanied by graphics, to help with quitting smoking: set a date, banish ashtrays, call the Quitline (with Quitline number prominently included, exercise, snack on healthy foods, drink water and have drinks with less alcohol. URL not provided as original source not able to be determined. Image: pet smoking Form the USA Truth campaign, image of the words “Fact: dogs and cats are twice as likely to get cancer if their owner smokes”. 11. 10. 11. 9. Page 6 of 21 Hefler et al. BMC Public Health (2019) 19:615 ontent options provided to peer researchers (Continued) Option A Option B Option C tea instead”, in your car, clean it out to get rid of the smell and keep healthy snacks with you” and “when you’re stressed, get enough sleep, stay active and talk about what is bothering you”. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. URL of poster not provided as original source not able to be determined. Department website: https://www.mass.gov/ massachusetts-tobacco-cessation- and-prevention-program-mtcp Image: appearance change. Shows photo of a woman with one side as a non-smoker aged 35 and the other side as a smoker of the same age, highlighting the differ- ences and appearance impacts of smoking. Queensland Health, Australia campaign. Image can be viewed at https://www.couriermail.com. au/news/queensland/smoking-makes-you-ugly- queensland-health-to-tell-young-women/news- story/d80e3e017b01d636d692ed884113ef41 Image: Quit tip Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitTip Ask friends who smoke to promise not to offer you any cigarettes”. Current poster URL not available. Campaign website: https://web.archive.org/web/ 20190304210745, https://campaigns. health.gov.au/smokes Image: smoking sarcasm Cartoon like image of a lit cigarette with arms ‘eating’ a person through the lit end of the cigarette. Accompanied by a caption “Share if you are against smoking”. URL not provided as original source not able to be determined. Data collection Image: Quit tip morning Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitTip Take it 1 day at a time. Every morning say, “I’m not going to smoke today””. Current poster URL not available. Campaign website: https://campaigns.health.gov.au/smokes Image: timebomb Photo of a bundle of cigarettes tide to a clock to resemble a time bomb, with a hand with a lighter about to light the fuse. Accompanied by the caption “Every breath you take will eventually destroy your future”. URL not provided as original source not able to be determined. Image: Butts are litter Image of a cigarette being stubbed out with the words “Cigarettes butts are LITTER too…” URL not provided as original source not able to be determined. Image: fish butts Image of internal cross-section of a fish with butts in the stomach cavity, accompanied by a piece of paper which resembles a shopping docket with information about how cigarette butts contaminate water. URL not provided as original source not able to be determined. Image: Four D’s Image of a poster with the heading “Practice the Four D’s to help you get through a craving. Each D is listed (Delay, Drink water, Deep breathing, Distract) with explanatory text and a graphic. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. URL of poster not provided as original source not able to be determined. Department website: https://www.mass.gov/massachusetts- tobacco-cessation-and-prevention- program-mtcp Video: Sugar sugar Produced for Make Smoking History campaign by Cancer Council WA, Western Australia. Campaign website: https:// makesmokinghistory.org.au/ https://www.youtube.com/ watch?v=0fXdLCTND1A 1 Weekly content options provided to peer researchers (Continued) Option A Option B Option C tea instead”, in your car, clean it out to get rid of the smell and keep healthy snacks with you” and “when you’re stressed, get enough sleep, stay active and talk about what is bothering you”. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. URL of poster not provided as original source not able to be determined. Department website: https://www.mass.gov/ massachusetts-tobacco-cessation- and-prevention-program-mtcp Image: appearance change. Shows photo of a woman with one side as a non-smoker aged 35 and the other side as a smoker of the same age, highlighting the differ- ences and appearance impacts of smoking. Queensland Health, Australia campaign. Image can be viewed at https://www.couriermail.com. Data collection au/news/queensland/smoking-makes-you-ugly- queensland-health-to-tell-young-women/news- story/d80e3e017b01d636d692ed884113ef41 Image: Quit tip Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitTip Ask friends who smoke to promise not to offer you any cigarettes”. Current poster URL not available. Campaign website: https://web.archive.org/web/ 20190304210745, https://campaigns. health.gov.au/smokes Image: smoking sarcasm Cartoon like image of a lit cigarette with arms ‘eating’ a person through the lit end of the cigarette. Accompanied by a caption “Share if you are against smoking”. URL not provided as original source not able to be determined. Image: Quit tip morning Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitTip Take it 1 day at a time Every morning Image: timebomb Photo of a bundle of cigarettes tide to a clock to resemble a time bomb, with a hand with a lighter about to light the Image: Butts are litter Image of a cigarette being stubbed out with the words “Cigarettes butts are LITTER Option C tea instead”, in your car, clean it out to get rid of the smell and keep healthy snacks with you” and “when you’re stressed, get enough sleep, stay active and talk about what is bothering you”. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. URL of poster not provided as original source not able to be determined. Department website: https://www.mass.gov/ massachusetts-tobacco-cessation- and-prevention-program-mtcp Image: smoking sarcasm Cartoon like image of a lit cigarette with arms ‘eating’ a person through the lit end of the cigarette. Accompanied by a caption “Share if you are against smoking”. URL not provided as original source not able to be determined. Image: Butts are litter Image of a cigarette being stubbed out with the words “Cigarettes butts are LITTER too…” URL not provided as original source not able to be determined. Video: Sugar sugar Produced for Make Smoking History campaign by Cancer Council WA, Western Australia. Campaign website: https:// makesmokinghistory.org.au/ https://www.youtube.com/ watch?v=0fXdLCTND1A Option C tea instead”, in your car, clean it out to get rid of the smell and keep healthy snacks with you” and “when you’re stressed, get enough sleep, stay active and talk about what is bothering you”. Produced by the Massachusetts Tobacco Cessation and Prevention Program, USA. URL of poster not provided as original source not able to be determined. Data collection com/watch?v=7ctaMwtHwUo Image: smoking gun Black and white photo of a hand holding a cigarette against a blank wall, with the shadow resembling a pointed gun. URL not provided as original source not able to be determined. Video: I will survive Video produced for HSE Ireland. Campaign website: www.quit.ie. https://www.youtube.com/watch?v= NSETW_zO9jc&feature=youtu.be&list= PLsQK32cdMW_ w6X1OiaNXOjwbD2XKbcS1r Video: Don’t make smokes your story. Australian Government campaign, Indigenous focus, with real local people. https://www.youtube.com/ watch?v=em_0c_Yzxyk Image: poor swimmers Photo of used matches resembling sperm swimming in an ashtray towards ash (representing an ovum). Produced for ASH UK, image can be viewed at https://www.flickr.com/ photos/ashuk/4459586151. Video: Message from the lungs Video produced for the Thai Health Promotion Foundation. https://vimeo.com/126220314 Image: cancer cures smoking Image of the words “Cancer cures smoking”. No other graphics or pictures are included. Taken from https://www. thefreshquotes.com/50-smoking- and-tobacco-quotes-and-slogans/ cancer-cures-smoking/. Image: baby bottle Photo of a baby’s bottle half-filled with cigarette butts and smoking coming out of the top, ac- companied with the words “How many ciga- rettes a day does your child smoke?” at the top, and “Prevent passive smoking” in smaller letters at the bottom. URL not provided as original source not able to be determined. Video: Plain packaging Video produced for World Health Organization for World No Tobacco Day 2016, which had the theme “Get ready for plain packaging.” https://www.youtube.com/watch?v= rXUCTSp2_58 Image: pregnant Poster with a photo of a pregnant belly with a lit cigarette held next to it and smoke appearing to come out of the belly button. URL to poster not available, organisation website: www.kindergesundheit.de. Image: squirrels Photo of a squirrel with the wording “Cigarettes are like squirrels: they’re perfectly harmless until you put one in your mouth and light it on fire.” Video: Quit smoking (Arrente) Animated video of a brain explaining the impact of smoking in Arrente, a central Australian Aboriginal language. Image: child labour pack Photo of a an open pack of cigarettes with children inside the pack instead of cigarettes, 1 Weekly content options provided to peer researchers (Continued) Option A Option B Option C Image: Quit fact Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitFact within a day of quitting, almost all the nicotine is out of your bloodstream”. Current poster URL not available. Campaign website: https://web.archive.org/web/ 20190304210745, https://campaigns.health.gov. Data collection au/smokes Video: Make smoking history (please help me quit) Produced for the Cancer Council WA Make Smoking History campaign, Western Australia. Campaign website: https://makesmokinghistory.org.au/ https://www.youtube.com/watch?v= 80gkwcx314w Video: lost child Video produced for Cancer Council Victoria, Australia. Information about the ad can be viewed here: https://www. cancervic.org.au/about/media- releases/2008-media-releases/ media-rel-october08/quit- launches-separation-08.html Link to video: https://www. bestadsontv.com/ad/17507/Quit- Victoria-Separation Image: No more killing Image of two burning cigarettes placed upright to resemble the twin towers on fire after the 9/ 11 attacks. Accompanied with the words “NO MORE Killing” and in small type “It is estimated that one person dies every 8 s from smoking. Stop smoking now!” URL not provided as original source not able to be determined. Image: Happy ending Poster with a photo of a young Indigenous girl in a T-shirt printed with an Aboriginal flag, with the caption “Stories about smokers never end with happily ever after”. Current poster URL not available. Poster includes the URL https://www.instagram.com/p/Bso_ iAvlvc4/ Video: Every cigarette rots you from the inside out NHS England Smoke Free campaign https://www.youtube. com/watch?v=7ctaMwtHwUo Image: smoking gun Black and white photo of a hand holding a cigarette against a blank wall, with the shadow resembling a pointed gun. URL not provided as original source not able to be determined. Video: I will survive Video produced for HSE Ireland. Campaign website: www.quit.ie. https://www.youtube.com/watch?v= NSETW_zO9jc&feature=youtu.be&list= PLsQK32cdMW_ w6X1OiaNXOjwbD2XKbcS1r Video: Don’t make smokes your story. Australian Government campaign, Indigenous focus, with real local people. https://www.youtube.com/ watch?v=em_0c_Yzxyk Image: poor swimmers Photo of used matches resembling sperm swimming in an ashtray towards ash (representing an ovum). Produced for ASH UK, image can be viewed at https://www.flickr.com/ photos/ashuk/4459586151. Video: Message from the lungs Video produced for the Thai Health Promotion Foundation. https://vimeo.com/126220314 Image: cancer cures smoking Image of the words “Cancer cures smoking”. No other graphics or pictures are included. Taken from https://www. thefreshquotes.com/50-smoking- and-tobacco-quotes-and-slogans/ cancer-cures-smoking/. Image: baby bottle Photo of a baby’s bottle half-filled with cigarette butts and smoking coming out of the top, ac- companied with the words “How many ciga- rettes a day does your child smoke?” at the top, and “Prevent passive smoking” in smaller letters at the bottom. URL not provided as original source not able to be determined. Data collection URL of poster not provided as original source not able to be determined. Department website: https://www.mass.gov/massachusetts- tobacco-cessation-and-prevention- program-mtcp Image: appearance change. Shows photo of a woman with one side as a non-smoker aged 35 and the other side as a smoker of the same age, highlighting the differ- ences and appearance impacts of smoking. Queensland Health, Australia campaign. Image can be viewed at https://www.couriermail.com. au/news/queensland/smoking-makes-you-ugly- queensland-health-to-tell-young-women/news- story/d80e3e017b01d636d692ed884113ef41 Image: Quit tip morning Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitTip Take it 1 day at a time. Every morning say, “I’m not going to smoke today””. Current poster URL not available. Campaign website: https://campaigns.health.gov.au/smokes Image: fish butts Image of internal cross-section of a fish with butts in the stomach cavity, accompanied by a piece of paper which resembles a shopping docket with information about how cigarette butts contaminate water. URL not provided as original source not able to be determined. 12. 14. 13. Page 7 of 21 Hefler et al. BMC Public Health (2019) 19:615 Weekly content options provided to peer researchers (Continued) Option A Option B Option C Image: Quit fact Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “#QuitFact within a day of quitting, almost all the nicotine is out of your bloodstream”. Current poster URL not available. Campaign website: https://web.archive.org/web/ 20190304210745, https://campaigns.health.gov. au/smokes Video: Make smoking history (please help me quit) Produced for the Cancer Council WA Make Smoking History campaign, Western Australia. Campaign website: https://makesmokinghistory.org.au/ https://www.youtube.com/watch?v= 80gkwcx314w Video: lost child Video produced for Cancer Council Victoria, Australia. Information about the ad can be viewed here: https://www. cancervic.org.au/about/media- releases/2008-media-releases/ media-rel-october08/quit- launches-separation-08.html Link to video: https://www. bestadsontv.com/ad/17507/Quit- Victoria-Separation Image: No more killing Image of two burning cigarettes placed upright to resemble the twin towers on fire after the 9/ 11 attacks. Accompanied with the words “NO MORE Killing” and in small type “It is estimated that one person dies every 8 s from smoking. Stop smoking now!” URL not provided as original source not able to be determined. Image: Happy ending Poster with a photo of a young Indigenous girl in a T-shirt printed with an Aboriginal flag, with the caption “Stories about smokers never end with happily ever after”. Current poster URL not available. Poster includes the URL https://www.instagram.com/p/Bso_ iAvlvc4/ Video: Every cigarette rots you from the inside out NHS England Smoke Free campaign https://www.youtube. Data collection Video: Plain packaging Video produced for World Health Organization for World No Tobacco Day 2016, which had the theme “Get ready for plain packaging.” https://www.youtube.com/watch?v= rXUCTSp2_58 Image: pregnant Poster with a photo of a pregnant belly with a lit cigarette held next to it and smoke appearing to come out of the belly button. URL to poster not available, organisation website: www.kindergesundheit.de. Image: squirrels Photo of a squirrel with the wording “Cigarettes are like squirrels: they’re perfectly harmless until you put one in your mouth and light it on fire.” Video: Quit smoking (Arrente) Animated video of a brain explaining the impact of smoking in Arrente, a central Australian Aboriginal language. Image: child labour pack Photo of a an open pack of cigarettes with children inside the pack instead of cigarettes, Video: Don’t make smokes your story. Australian Government campaign, Indigenous focus, with real local people. https://www.youtube.com/ watch?v=em_0c_Yzxyk Image: cancer cures smoking Image of the words “Cancer cures smoking”. No other graphics or pictures are included. Taken from https://www. thefreshquotes.com/50-smoking- and-tobacco-quotes-and-slogans/ cancer-cures-smoking/. Image: pregnant Poster with a photo of a pregnant belly with a lit cigarette held next to it and smoke appearing to come out of the belly button. URL to poster not available, organisation website: www.kindergesundheit.de. Image: child labour pack Photo of a an open pack of cigarettes with children inside the pack instead of cigarettes, Video: Don’t make smokes your story. Australian Government campaign, Indigenous focus, with real local people. https://www.youtube.com/ watch?v=em_0c_Yzxyk Image: cancer cures smoking Image of the words “Cancer cures smoking”. No other graphics or pictures are included. Taken from https://www. thefreshquotes.com/50-smoking- and-tobacco-quotes-and-slogans/ cancer-cures-smoking/. Image: pregnant Poster with a photo of a pregnant belly with a lit cigarette held next to it and smoke appearing to come out of the belly button. URL to poster not available, organisation website: www.kindergesundheit.de. Image: child labour pack Photo of a an open pack of cigarettes with children inside the pack instead of cigarettes, Image: Happy ending Poster with a photo of a young Indigenous girl in a T-shirt printed with an Aboriginal flag, with the caption “Stories about smokers never end with happily ever after”. Current poster URL not available. Poster includes the URL https://www.instagram.com/p/Bso_ iAvlvc4/ Video: I will survive Video produced for HSE Ireland. Campaign website: www.quit.ie. https://www.youtube.com/watch?v= NSETW_zO9jc&feature=youtu.be&list= PLsQK32cdMW_ w6X1OiaNXOjwbD2XKbcS1r Video: Message from the lungs Video produced for the Thai Health Promotion Foundation. Data collection This can prevent you from having an erection”. The image was used by Health Canada as a cigarette warning label, 2010– 2011. The image can be viewed at https://www.who.int/tobacco/ healthwarningsdatabase/ tobacco_large_canada_ impotence_01_en/en/ Video: stop smoking, start repairing Australian Government video to promote the My QuitBuddy app. https://www.youtube.com/ watch?v=e7iGNrdST6E&feature= youtu.be Image: pet 2nd hand smoke Photo of a dog and cat with a sign hanging around their necks with the words “dogs/cats against smoke” and a non- smoking symbol. At the top are the words “Secondhand smoke hurts them too. Keep your whole family safe…keep your car and home smoke-free.” URL not pro- vided as original source not able to be determined. Image: not our culture Option C and in place of the health warning label, the words “Made with child labor”. Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis. This can prevent you from having an erection”. The image was used by Health Canada as a cigarette warning label, 2010– 2011. The image can be viewed at https://www.who.int/tobacco/ healthwarningsdatabase/ tobacco_large_canada_ impotence_01_en/en/ Video: stop smoking, start repairing Australian Government video to promote the My QuitBuddy app. https://www.youtube.com/ watch?v=e7iGNrdST6E&feature= youtu.be Image: pet 2nd hand smoke Photo of a dog and cat with a sign hanging around their necks with the words “dogs/cats against smoke” and a non- smoking symbol. At the top are the words “Secondhand smoke hurts them too. Keep your whole family safe…keep your car and home smoke-free.” URL not pro- vided as original source not able to be determined. Image: not our culture Option C and in place of the health warning label, the words “Made with child labor”. Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis. This can prevent you from having an erection”. The image was used by Health Canada as a cigarette warning label, 2010– 2011. Data collection URL not provided as original source not able to be determined. Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis. This can prevent you from having an erection”. The image was used by Health Canada as a cigarette warning label, 2010– 2011. The image can be viewed at https://www.who.int/tobacco/ healthwarningsdatabase/ tobacco_large_canada_ impotence_01_en/en/ Image: Piggy bank Photo of a child’s piggy bank with cigarette butts stuffed into the slot where money is ll l d A h d ddi i l i Image: dirty lungs Photo of two sets of preserved lungs – one white, one partially blackened due N Video: stop smoking, start repairing Australian Government video to h M Q B dd Hefler et al. BMC Public Health (2019) 19:615 able 1 Weekly content options provided to peer researchers (Continued) eek Option A Option B Option C URL not provided as original source not able to be determined. Available at: http://nosmokes.com.au/teachers-and- health-workers/language-translations/ central-arrernte/ and in place of the health warning label, the words “Made with child labor”. Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms . Video: Tips from former smokers (gum disease) Video produced from the “Tips from Former Smokers” campaign by the USA Centers for Disease Control. https://www.youtube.com/watch?v= y70V5HGhpHs Image: Weight loss Image of the words “Smoking reduces weight” in large print with “(one lung at a time)” in smaller print below. The image has the logo and name of Cancer Patients Aid Association. URL not provided as original source not able to be determined. Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis This can prevent you from 1 Weekly content options provided to peer researchers (Continued) Option A Option B Option C URL not provided as original source not able to be determined. Available at: http://nosmokes.com.au/teachers-and- health-workers/language-translations/ central-arrernte/ and in place of the health warning label, the words “Made with child labor”. Data collection At the top are the words “Secondhand smoke (Continued) Option B Option C ble to Available at: http://nosmokes.com.au/teachers-and- health-workers/language-translations/ central-arrernte/ and in place of the health warning label, the words “Made with child labor”. Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms ease) mer or Image: Weight loss Image of the words “Smoking reduces weight” in large print with “(one lung at a time)” in smaller print below. The image has the logo and name of Cancer Patients Aid Association. URL not provided as original source not able to be determined. Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis. This can prevent you from having an erection”. The image was used by Health Canada as a cigarette warning label, 2010– 2011. The image can be viewed at https://www.who.int/tobacco/ healthwarningsdatabase/ tobacco_large_canada_ impotence_01_en/en/ te s rette ds are d he ght to be Image: dirty lungs Photo of two sets of preserved lungs – one white, one partially blackened due to tar. Not caption or accompanying text. URL not provided as original source not able to be determined. Video: stop smoking, start repairing Australian Government video to promote the My QuitBuddy app. https://www.youtube.com/ watch?v=e7iGNrdST6E&feature= youtu.be with nging mouth. ble to Image: smoking kids Portrait style photograph of a young red-haired girl in a blue dress lighting a cigarette from another one. Image avail- able at http://frieke.com/smoking-kids/. Image: pet 2nd hand smoke Photo of a dog and cat with a sign hanging around their necks with the words “dogs/cats against smoke” and a non- smoking symbol. At the top are the words “Secondhand smoke hurts them too. Keep your whole family safe…keep your car and home smoke-free.” URL not pro- vided as original source not able to be determined. ory) Image: savings Image: not our culture Option C and in place of the health warning label, the words “Made with child labor”. Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis. Data collection https://vimeo.com/126220314 Video: Plain packaging Video produced for World Health Organization for World No Tobacco Day 2016, which had the theme “Get ready for plain packaging.” https://www.youtube.com/watch?v= rXUCTSp2_58 Video: Quit smoking (Arrente) Animated video of a brain explaining the impact of smoking in Arrente, a central Australian Aboriginal language. Image: No more killing Image of two burning cigarettes placed upright to resemble the twin towers on fire after the 9/ 11 attacks. Accompanied with the words “NO MORE Killing” and in small type “It is estimated that one person dies every 8 s from smoking. Stop smoking now!” URL not provided as original source not able to be determined. Image: smoking gun Black and white photo of a hand holding a cigarette against a blank wall, with the shadow resembling a pointed gun. URL not provided as original source not able to be determined. Image: poor swimmers Photo of used matches resembling sperm swimming in an ashtray towards ash (representing an ovum). Produced for ASH UK, image can be viewed at https://www.flickr.com/ photos/ashuk/4459586151. Image: baby bottle Photo of a baby’s bottle half-filled with cigarette butts and smoking coming out of the top, ac- companied with the words “How many ciga- rettes a day does your child smoke?” at the top, and “Prevent passive smoking” in smaller letters at the bottom. URL not provided as original source not able to be determined. Image: squirrels Photo of a squirrel with the wording “Cigarettes are like squirrels: they’re perfectly harmless until you put one in your mouth and light it on fire.” 17. 20. 16. 18. 19. Hefler et al. BMC Public Health (2019) 19:615 Page 8 of 21 ble 1 Weekly content options provided to peer researchers (Continued) ek Option A Option B Option C URL not provided as original source not able to be determined. Available at: http://nosmokes.com.au/teachers-and- health-workers/language-translations/ central-arrernte/ and in place of the health warning label, the words “Made with child labor”. Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms Video: Tips from former smokers (gum disease) Video produced from the “Tips from Former Smokers” campaign by the USA Centers for Disease Control. https://www.youtube.com/watch?v= y70V5HGhpHs Image: Weight loss Image of the words “Smoking reduces weight” in large print with “(one lung at a time)” in smaller print below. The image has the logo and name of Cancer Patients Aid Association. Data collection Image can be viewed at https://www.hrw.org/ news/2014/05/14/us-child- workers-danger-tobacco-farms Video: Tips from former smokers (gum disease) Video produced from the “Tips from Former Smokers” campaign by the USA Centers for Disease Control. https://www.youtube.com/watch?v= y70V5HGhpHs Image: Weight loss Image of the words “Smoking reduces weight” in large print with “(one lung at a time)” in smaller print below. The image has the logo and name of Cancer Patients Aid Association. URL not provided as original source not able to be determined. Image: impotent Photo of a partially burned cigarette half ash, with the word “Warning” in red, followed by “Tobacco use can make you impotent” in large letters, and in smaller letters “Cigarettes may cause sexual impotence tdue to decreased blood flow to the penis. This can prevent you from having an erection”. The image was used by Health Canada as a cigarette warning label, 2010– 2011. The image can be viewed at https://www.who.int/tobacco/ healthwarningsdatabase/ tobacco_large_canada_ impotence_01_en/en/ Image: Piggy bank Photo of a child’s piggy bank with cigarette butts stuffed into the slot where money is usually placed. Ash and an additional cigarette butt are around the piggy bank. The words “Daddy couldn’t give me pocket money” are below the image in handwritten, coloured crayon style. The image has the logo of the British Heart Foundation in the bottom right hand corner, current poster URL not able to be located. Image: dirty lungs Photo of two sets of preserved lungs – one white, one partially blackened due to tar. Not caption or accompanying text. URL not provided as original source not able to be determined. Video: stop smoking, start repairing Australian Government video to promote the My QuitBuddy app. https://www.youtube.com/ watch?v=e7iGNrdST6E&feature= youtu.be Image: smoke mouth Stylised mage of a woman’s open mouth with glossy bright lipstick. The mouth is full of cigarette butts, and there is a cigarette hanging from the mouth with the ash end in the mouth. URL not provided as original source not able to be determined. Image: smoking kids Portrait style photograph of a young red-haired girl in a blue dress lighting a cigarette from another one. Image avail- able at http://frieke.com/smoking-kids/. Image: pet 2nd hand smoke Photo of a dog and cat with a sign hanging around their necks with the words “dogs/cats against smoke” and a non- smoking symbol. Data collection Video: Tobacco, a threat to development Video produced for World Health Organization for World No Tobacco Day 2016, which had the theme “Tobacco: a threat to development.” https://www.youtube.com/ watch?v=B9xqy6g8SIw Video: Miwatj – cost of smoking Video created by Aboriginal community controlled health service for World No Tobacco Day 2017. https://vimeo.com/220741839 Video: Apunipima – Selena’s story Video produced by a local Aboriginal community controlled health service https://www.youtube.com/watch?v= LMPcoA6cz_Q Image: diabetes Poster with graphic of cigarette on brown background on left side, and finger prick blood test on red background on right side. In the middle, inside a circle ekly content options provided to peer researchers (Continued) Option A Option B Option C Produced for the Cancer Council WA Make Smoking History campaign, Western Australia. Campaign website: https://makesmokinghistory. org.au/ https://www.youtube.com/watch?v= r8yvgQFWObA Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “By quitting smoking, a daily smoker could save $8500 a year”. Current poster URL not available. Campaign website: https:// web.archive.org/web/20190304210745, https://campaigns.health.gov.au/smokes Image of words “Not Our Culture” next to images associated with Aboriginal culture and a cigarette crossed out. Image can be viewed at https://nacchocommunique.com/ tag/ahl/ Image: blindness Photo of a public rubbish bin with top designed to resemble an eye and a cigarette being stubbed out on the iris. Around the bottom of the eye white are the words “Smoking causes blindness” with a Quit logo. URL not provided as original source not able to be determined. Image: baby lungs Poster with a photo of a baby in a light coloured jumpsuit, with blackened out lungs on the jumpsuit. At the bottom are the words “You smoke, your child smokes! Take your smoke outside. By the time he is 6 years old your child will have inhaled the equivalent of 102 packs of cigarettes”. Produced for Tobacco Free Futures (http://www. tobaccofreefutures.org/), current URL for poster not located. Video: Tobacco, a threat to development Video produced for World Health Organization for World No Tobacco Day 2016, which had the theme “Tobacco: a threat to development.” https://www.youtube.com/ watch?v=B9xqy6g8SIw Video: Miwatj – cost of smoking Video created by Aboriginal community controlled health service for World No Tobacco Day 2017. Data collection Image: baby lungs Poster with a photo of a baby in a light coloured jumpsuit, with blackened out lungs on the jumpsuit. At the bottom are the words “You smoke, your child smokes! Take your smoke outside. By the time he is 6 years old your child will have inhaled the equivalent of 102 packs of cigarettes”. Produced for Tobacco Free Futures (http://www. tobaccofreefutures.org/), current URL for poster not located. Video: Tobacco, a threat to development Video produced for World Health Organization for World No Tobacco Day 2016, which had the theme “Tobacco: a threat to development.” https://www.youtube.com/ watch?v=B9xqy6g8SIw 26. Video: Miwatj – cost of smoking Video created by Aboriginal community controlled health service for World No Tobacco Day 2017. https://vimeo.com/220741839 Video: Apunipima – Selena’s story Video produced by a local Aboriginal community controlled health service https://www.youtube.com/watch?v= LMPcoA6cz_Q Image: diabetes Poster with graphic of cigarette on brown background on left side, and finger prick blood test on red background on right side. In the middle, inside a circle Weekly content options provided to peer researchers (Continued) Option A Option B Option C Produced for the Cancer Council WA Make Smoking History campaign, Western Australia. Campaign website: https://makesmokinghistory. org.au/ https://www.youtube.com/watch?v= r8yvgQFWObA Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “By quitting smoking, a daily smoker could save $8500 a year”. Current poster URL not available. Campaign website: https:// web.archive.org/web/20190304210745, https://campaigns.health.gov.au/smokes Image of words “Not Our Culture” next to images associated with Aboriginal culture and a cigarette crossed out. Image can be viewed at https://nacchocommunique.com/ tag/ahl/ Image: blindness Photo of a public rubbish bin with top designed to resemble an eye and a cigarette being stubbed out on the iris. Around the bottom of the eye white are the words “Smoking causes blindness” with a Quit logo. URL not provided as original source not able to be determined. Image: baby lungs Poster with a photo of a baby in a light coloured jumpsuit, with blackened out lungs on the jumpsuit. At the bottom are the words “You smoke, your child smokes! Take your smoke outside. By the time he is 6 years old your child will have inhaled the equivalent of 102 packs of cigarettes”. Produced for Tobacco Free Futures (http://www. tobaccofreefutures.org/), current URL for poster not located. Data collection The image can be viewed at https://www.who.int/tobacco/ healthwarningsdatabase/ tobacco_large_canada_ impotence_01_en/en/ Video: stop smoking, start repairing Australian Government video to promote the My QuitBuddy app. https://www.youtube.com/ watch?v=e7iGNrdST6E&feature= youtu.be Image: pet 2nd hand smoke Photo of a dog and cat with a sign hanging around their necks with the words “dogs/cats against smoke” and a non- smoking symbol. At the top are the words “Secondhand smoke hurts them too. Keep your whole family safe…keep your car and home smoke-free.” URL not pro- vided as original source not able to be determined. Image: not our culture Image: Piggy bank Photo of a child’s piggy bank with cigarette butts stuffed into the slot where money is usually placed. Ash and an additional cigarette butt are around the piggy bank. The words “Daddy couldn’t give me pocket money” are below the image in handwritten, coloured crayon style. The image has the logo of the British Heart Foundation in the bottom right hand corner, current poster URL not able to be located. Image: smoke mouth Stylised mage of a woman’s open mouth with glossy bright lipstick. The mouth is full of cigarette butts, and there is a cigarette hanging from the mouth with the ash end in the mouth. URL not provided as original source not able to be determined. Video: Make smoking history (Darleen’s story) 22. 24. 23. Page 9 of 21 Hefler et al. BMC Public Health (2019) 19:615 Table 1 Weekly content options provided to peer researchers (Continued) Week Option A Option B Option C Produced for the Cancer Council WA Make Smoking History campaign, Western Australia. Campaign website: https://makesmokinghistory. org.au/ https://www.youtube.com/watch?v= r8yvgQFWObA Image from Australian Government campaign Don’t Make Smokes Your Story, with the words “By quitting smoking, a daily smoker could save $8500 a year”. Current poster URL not available. Campaign website: https:// web.archive.org/web/20190304210745, https://campaigns.health.gov.au/smokes Image of words “Not Our Culture” next to images associated with Aboriginal culture and a cigarette crossed out. Image can be viewed at https://nacchocommunique.com/ tag/ahl/ 25. Image: blindness Photo of a public rubbish bin with top designed to resemble an eye and a cigarette being stubbed out on the iris. Around the bottom of the eye white are the words “Smoking causes blindness” with a Quit logo. URL not provided as original source not able to be determined. Data collection https://vimeo.com/220741839 Video: Apunipima – Selena’s story Video produced by a local Aboriginal community controlled health service https://www.youtube.com/watch?v= LMPcoA6cz_Q Image: diabetes Poster with graphic of cigarette on brown background on left side, and finger prick blood test on red background on right side. In the middle, inside a circle made up of 2 arrows are the words “Smokers have a 30 to 40% higher risk of diabetes than nonsmokers”. The poster has a US Centers for Disease Control and one other logo, and has appeared in campaigns and on social media for several US health groups. (Eg see https:// twitter.com/tobaccofreekids/ status/978745983923142657 and http://www.coprevent.org/2014/ 11/national-diabetes-month- smoking-and.html). URL not provided as original link not able to be determined. 26. 26. 25. Hefler et al. BMC Public Health (2019) 19:615 Page 10 of 21 Table 2 Content options by popularity No. of participants for week Post ID (week/ choice) Title No. Data collection of participants who posted this content option % of participants who posted this content option Total number of posts by participants for the weeka Content characteristics/ themes 10 WEEK 12A Appearance change: IMAGE 9 90 14 Personal impact, visible damage 10 Week 6A Thai Smoking Kids: VIDEO 8 80 12 Kid focused, unique/fresh take on message, role modelling 9 WEEK 13A Quit Tip Morning: IMAGE 8 89 13 Practical, quit tip 11 Week 4A Don’t Make Smokes yr story: VIDEO 7 64 12 Testimonial, Aboriginal, family/kids focus, positive 8 Week 21C Impotent: IMAGE 7 88 15 Humorous approach, sexual health, male focus 8 Week 26A Miwatj money: VIDEO 7 88 14 Personal, Local NT Aboriginal (Yolngu) content, finance 12 Week 5B Quit Timeline: VIDEO 6 50 14 Positive, benefits of quitting 9 Week 7B Child Labour Tobacco: VIDEO 6 67 13 Child-focused, new information, targets industry 8 WEEK 11B Alice Springs: VIDEO 6 75 12 Testimonial, Local NT Aboriginal content, family/ kids focus, positive 9 WEEK 15A Quit Fact: IMAGE 6 67 13 Positive benefits/fact re quitting 8 WEEK 16B Happy Ending: IMAGE 6 75 12 Aboriginal content, child focused 9 Week 22C Quit Buddy App: VIDEO 6 67 15 Practical, quit tip, based on personal experience 8 Week 24B Savings: IMAGE 6 75 14 Financial focus 9 Week 25A Blindness: IMAGE 6 67 14 Health impact (new/lesser known) 12 Week 5C Dream boy: IMAGE 5 42 14 Aboriginal content, child focused 8 Week 8B Culture or Killer: VIDEO 5 63 10 Aboriginal content, culture focus 7 Week 10A Quit Steps: IMAGE 5 71 11 Practical quit tips 8 WEEK 11C Trigger: IMAGE 5 63 12 Practical quit tips 8 WEEK 14B Four D’s: IMAGE 5 63 11 Practical quit tips 9 WEEK 15C Lost Child: VIDEO 5 56 13 Child-focused, family 8 Week 18A Poor Swimmers: IMAGE 5 63 9 Humorous/fresh approach, sexual health, male focus 8 Week 20C Child labour pack: IMAGE 5 63 11 Child-focused, new information, targets industry 8 Week 21B Weightloss: IMAGE 5 63 15 Negative, health impacts, non-obvious message Table 2 Content options by popularity No. of participants for week Post ID (week/ choice) Title No. Data collection of p who po content 9 Week 25B Baby lungs: IMAGE 5 8 Week 26B Selena Story: VIDEO 5 8 Week 1C Lifemeter: IMAGE 4 11 Week 3B 5 Things, Tobacco Industry: VIDEO 4 11 Week 3C Smokers teeth: IMAGE 4 9 Week 7A Everybody Knows, Quitline: VIDEO 4 8 Week 9B Damage Lesson: VIDEO 4 7 Week 10B Start Repairing: IMAGE 4 8 Week 14A Fish Butts: IMAGE 4 8 Week 16A No More Killing: IMAGE 4 9 Week 17B I Will Survive: VIDEO 4 9 Week 17C Kirra Story: VIDEO 4 9 Week 19A Baby bottle: IMAGE 4 9 Week 19C Pregnant: IMAGE 4 8 Week 20B Arrernte: VIDEO 4 9 Week 22B Dirty Lungs: IMAGE 4 8 Week 23C Pet 2nd hand smoke: IMAGE 4 8 Week 24A Personal story: VIDEO 4 8 Week 24C Not our culture: IMAGE 4 8 Week 1A #Catmaggedon: VIDEO 3 8 Week 2B Bolypingu, Skinnyfish: VIDEO 3 8 Week 2C Smoking burns money: IMAGE 3 11 Week 3A Break the Chain: VIDEO 3 11 Week 4B Quit Smoking, Allen Carr:VIDEO 3 12 Week 5A Smoking Kills, Bryan Curtis: 3 Table 2 Content options by popularity (Continued) No. of participants for week Post ID (week/ choice) Title No. Data collection of participan who posted this content option 10 WEEK 12A Appearance change: IMAGE 9 10 Week 6A Thai Smoking Kids: VIDEO 8 9 WEEK 13A Quit Tip Morning: IMAGE 8 11 Week 4A Don’t Make Smokes yr story: VIDEO 7 8 Week 21C Impotent: IMAGE 7 8 Week 26A Miwatj money: VIDEO 7 12 Week 5B Quit Timeline: VIDEO 6 9 Week 7B Child Labour Tobacco: VIDEO 6 8 WEEK 11B Alice Springs: VIDEO 6 9 WEEK 15A Quit Fact: IMAGE 6 8 WEEK 16B Happy Ending: IMAGE 6 9 Week 22C Quit Buddy App: VIDEO 6 8 Week 24B Savings: IMAGE 6 9 Week 25A Blindness: IMAGE 6 12 Week 5C Dream boy: IMAGE 5 8 Week 8B Culture or Killer: VIDEO 5 7 Week 10A Quit Steps: IMAGE 5 8 WEEK 11C Trigger: IMAGE 5 8 WEEK 14B Four D’s: IMAGE 5 9 WEEK 15C Lost Child: VIDEO 5 8 Week 18A Poor Swimmers: IMAGE 5 8 Week 20C Child labour pack: IMAGE 5 8 Week 21B Weightloss: IMAGE 5 9 Week 22A Piggy Bank: IMAGE 5 8 Week Smoke mouth: 5 Table 2 Content options by popularity Hefler et al. BMC Public Health (2019) 19:615 Page 11 of 21 Table 2 Content options by popularity (Continued) No. of participants for week Post ID (week/ choice) Title No. Data collection of participants who posted this content option % of participants who posted this content option Total nu by partic weeka 9 Week 25B Baby lungs: IMAGE 5 56 14 8 Week 26B Selena Story: VIDEO 5 63 14 8 Week 1C Lifemeter: IMAGE 4 50 8 11 Week 3B 5 Things, Tobacco Industry: VIDEO 4 36 11 11 Week 3C Smokers teeth: IMAGE 4 36 11 9 Week 7A Everybody Knows, Quitline: VIDEO 4 44 13 8 Week 9B Damage Lesson: VIDEO 4 50 11 7 Week 10B Start Repairing: IMAGE 4 57 11 8 Week 14A Fish Butts: IMAGE 4 50 11 8 Week 16A No More Killing: IMAGE 4 50 12 9 Week 17B I Will Survive: VIDEO 4 44 11 9 Week 17C Kirra Story: VIDEO 4 44 11 9 Week 19A Baby bottle: IMAGE 4 44 11 9 Week 19C Pregnant: IMAGE 4 44 11 8 Week 20B Arrernte: VIDEO 4 50 11 9 Week 22B Dirty Lungs: IMAGE 4 44 15 8 Week 23C Pet 2nd hand smoke: IMAGE 4 50 11 8 Week 24A Personal story: VIDEO 4 50 14 8 Week 24C Not our culture: IMAGE 4 50 14 8 Week 1A #Catmaggedon: VIDEO 3 38 8 8 Week 2B Bolypingu, Skinnyfish: VIDEO 3 38 8 8 Week 2C Smoking burns money: IMAGE 3 38 8 11 Week 3A Break the Chain: VIDEO 3 27 11 11 Week 4B Quit Smoking, Allen Carr:VIDEO 3 27 12 12 Week 5A Smoking Kills, Bryan Curtis: 3 25 14 Table 2 Content options by popularity (Contin No. of participants for week Post ID (week/ choice) Title No. Data collection of participants who posted this content option % of participants who posted this content option Total n by part weeka 10 Week 6C Smokers Funeral: IMAGE 3 30 12 9 Week 7C Cup of butts: IMAGE 3 33 13 8 Week 8C Quit gains: IMAGE 3 38 10 8 Week 9A Exercise: VIDEO 3 38 11 8 Week 9C Stress: IMAGE 3 38 11 10 WEEK 12B Quit Tip: IMAGE 3 30 14 9 WEEK 13B Timebomb: IMAGE 3 33 13 9 WEEK 17A Smoking Gun: IMAGE 3 33 11 8 Week 18C Cancer cures smoking: IMAGE 3 38 9 9 Week 19B Plain packaging: VIDEO 3 33 11 8 Week 20A Squirrels: IMAGE 3 38 11 8 Week 21A Gum Disease: VIDEO 3 38 15 9 Week 25C World No Tobacco Day: VIDEO 3 33 14 8 Week 2A Quit for you, Quit for Two: VIDEO 2 25 8 11 Week 4C Smoking good for environment: IMAGE 2 18 12 8 Week 8A Smoking and drinking: VIDEO 2 25 10 7 Week 10C Real Cost - Your skin: VIDEO 2 29 11 10 Week 12C Smoking sarcasm: IMAGE 2 20 14 9 Week 13C Butts are Litter: IMAGE 2 22 13 8 Week 14C Sugar Sugar: VIDEO 2 25 11 9 Week 15B Smoking History: VIDEO 2 22 13 8 Week 16C Rotting: VIDEO 2 25 12 8 Week 23B Smoking kids: IMAGE 2 25 11 8 Week 26C Diabetes: IMAGE 2 25 14 8 Week 1B 1200 people die: VIDEO 1 13 8 Table 2 Content options by popularity (Continued) No. of participants for week Post ID (week/ choice) Title No. Data collection of participants who posted this content option % of participants who posted this content option Total number of posts by participants for the weeka Content characteristics/ themes 9 Week 25B Baby lungs: IMAGE 5 56 14 Child-focused, protection from smoke impact 8 Week 26B Selena Story: VIDEO 5 63 14 Testimonial, Aboriginal content, family/kids focus, Top End 8 Week 1C Lifemeter: IMAGE 4 50 8 Negative, health impacts 11 Week 3B 5 Things, Tobacco Industry: VIDEO 4 36 11 Negative, health industry focus, uninteresting thumbnail 11 Week 3C Smokers teeth: IMAGE 4 36 11 Negative, appearance focused 9 Week 7A Everybody Knows, Quitline: VIDEO 4 44 13 Negative, health impacts 8 Week 9B Damage Lesson: VIDEO 4 50 11 Negative, health impacts 7 Week 10B Start Repairing: IMAGE 4 57 11 Health impacts 8 Week 14A Fish Butts: IMAGE 4 50 11 Environmental focus, negative 8 Week 16A No More Killing: IMAGE 4 50 12 Indirect/non-obvious message 9 Week 17B I Will Survive: VIDEO 4 44 11 Negative, health impacts 9 Week 17C Kirra Story: VIDEO 4 44 11 Local content, testimonial, child focused 9 Week 19A Baby bottle: IMAGE 4 44 11 Negative, indirect 9 Week 19C Pregnant: IMAGE 4 44 11 Pregnancy focus 8 Week 20B Arrernte: VIDEO 4 50 11 Local content 9 Week 22B Dirty Lungs: IMAGE 4 44 15 Gross, biomedical focus 8 Week 23C Pet 2nd hand smoke: IMAGE 4 50 11 Pet focused 8 Week 24A Personal story: VIDEO 4 50 14 Local content, testimonial 8 Week 24C Not our culture: IMAGE 4 50 14 Aboriginal, culture focus 8 Week 1A #Catmaggedon: VIDEO 3 38 8 Pet focused, humorous 8 Week 2B Bolypingu, Skinnyfish: VIDEO 3 38 8 Local Aborigjnal content, indirect message 8 Week 2C Smoking burns money: IMAGE 3 38 8 Financial focus, cartoon 11 Week 3A Break the Chain: VIDEO 3 27 11 Aboriginal focus, testimonial 11 Week 4B Quit Smoking, 3 27 12 How to quit focus Table 2 Content options by popularity (Continued) Aboriginal focus, testimonial Hefler et al. BMC Public Health (2019) 19:615 Page 12 of 21 Table 2 Content options by popularity (Continued) No. of participants for week Post ID (week/ choice) Title No. Data collection of participants who posted this content option % of participants who posted this content option Total number of posts by participants for the weeka Content characteristics/ themes 10 Week 6C Smokers Funeral: IMAGE 3 30 12 Negative, unclear message 9 Week 7C Cup of butts: IMAGE 3 33 13 Negative, focus on taste/smell 8 Week 8C Quit gains: IMAGE 3 38 10 Indigenous content, unclear message 8 Week 9A Exercise: VIDEO 3 38 11 Practical tips 8 Week 9C Stress: IMAGE 3 38 11 Indirect, stress focus, unclear message 10 WEEK 12B Quit Tip: IMAGE 3 30 14 Practical tips 9 WEEK 13B Timebomb: IMAGE 3 33 13 Negative focus 9 WEEK 17A Smoking Gun: IMAGE 3 33 11 Negative, unclear/indirect message 8 Week 18C Cancer cures smoking: IMAGE 3 38 9 Sarcasm, negative 9 Week 19B Plain packaging: VIDEO 3 33 11 Official, tobacco packaging focus 8 Week 20A Squirrels: IMAGE 3 38 11 ‘Silly’ humour 8 Week 21A Gum Disease: VIDEO 3 38 15 Negative, health impacts, gross 9 Week 25C World No Tobacco Day: VIDEO 3 33 14 Boring, no new information 8 Week 2A Quit for you, Quit for Two: VIDEO 2 25 8 Pregnancy focus 11 Week 4C Smoking good for environment: IMAGE 2 18 12 Environment focus, sarcastic, insensitive, cruel 8 Week 8A Smoking and drinking: VIDEO 2 25 10 Health impacts, biomedical 7 Week 10C Real Cost - Your skin: VIDEO 2 29 11 Cosmetic impacts, gross 10 Week 12C Smoking sarcasm: IMAGE 2 20 14 Cartoonish, potentially hypocritical 9 Week 13C Butts are Litter: IMAGE 2 22 13 Environmental focus, message not important 8 Week 14C Sugar Sugar: VIDEO 2 25 11 health damage, sad and tragic 9 Week 15B Smoking History: VIDEO 2 22 13 Clashes with idea of respect for others, quitting own responsibility 8 Week 16C Rotting: VIDEO 2 25 12 health impacts, gross 8 Week 23B Smoking kids: IMAGE 2 25 11 Indirect, Image looked like it was promoting kids smoking 8 Week Diabetes: IMAGE 2 25 14 Health impacts Table 2 Content options by popularity (Continued) Hefler et al. BMC Public Health (2019) 19:615 Page 13 of 21 Table 2 Content options by popularity (Continued) No. of participants for week Post ID (week/ choice) Title No. Results Thai Health Promotion ad with subtitles (week 6, option A). It shows footage of young children asking for a light from adult smokers, all of whom refuse and cite a range of reasons why the children should not smoke. The chil- dren then hand a note to the adults, reminding them that these reasons apply equally to themselves. It was seen to have universal relevance, evoked strong emo- tions, and highlighted for many people the importance of both role modelling and being consistent in their ac- tions and advice to young people about smoking. This post was shared by eight of the 10 participants who posted that week. The average number of participants for each week of the study was 8.6 (range 7–12). One community researcher withdrew from the study at 5 weeks, and one posted only one once; all others continued for the duration of the study, with some weeks of non-participation decided by individual participants according to their personal circumstances. Data collection of participants who posted this content option % of participants who posted this content option Total number of posts by participants for the weeka Content characteristics/ themes 10 Week 6B Social Farting/ Smoking: VIDEO 1 10 12 Humorous, impolite, crass 8 Week 11A Pet Smoking: IMAGE 1 13 12 Pet focused 8 Week 18B Black Ink: VIDEO 1 13 9 Obscure message, thumbnail not appealing aThe total number of posts exceeds the number of participants in some weeks. This is due to some participants choosing to post more than one content option for that week Table 2 Content options by popularity (Continued) Characteristics of highly-posted content Overall, the most popular posts were child-focused, closely followed by Aboriginal-focused posts, with these two themes frequently overlapping. Practical information in the form of quit tips was also a popular choice. Table 2 shows posts by popularity. (Gammon is used in the Australian Indigenous context meaning to joke or make a token gesture) Content Seventy eight content options were offered: three per week for 26 weeks. Each option was offered only once. In the first 3 weeks of the study, the total number of posts matched the number of participants (for example eight participants each posted once for a total of eight posts). Thereafter, in every week, the total number of posts exceeded the number of participants (i.e. some par- ticipants posted more than one option). No participant created their own content, and only one posted content (once) not supplied by the research team. All other posts were taken from the content choices provided. “I have always told my sister’s children not to smoke...it was a good case of do as I say not as I do...my coughing around them was, I thought, testament to that and I thought my li’l gammon admonishments were enough. No good ...two out of the five children smoke...I did what I could at the time. Three outta five ain’t bad. But at the time of turning 18...it’s an adult choice. But if I found my 14 year old niece smoking...I think I would cry. My niece always used to look at me quizzically as to me telling her not to smoke and puffing away same time. This time I want to prove to her I’m important too...and I shouldn’t smoke either.” (D6, discussing week 6 option A) Health impacts, gross imagery The posts that were least shared about health impacts tended to have a biomedical focus, were not considered en- gaging or did not present new information. ‘Gross’ content (posts which used imagery participants considered disgust- ing to communicate the harms of smoking) generated very negative responses which turned people off posting. A clear example was week 16 option C, a video showing that ‘every cigarette rots you from the inside out’ which was seen as disgusting, and without a strong motivating element or new information. Similarly, content about how smoking In this case, the personal connection of the message to D1’s own family was an important motivator for posting. Child-focused messages The impact of having children in messages was per- haps best encapsulated by a former smoker: Child-focused messages included: the importance of chil- dren and family as a motivator for quitting, the need to protect children from tobacco smoke, and tobacco indus- try exploitation of child labour. Discussions with peer re- searchers throughout the project highlighted that a focus on children is a non-threatening way to communicate messages - even smokers who don’t want to quit can agree on the message of valuing and protecting children. “I think using children and emotions is the best thing to tackle that because when I talk to a lot of my friends about why they gave up smoking, and why I gave up smoking, the reason is because of our children.” (D4) While the participants all reported valuing and feeling comfortable sharing messages about children, there was some child-focused content that was poorly received and not selected for sharing. Among the less popular content While an Aboriginal and/or local focus was valued by participants and made it more likely that a post would be shared, it was not a necessary pre-condition for shar- ing child-focused messages. The most shared post was a Hefler et al. BMC Public Health (2019) 19:615 Page 14 of 21 Page 14 of 21 Page 14 of 21 featuring children were advertisements in which the anti-smoking or ‘protect children’ message was not explicit. One post option in particular - a parody of a tobacco adver- tisement featuring a child imitating adults by smoking (week 23 option B)– was highlighted by a participant as looking like it “was designed to promote smoking to children”. (D7) perhaps reflective of the fact that many participants were smokers who wanted to quit. Information that people could use to change behaviour was highly valued, particu- larly if it had been personally helpful and was therefore considered credible. It was less important that quit tips contained new information than for other types of con- tent. If community researchers were already aware of a tip, it was considered a good reminder/reinforcement for their own behaviour, and also helpful to share with others. Indirect, obscure and sarcastic messages Messages which had an unexpected twist in the message were understood in different ways by different people, and frequently misinterpreted. For example, week 21 option B ‘Smoking reduces weight’ (with ‘one lung at a time’ in small print) was understood by several people as simply being a straightforward message that smoking can be helpful for keeping weight off. Similarly, week 20 option A – likening cigarettes to squirrels because both are only dangerous if you put them in your mouth and set them on fire – was seen as silly, but without a clear anti-smoking message. Content featuring an Indigenous person, telling a story that was relatable and perceived to be genuine, were popular regardless of a local connection, as demon- strated by D1, discussing the reasons for posting content from another state: “The reason for me to choose this option was to connect followers from [my home area]. Her story people I know could relate to, non-smoker being surrounded by people who are smoking, ended up smoking because of people around her.” (D1, discussing week 26, option C) Characteristics of least popular content p p The least popular content featured sarcastic, indirect or obscure messages. Content considered likely to have the potential for shame, embarrassment or disgust was also shared less frequently, as was content which placed less importance on people such as the environmental impact of tobacco, or the effect of second-hand smoke on pets. If people had a personal connection to the person fea- tured (for example, it featured a person known in their friendship or family circles), the importance of supporting local community, and encouraging others was an import- ant motivator for posting - sometimes above the smoke-free message. However, personal connections alone were not sufficient –the content still needed to be per- ceived as relevant to the lives of both the community re- searchers themselves and people within their networks. Aboriginal and local content An Aboriginal focus, or content produced in the North- ern Territory, was seen as more relatable and relevant to people’s lives than other content. However, an Indigen- ous or Northern Territory focus was itself not sufficient – if the content was not perceived to be high quality, credible or relevant, it was less likely to be shared. Sev- eral messages which had an Indigenous focus were less popular for these reasons. Credibility was particularly important – for example, one Aboriginal-specific option was rejected by several participants who were aware that the woman featured was an actress rather than a person discussing her real life, particularly if they believed she was a never-smoker. “As a smoker, I personally experience and became aware of the triggers for me. Triggers seem to be habits for me. A few triggers for me is, jumping in the car to drive and after a meal or having a coffee. I would normally light up a cigarette after these activities… I have cut back dramatically; in turn I don’t drink as much tea and coffee. I have inadvertently cut back on tea and coffee because they go hand in hand with my smoking. I think people would benefit from little reminders like this image to help break the habit.” (D7, discussing week 11, option 7) “The other image with the woman telling ‘ her story’. I know the actor and she has never smoked so that illusion didn’t work for me.” (D6) Environmental focus New/different information, well presented Information that was considered to be new was particu- larly important for posts about health impacts of smok- ing; for example the connection between blindness and smoking (week 25, option A). Posts about the impact on sexual health (for example, week 21 option C – impo- tence, and week 18 option A – poor swimmers), were also chosen for this reason. Conversely, some content about smoking in pregnancy was considered to be re- peating a message that participants believed already re- ceives disproportionate attention, and was perceived as potentially adding more stress at a time that can already be difficult for some women. Many participants had close personal experience of smoking during pregnancy, and appreciated the complexity of the lives of women who may find it difficult to quit at that time. Content which focused on the environmental impact of cigarettes and tobacco were typically not popular, largely being seen as a secondary issue to human health. A direct, but sarcastic, message that ‘smoking is good for the envir- onment’ (because it kills people) (Week 4 Option C) was rejected because of both the sarcasm, and the fact that it appeared to value the environment over people. “I felt that this image wasn’t aiming at the effects of smoking with our bodies but aimed at the environment…I didn’t think it would be effective enough for my targeted audience, unless we were more worried about our surroundings more than our health for our bodies…My focus though is us humans, the effects of cigarette smoking on the body.”(A1) Presentation was particularly important; some posts were considered to have good information and content, but were not chosen because they were presented in a way that was boring, not relevant to peoples’ lives, or the message was too long (for videos). The thumbnails and titles for videos were also very important - if they were not clearly related to the topic or sparked interest, they were usually not selected. Participants noted they would not have watched some videos if they were not involved in the project – for example, the ‘1200 people die’ video (week 1 option B), which depicts the fact that 1200 people die from smoking each day in the USA, in a portrayal reminiscent of a mass terrorist event. Several participants remarked they would not have realised it was related to smoking from the thumbnail and title. Environmental focus Similarly, one participant chose week 14 option A showing a fish full of cigarette butts. However, the reason was that the participant enjoys eating fish as part of a healthy diet: “I love cooking fish and I’d be angry if I would find my fish full of cigarettes” (D1) Practical, useful information Quit tips were well regarded because participants reported that people know the health impacts of smoking and more attention needs to be paid to helping in a practical way – Page 15 of 21 Page 15 of 21 Hefler et al. BMC Public Health (2019) 19:615 Hefler et al. BMC Public Health (2019) 19:615 appropriate to post to influence others. It was important to this participant to keep their own quit journey private. affects lungs and general ‘smoking kills’ or images of sick people were less likely to be shared. affects lungs and general ‘smoking kills’ or images of sick people were less likely to be shared. Decision-making Apart from the content itself, several other factors and processes influenced decision-making. It should be noted that in some cases the reasons for why a post was chosen were clearer than why some posts weren’t chosen. In some weeks, people indicated that they would have been happy to share all options, but did not want to post multiple times in 1 week to avoid tobacco-related posts dominating their feed. In other weeks, the posts overall had little ap- peal, and it was a case of choosing the ‘least-worst’ option. In contrast, some participants avoided posting content that had the most impact on them personally, particularly if they were smokers: Credibility, personal relevance and experience Credibility, personal relevance and experience Messages that had personal relevance and credibility were seen as authentic and were highly valued by partic- ipants, both when deciding what to share, as well as when discussing the personal impact of the content on themselves. This was true for participants at different stages of their quit journeys. “I believe getting real life stories from smokers telling their journey to quitting is inspirational and works. Real people, real stories, real victories.” (A1, trying to quit and interested in motivating others.) “Just watched the video and wow that scared me! I wouldn’t have shared it. It does have a strong message but me as a smoker trying to quit its conflicting to share…I know that the consequences and reality of this are real but so real it scares me...maybe if I wasn’t a smoker I would share it to encourage people to give up.” (A3, current smoker trying to quit) “[Good to] post onto my timeline with Facebook because telling yourself I’m not going to smoke today is a great motivational seed to plant so you think twice before smoking during the day.” (A5, reflecting on what had been personally helpful to quit and might be useful to others]. Throughout the study this participant reported being strongly impacted by some content in relation to their own quit journey, but separated this from what they considered Page 16 of 21 Hefler et al. BMC Public Health (2019) 19:615 Hefler et al. BMC Public Health (2019) 19:615 Hefler et al. BMC Public Health (2019) 19:615 away and it was similar stories so that’s why I didn’t post cause I didn’t want to make people feel sad.” (A3) Conversely, for some participants, the images that had the most personal impact were less likely to be shared. During a reflection interview at the conclusion of the study, one participant discussed the post that had the most dramatic personal impact, and was the first image recalled when asked about content options offered. The image strongly reminded this person of the loss of a loved one; for this reason they had deliberately not posted it, despite its resonance and the fact that it had helped prompt the person to try to quit smoking. People separated the importance and relevance of messages to their own lives from the expected likely re- action and interactions from their Facebook friends. Alignment of messages with existing online identity There was considerable diversity in the extent to which people considered their existing online identity when de- ciding on appropriate posts. For some participants, their online activity was a mixture of both their personal and professional lives, and this had a significant influence on how they framed messages, as well as when they posted. For others, participation in this study changed their per- ceptions about how social media can be used, and im- pacted significantly on their online identity. Several participants analysed the relevance of content to their own lives and contrasted it with those in their network, particularly in relation to age differences, smoking status and how long different people had been smoking. In some cases, posts were chosen by older par- ticipants that they did not think was relevant to them and others of their age, but might appeal to younger people and help stop them smoking. In other cases, the participant considered that content might be effective in particular age groups, but not for their networks: Participants who had an established online identity tended to choose content based on how it integrated with their timeline, particularly if their online identity was a mix of personal and professional. One participant who has a Facebook profile with a large following con- nected to their professional identity explained that al- though they believed some of the more ‘hard-hitting’ content was likely to be effective, it was incompatible with their own Facebook feed, which was deliberately kept to a friendly and positive tone. “I like to keep my posts…kinda like Facebook-friendly to many family and friends back home.” (D1) That researcher also timed posts for this research project to fit appropriately with a range of other posts throughout the week, and devel- oped a weekly ‘theme’ for their posts, which were posted on the same day each week. “I didn’t share this for a couple of reasons…my friends and family don’t tend to view my quitting videos (unless I specifically tag them) and most of my friends and family is a different target group. I could not fault the video and as mentioned would be really good for young to mid age mothers and fathers groups where the guilt might hit them for their young family.”(D7, discussing week 2 option A) Change over time during project, impact on personal attitude Several participants were nervous and apprehensive about posting content at the start of the study, especially smokers. Some were initially concerned about appearing to be hypocritical by posting messages about smoking, when many in their networks knew they were smokers. Both smokers and non-smokers also expressed concerns about potentially upsetting friends or feared they may be perceived as ‘preaching’, particularly in relation to dis- turbing or confronting images. Some participants be- lieved that sharing the posts might be generating negative reactions which were not reflected in online in- teractions, with some suggesting that they had been ‘un- friended’ by people within their network.. Credibility, personal relevance and experience In some cases, health information was already known by the participant, but they chose it because they thought it was probably not well known by people in their net- works. This was also the case for some quit tips. On the other hand, sometimes the choice of post was deliber- ately chosen by the participant as a reminder to them- selves in their own quitting journeys. Considering potential reactions, likelihood of viewing Considering potential reactions, likelihood of viewing The potential sensitivity of messages for others was an important consideration for some participants. For ex- ample, there was a high level of awareness about when a message might ‘hit a bit too close to home’ for people dealing with a recent bereavement, especially for images that were particularly realistic. Certain times of the year also influenced choices; Christmas was considered a time when people may be thinking of absent family and friends, and therefore an important time to be sensitive to issues of grief and loss. “Some people have said, oh boring you’re not the same…..cause usually….I used to be putting up all the jokes…I think this [participation in this study] has changed me….I’ll put up a joke every now and then…but “…it was just too sad for me...it’s the harsh reality I know but I just didn’t feel comfortable posting it. So many people over the Christmas period were posting how much they missed their mums who have passed Page 17 of 21 Page 17 of 21 Hefler et al. BMC Public Health (2019) 19:615 its more the serious side of things….Close to 10 people have unfriended me” (A1) and sometimes for participants themselves. At times, this meant that longer videos were less likely to be shared, even if the content was considered good and interesting. One participant highlighted this as an issue that they considered when deciding whether to post a video about the (Austra- lian government initiative) My Quit Buddy app: However, over time, some people became bolder and less concerned about their choices, particularly when they did not receive expected negative reactions. In these cases, there was a greater willingness to post messages that might be considered confronting and harsh as the study progressed. Several found that the reactions from their networks were ei- ther minimal or more positive than expected, and they be- came more willing to post confronting images. Consistent with this, some people started to post more than once per week, and also included personal messages to try and gener- ate reactions. This was consistent with reports from friends and family within participants’ social networks in face-to-face interviews. Overall, friends and family reported that they per- ceived posts as being contextualised within the relationship they had with participants, generally as part of caring rela- tionships. Discussion This study has used community researchers to dissemin- ate smoking prevention messages through their existing Facebook networks. The results show that posts which are child-focused, feature Indigenous content, and are perceived as practical, relevant and credible, with a dir- ect and unambiguous message, are the most likely to be shared. Indirect, obscure, sarcastic and disgusting con- tent without a clear message were less likely to be shared. Given that social media largely shifts the power to consumers to decide what content will be seen, this provides important lessons for planning social media- based smoking prevention strategies. The extent to which people were posting for their own purposes, rather than with the intention of influencing others, also changed over the course of the project. Particularly for participants who were trying to quit smoking, choices some- times reflected a particularly critical time in their quit journey – for example, posting quit tips that were a reminder for themselves of practical steps they needed to take. “After watching a video I noticed the cigarette tastes really funny. It does something and I don’t feel like smoking so much. Could be subconsciously…I’ve mentioned that to my [spouse]…” (A1, reflecting on videos viewed during the project) The popularity of child-focused messages might reflect the ambivalence many participants felt about at different stages of their participation in the study. Particularly for smokers, content focused on children represented ‘safe ground’ for posting, did not raise potential issues of hyp- ocrisy, and reflects priorities that are common across most demographics, regardless of smoking status. In general, new information or a ‘fresh take’ on information was pre- ferred; this was particularly true for some of the child-fo- cused content. A good example was the popularity of the Thai Health advertisement; participants suggested that an Australian version of this ad featuring Indigenous people would be very well received, and likely to generate high levels of engagement. The study reinforces that inter- national tobacco control content can be adapted for local messages on social media, as has been done over many years for mass media campaigns [41, 42]. For those participants who were finding it challenging to quit, the desire to post confronting images was often con- nected to their own perceived need for greater shock tactics. “I picked the image I did because it was the most shocking and hardcore. I have to take this more serious. Considering potential reactions, likelihood of viewing Often, they gave no visible indication of interacting with posts, even if the post was personally impactful (to be reported separately; manuscript in preparation). “Although this is a beneficial video promoting the Quit Buddy app, that can be downloaded for free & has helped a lot of people I know of…. I’m thinking some of my family & friends have cheap phones and we love our music, videos, games and not to mention selfies, making no storage space on our phones and even with a memory card that space is used up. I’ve noticed this with a lot of young people in my family. Otherwise a great interactive app to have.” (A1, discussing week 22 option C) Discussion I’m traumatised by the image as I imagine many of my [Facebook friends and family] are. And I guess it’s one of my strategic moves to help me on my journey to quit. I need to. I didn’t choose the others because they weren’t as direct. I may change my mind and say it’s too close to home. My mum died of lung cancer at 66 years old. I am so scared. I need help.” (D6, discussing week 1 option C) The results raise questions about the potential effective- ness of using social media to disseminate confronting messages that arouse strong negative emotions, which have been proven to be successful in traditional media [33]. Although limited, previous research has found that Impact on phone credit/data usage An important finding is the reported strong personal impact of some messages on participants, which con- trasts with the decision to not post these same impactful messages based on concern for potential sensitivities among their social media friends. This appears to be due to two separate processes. On the one hand, participants who were smokers appeared to be challenged about their own smoking, through the process of having to engage intensively with the material provided. y p On the other hand, when deciding what messages to share, participants appeared to be guided by the fact that social media is used to build social capital, contribute to psychological well-being and construct a positive self-image [22]. In particular, having Facebook networks comprised of close social ties (including close offline rela- tionships as in our study) is likely to influence the content people choose to share, as emotional support is an import- ant component of such networks [23]. In this context of prioritising relationships, it makes sense that participants would choose messages that would not expose their friends to content which they found most uncomfortably challenging or which aroused strong negative emotions. These findings suggest that messages designed for sharing on social media need to be complementary to strong negative arousal messages that are spread through trad- itional broadcast media, point of sale warnings and graphic health warnings on tobacco packaging. An important insight from this study was the extent to which people’s attitudes to posting content changed over time. Despite initial reticence, both smokers and non-smokers showed an increasing willingness to post a range of content, particularly as fears about anticipated negative reactions proved to be unfounded. This is con- sistent with insights from the neuroscience of social media, which shows that posting involves a self-referential cognition network loop consisting of thinking about one- self, sharing experiences and opinions, and receiving feed- back which then prompts further self-appraisal. Posts that receive even minimalistic positive cues such as likes, fur- ther activate the sense of reward, which in turn provides further incentive to post [24]. This process may explain participants’ confidence increasing during the course of the study, along with personal investment and engage- ment in the study. A surprising finding was the willingness to share posts about the impact of smoking on male fertility, given the gender-specific cultural sensitivity around reproductive health [45]. Impact on phone credit/data usage The potential impact on data usage and phone credit for people in their networks was a concern for some people, Hefler et al. BMC Public Health (2019) 19:615 Page 18 of 21 Page 18 of 21 Page 18 of 21 include the impact of smoking on male fertility in mes- sages, although care would need to be taken to consider appropriateness related to gender relations and specific socio-cultural and geographical contexts. mainstream Australian anti-smoking advertisements with strong graphic and emotive narratives are likely to be highly motivating for Indigenous smokers [43]. However, participants in this study wavered in their willingness for posting such messages. When they did share them, the personal relevance and content in the message needed to have additional motivating features beyond disgust. In in- terviews, they reported wanting content that was both practical and positive, which may reflect the fact that many of the study participants wanted to quit smoking. This contrasts with recent research among socially disad- vantaged smokers which suggests that ‘why to’ quit mes- sages are perceived as more effective than ‘how to’ messages [44]. However, although qualitative information suggested a strong preference for ‘how to’, much of the content selected by participants was ‘why to’. Similarly surprising was the lack of interest in posts that used environmental themes, as this was highlighted as an important Indigenous health issue in our first ex- ploratory study for this project [36]. Even participants who consider the environment a high-priority issue only selected environmentally-focused messages when the issue was framed as concern for human health. The concerns about data and credit usage are signifi- cant for planning social media-based health promotion campaigns, particularly in communities where most ac- cess to social media relies on expensive mobile phone data. It should be noted that in this study, we did not have the ability for participants to embed videos in Face- book stream for automatic play, which minimises the impact on data. Being able to do so may make it likely for some videos to be more widely shared. However, given the risk of people scrolling past content even if video plays automatically, care should be taken to ensure the first frames and/or thumbnails of a video are en- gaging and attention-grabbing. Our results show that the thumbnail and title can be a deciding factor in engaging with or sharing content. Conclusions The authors declare that they have no competing interests. The results from this study suggest that anti-smoking con- tent designed to be shared on social media should com- plement, rather than attempt to replicate, messages from tobacco control mass media campaigns. In particular, con- tent should take into account the role of social media in strengthening relationships and developing social capital, both of which are likely to be prioritised above posting messages which are confronting and designed to arouse strong negative emotions. Participants in this study needed to perceive content as likely to be helpful to their personal networks, preferably because it contained new and/or supportive information. Tobacco control social media messages that include a focus on children, feature culturally-specific and locally tailored content, are prac- tical, relevant, credible, and have direct and unambiguous messages were the most likely to be shared. Indirect, ob- scure, sarcastic and disgusting content without a clear message are less likely to be shared. This study shows the potential for health services to incorporate a strategy of using paid local social media ‘champions’ or ‘ambassadors’ to disseminate tobacco control messages on Facebook through community networks. Availability of data and materials The datasets generated during the current study are not publicly available due to participant confidentiality but are available from the corresponding author on reasonable request. Author details 1Tobacco Control Research Program, Wellbeing & Preventable Chronic Diseases Division, Menzies School of Health Research, PO Box 41096, Casuarina, NT 0811, Australia. 2Prevention Research Collaboration, School of Public Health, Charles Perkins Centre, Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia. 3Miwatj Health Aboriginal Corporation, PO Box 519, Nhulunbuy, NT 0881, Australia. Received: 29 November 2018 Accepted: 29 April 2019 Strengths and limitations The strengths of this study include the detailed context- ual information about decision-making for sharing to- bacco control content within real-world networks, and the diversity of participants in terms of age range, smok- ing status, socioeconomic status, and initial attitudes to the study. A limitation of the study is that participants were from two small regional cities, which may limit generalisability to other services and geographical areas. Furthermore, the fact that participants were paid limits the applicability to the study findings to generating ‘or- ganic’ sharing of messages on Facebook. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors’ contributions MH conceived of the study. MH and DPT designed the study and obtained funding. VK managed the study and contributed to developing the methods. MH and VK were responsible for data collection and initial coding of data. MH, VK and DPT were responsible for data analysis. MH, VK, BF, GB and DPT contributed to data interpretation. MH prepared the first draft of this article. VK, BF, GB and DPT contributed to revising the manuscript. All authors approved the final version and agree to be accountable for all aspects of the work. Impact on phone credit/data usage Most of the community researchers reported this focus being of interest because it was completely new information. This may be a case of deflection, as the majority of our participants were female and had ei- ther direct or indirect experience of smoking in preg- nancy. Given the strong focus on smoking in pregnancy in tobacco control [46–48], particularly for Australian Indigenous women [49], there may be potential to The use of paid social media ‘influencers’ is a strategy used by the tobacco industry in a range of contexts [50]; our results show that tobacco control social media ‘cham- pions’ and ‘influencers’, can be nurtured. This approach has several benefits: in addition to boosting active com- munity engagement and disseminating messages through targeted networks, it is relatively low cost, while also pro- viding casual employment opportunities to local commu- nity members. Tobacco control messages were a small proportion of health messages observed in the first ex- ploratory study for this project [36]; this study shows that Page 19 of 21 Hefler et al. BMC Public Health (2019) 19:615 Page 19 of 21 Page 19 of 21 it is possible to cultivate a network of local community ‘influencers’ to disseminate tobacco control messages through personal social media networks. APP1089403. The funding bodies had no role in the design of the study, data collection, analysis and interpretation, or in writing the manuscript. References 1. McPhail-Bell K, Appo N, Haymes A, et al. Deadly choices empowering indigenous Australians through social networking sites. Health Promot Int. 2017. https://doi.org/10.1093/heapro/dax014 published Online First: Epub Date 2. Sweet M, Geia L, Dudgeon P, et al. #IHMayDay: tweeting for empowerment and social and emotional wellbeing. Australas Psychiatry. 2015;23(6):636–40. https://doi.org/10.1177/1039856215609762 published Online First: Epub Date. 3. Carlson B, Frazer R. Social media mob: being indigenous online. Sydney: Macquarie University; 2018. 4. McNair Ingenuity Research. 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https://openalex.org/W1505840264	https://figshare.com/articles/thesis/Does_Non-Suicidal_Self-Injury_Function_Primarily_as_an_Experientially_Avoidant_Behaviour_within_Aotearoa_New_Zealand_/16998277/3/files/31444933.pdf	English	null	Does Non-Suicidal Self-Injury Function Primarily as an Experientially Avoidant Behaviour within Aotearoa New Zealand?	null	2,021	cc-by	131,275	Does non-suicidal self-injury function primarily as an experientially avoidant behaviour within Aotearoa New Zealand? ABSTRACT Theoretical, empirical, and experiential attempts at disentangling the functions of Non-Suicidal Self-Injury (NSSI) have been driven by the desire to answer the complex question: Why do people engage in self-injurious behaviours? A recently developed behavioural model of NSSI—the Experiential Avoidance Model (EAM; Chapman, Gratz, & Brown, 2006)—proposes that self-injury functions primarily as a form of negatively reinforced, experiential avoidance and places particular emphasis on emotional avoidance. Experiential avoidance is conceptualised as a behavioural process whereby people are unwilling to tolerate distressing emotions, thoughts, memories, or physical sensations and engage in behaviours to change, avoid, or escape from these aversive, intrapersonal experiences (Hayes, Wilson, Gifford, Follette, & Strosahl, 1996). Although the results of international studies support the key assumptions of this model to varying degrees (Klonsky, 2007; Klonsky & Glenn, 2008; Nock & Prinstein, 2004), the EAM has never been empirically evaluated within Aotearoa New Zealand. To determine whether experiential avoidance is the primary function of NSSI for people living within Aotearoa New Zealand, I designed and conducted three studies. For my first study, I interviewed 24 people who had engaged in non- suicidal self-injurious behaviours in the previous 12 months about the antecedents and consequences of their most recent episode of self-injury. The interviews were analysed using a framework based on behavioural principles, which I developed for the purpose of this research. This method of analysis, which I called Interpretative Functional Analysis, allowed me to identify, and then compare, the functions served by discrete self-injurious episodes. Results supported the EAM (Chapman et al., 2006) in that self-injury episodes functioned predominantly as attempts to avoid or escape from intense, negative emotional experiences. Cognitive avoidance, however, also played a significant role in the self-injury trajectory, which highlighted the importance of investigating unwanted thoughts in subsequent studies. ii The second study involved surveying 198 people across Aotearoa New Zealand who had self-injured in the previous 12 months to further test whether the key assumptions of the EAM (Chapman et al., 2006) apply to a New Zealand-based population. Quantitative findings supported the model and were consistent with extant international studies in that experientially avoidant, intrapersonal functions (i.e., affect regulation and self-punishment) were identified as primary to the reinforcement and maintenance of NSSI. Intrapersonal functions, in general, were more highly endorsed than interpersonal functions. Finally, both negative affect and cognitions decreased following episodes of self-injury, while joviality increased. Does non-suicidal self-injury function primarily as an experientially avoidant behaviour within Aotearoa New Zealand? The increase in positive emotions undermines the EAM’s (Chapman et al., 2006) exclusive focus on negative reinforcement, suggesting that positive reinforcement also has an important role to play in the continued use of NSSI. Analyses of the open-ended, survey responses highlighted the impact of particular contextual factors (such as interpersonal conflict and community norms) on the incidence and maintenance of NSSI. Conducting a thematic analysis of the consequences of people’s most recent episode of NSSI allowed me to identify two distinct themes within this data corpus. Specifically, through self-injury participants assumed two paradoxical roles, that of transgressor and helper. For my final study, I surveyed university students across two time-points (Time 1 N = 408, Time 2 N = 224) about their general intrapersonal experiences (i.e., emotions and thoughts) and dispositional coping styles (e.g., global experiential avoidance, thought suppression). Negative intrapersonal experiences and avoidant coping styles were found to vary as a function of NSSI history and recency. Negative automatic thoughts and guilt at Time 1 also predicted new episodes of self-injury at Time 2. Additionally, thought suppression, not global experiential avoidance, was identified as a partial mediator of Time 1 relationships between negative intrapersonal experiences and NSSI. To conclude, the findings from this thesis are situated within a global context, and implications for clinical practice and future research studies are discussed. iii ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Top billing in this list of acknowledgements undoubtedly belongs to my husband, George. In the almost 14 years we have spent together, I have always considered myself unimaginably lucky to have found him and for him to have chosen me. This PhD odyssey has been incredibly difficult at times and without George, I do not know that I would have reached the end. If I had, I certainly would have been malnourished and absolutely miserable by now. His support has been unwavering and I am still amazed that he never once asked me, in the past four and a half years, when I was going to be finished. Despite the enormous impact this project has had on our lives and my constant anxiety about completing it, he has always simply said that it will be done when it is done. And now it is. Thank you my Gorgeous, I love you more each day. I am indebted to my participants who trusted me with their stories, took the time to fill in my surveys, and who believed that this research would be of benefit to others. I hope I have done justice to their experiences and reflections. I am also grateful to all the individuals, groups, and organisations that disseminated information about my research to potential participants on my behalf. My supervisor, Marc Wilson, has also been unfailingly supportive. He has patiently listened to me, constructively challenged me, advocated for me, and taught me. There were many times when, not having enough faith of my own, I had to rely on his faith in my abilities to sustain me. His consistent affirmation and CBT’esque objections to my self-doubt have been invaluable. Certainly, our afternoon sessions, spent discussing self-injury and many other vaguely relevant (but often tangential) topics, have been a highlight of this process for me. Thank you also to my family; my parents, Joan and Graeme Anderson, who instilled in me a love of education and always supported my desire to continue learning, and my sisters, Lara and Kate, whose friendship I cherish. My parents-in- law, Jane and Simon Langlands, also played an important support role in choosing iv to invest in a house with George and I, thus providing us with a very comfortable roof over our heads and a savings plan to boot. ACKNOWLEDGEMENTS I am also fortunate enough to have met and held onto some amazingly warm, smart, and funny friends. Thanks to Prabhat Sethi, Linda Rodenburg, Phil Cook, and Wendy Faust for being in my life. Special mention goes to Jessie Wilson and Erica Chadwick–kindred spirits whom I met while completing my PhD. I know we will be lifelong friends. At the risk of revealing myself as a “crazy cat lady”, I have also appreciated my cats’ love and company. Thanks to Wol, Fisher, and Tandem for all the times you distracted me from myself and this thesis by mewing for attention and affection; walking over my keyboard and typing random, strings of letters; and chasing the cursor. Thanks to all the numerous other people who have helped me in various ways, shapes, and forms including: Monica Cartner, Bridget Greaney, Gaynor Parkin, Murray Patton, Egan Bidois, Gerard Hoffman, Moana Kerr, Garth Healey, Kuni Shepherd, Clive Banks, Lynne Russell, John McDowall, and Tony Ward. I also appreciated the insightful comments and suggestions provided by my three examiners—Dr Deirdre Brown (Victoria University of Wellington), Dr Cate Curtis (University of Waikato), and Dr David Klonsky (University of British Columbia). Finally, I will always be grateful to the Tertiary Education Commission, the Freemasons Charity, and Victoria University of Wellington for the generous financial support I received to complete my PhD. v v TABLE OF CONTENTS ee i i i a io 3.4 Summary and limitations 4. SELF-REPORTED REASONS AND MOTIVATIONS FOR SELF-INJURY 4.1 Intrapersonal Reasons 4.1.1 Emotional reasons 4.1.1.1 Specific emotions 4.1.1.2 Specific affect states 4.1.1.3 General emotional experiences 4.1.2 Cognitive reasons 4.1.2.1 Specific cognitions 4.1.2.2 General cognitive states 4.1.3 Physiological reasons 4.1.3.1 Releasing pressure or tension 4.1.3.2 Inducing stimulation 4.1.4 Self-punishment 4.2 Interpersonal Reasons 4.2.1 Support-seeking 4.2.2 Avoidance 4.3 Limitations 4.4 Summary 5. SELF-REPORTED ANTECEDENTS AND CONSEQUENCES OF SELF- INJURY 5.1 Retrospective studies 5.2 Prospective studies 5.3 Summary and limitations 6. LABORATORY STUDIES 7. CONCLUSION Chapter three: The functions of self-injury: Theoretical conceptualisations 1. INTRODUCTION 2. SINGLE-FUNCTION MODELS OF SELF-INJURY 2.1 Affect regulation 2.2 Self-punishment 2.3 Anti-dissociation 2.4 Anti-suicide 2.5 Sexual 2.6 Sensation-seeking 2.7 Interpersonal boundaries 43 44 45 45 46 47 48 48 48 49 50 50 51 52 53 53 54 54 56 57 58 59 60 61 63 66 66 68 68 70 71 72 73 74 74 4. SELF-REPORTED REASONS AND MOTIVATIONS FOR SELF-INJURY 4.1 Intrapersonal Reasons 4.1.1 Emotional reasons 4.1.1.1 Specific emotions 4.1.1.2 Specific affect states 4.1.1.3 General emotional experiences 4.1.2 Cognitive reasons 4.1.2.1 Specific cognitions 4.1.2.2 General cognitive states 4.1.3 Physiological reasons 4.1.3.1 Releasing pressure or tension 4.1.3.2 Inducing stimulation 4.1.4 Self-punishment 4.2 Interpersonal Reasons 4.2.1 Support-seeking 4.2.2 Avoidance 4.3 Limitations 4.4 Summary 5. SELF-REPORTED ANTECEDENTS AND CONSEQUENCES OF SELF- INJURY 5.1 Retrospective studies 5.2 Prospective studies 5.3 Summary and limitations 6. LABORATORY STUDIES 7. CONCLUSION Chapter three: The functions of self-injury: Theoretical conceptualisations 1. INTRODUCTION 2. SINGLE-FUNCTION MODELS OF SELF-INJURY 2.1 Affect regulation 2.2 Self-punishment 2.3 Anti-dissociation 2.4 Anti-suicide 2.5 Sexual 2.6 Sensation-seeking 2.7 Interpersonal boundaries 44 45 45 46 47 48 48 48 49 50 50 51 52 53 53 54 54 56 57 58 59 60 61 63 66 66 68 68 70 71 72 73 74 74 TABLE OF CONTENTS List of tables List of figures Thesis overview Chapter one: What is non-suicidal self-injury? 1. INTRODUCTION 2. SELF-INJURY DEFINITIONS ARE SOCIO-CULTURALLY BOUND 2.1 Historical perspectives 2.2 Cross-cultural perspectives 3. WHAT COUNTS AS SELF-INJURY WITHIN WESTERN CULTURES? 3.1 Definitional debates 3.2 Differentiating non-suicidal self-injury from suicide attempts 3.2.1 Consumer Perspectives 3.2.2 Professional Perspectives 3.3 Should self-injury be classified as a symptom or a syndrome? 3.3.1 Self-injury as a symptom 3.3.2 Self-injury as a syndrome 3.3.2.1 Proposed diagnostic criteria 3.3.2.2 Is taxonomising self-injury within the DSM useful or necessary? 3.3.2.3 How would a NSSI disorder impact on non-Western cultures? 4. DEFINING SELF-INJURY FOR THE PURPOSE OF THIS THESIS Chapter two: Why do people self-injure? 1. INTRODUCTION 2. HOW PREVALENT IS SELF-INJURY? 3. RISK FACTORS AND CORRELATES OF SELF-INJURY 3.1 Individual characteristics 3.1.1 Gender 3.1.2 Ethnicity 3.1.3 Sexuality 3.2 Psychological characteristics 3.2.1 Mental health disorders 3.2.2 Suicidality 3.2.3 Temperament xii xiii 1 4 4 5 5 7 8 10 14 15 16 18 19 21 21 23 25 26 27 27 28 29 29 29 31 33 34 34 35 38 3.1 Individual characteristics 3.1.1 Gender 3.1.2 Ethnicity 3.1.3 Sexuality 3.2 Psychological characteristics 3.2.1 Mental health disorders 3.2.2 Suicidality 3.2.3 Temperament vi 3.2.4 Alexithymia 3.3 Environmental characteristics 3.3.1 Child maltreatment 3.3.2 Peer victimisation 3.4 Summary and limitations 4. SELF-REPORTED REASONS AND MOTIVATIONS FOR SELF-INJURY vii 2.8 Interpersonal influence 2.9 Summary and limitations 3. MULTI-FUNCTION MODELS OF SELF-INJURY 3.1 The Four Functions Model 3.1.1 Assumptions of the FFM 3.1.2 Empirical support for the FFM 3.2 The Experiential Avoidance Model 3.2.1 Assumptions of the EAM 3.2.2 Empirical support for the EAM 3.3 Evaluating the FFM and the EAM 3.3.1 Predictive accuracy 3.3.2 Internal coherence 3.3.3 External consistency 3.3.4 Unifying power 3.3.5 Fertility 3.3.6 Simplicity 4. THE CURRENT THESIS Chapter four: An Interpretative Functional Analysis of self- injury 1. INTRODUCTION 2. STUDY OVERVIEW 3. STAGE ONE: SCREENING FOR SELF-INJURIOUS BEHAVIOURS 3.1 Method 3.1.1 Recruitment strategy 3.1.2 Participants and procedure 3.1.3 The Deliberate Self-harm Inventory 3.2 Results 3.2.1 Demographic and diagnostic information 3.2.2 Prevalence of different types of self-injury 4. STAGE TWO: THE INTERVIEWS 4.1 Participants and procedure 4.2 Analysing the interview transcripts 4.2.1 What is a functional analysis? 4.2.2 Utilising functional analyses with typically developing populations 4.2.3 Interpretative Functional Analysis 4.2.3.1 Rationale 4.2.3.2 Epistemological assumptions 4.2.3.3 Coding system 4.2.3.3.1 Relevant learning history 4.2.3.3.2 Antecedents 4 2 3 3 3 T t B h i 74 75 76 76 76 78 80 80 83 86 86 87 89 89 90 90 91 94 94 96 97 97 97 99 100 101 101 101 105 105 109 110 110 112 112 114 114 115 115 117 vii 2.8 Interpersonal influence 2.9 Summary and limitations 2.8 Interpersonal influence 2.8 Interpersonal influence 2.8 Interpersonal influence 2.9 Summary and limitations 2.9 Summary and limitations 3. MULTI-FUNCTION MODELS OF SELF-INJURY 3.1 The Four Functions Model 3.1.1 Assumptions of the FFM 3.1.2 Empirical support for the FFM 3.2 The Experiential Avoidance Model 3.2.1 Assumptions of the EAM 3.2.2 Empirical support for the EAM 3.3 Evaluating the FFM and the EAM 3.3.1 Predictive accuracy 3.3.2 Internal coherence 3.3.3 External consistency 3.3.4 Unifying power 3.3.5 Fertility 3.3.6 Simplicity 4. THE CURRENT THESIS Chapter four: An Interpretative Functional Analysis of self- injury 1. INTRODUCTION 2. STUDY OVERVIEW 3. STAGE ONE: SCREENING FOR SELF-INJURIOUS BEHAVIOURS 3.1 Method 3.1.1 Recruitment strategy 3.1.2 Participants and procedure 3.1.3 The Deliberate Self-harm Inventory 3.2 Results 3.2.1 Demographic and diagnostic information 3.2.2 Prevalence of different types of self-injury 4. STAGE TWO: THE INTERVIEWS 4.1 Participants and procedure 4.2 Analysing the interview transcripts 4.2.1 What is a functional analysis? 4.2.2 Utilising functional analyses with typically developing populations 4.2.3 Interpretative Functional Analysis 4.2.3.1 Rationale 4.2.3.2 Epistemological assumptions 4.2.3.3 Coding system 4.2.3.3.1 Relevant learning history 4.2.3.3.2 Antecedents 4.2.3.3.3 Target Behaviour 94 94 96 97 97 97 99 100 101 101 101 105 105 109 110 110 112 112 114 114 115 115 117 viii 4.2.3.3.4 Consequences 4.2.3.3.5 Escape or access: Functions of the target behaviour 4.2.3.4 Steps in the Interpretative Functional Analysis 4.3 Interview results 4.3.1 Intrapersonal escape/avoidance 4.3.1.1 Establishing operations 4.3.1.2 Discriminative stimuli 4.3.1.3 Consequences 4.3.1.4 Summary 4.3.2 Interpersonal escape/avoidance 4.3.3 Intrapersonal access 4.3.3.1 Establishing operations and discriminative stimuli 4.3.3.2 Consequences 4.3.3.3 Summary 4.3.4 Interpersonal access 4.3.4.1 Establishing operations 4.3.4.2 Discriminative stimuli 4.3.4.3 Consequences 4.3.4.4 Summary 4.3.5 Aversive consequences 4.3.5.1 Negative emotions and thoughts 4.3.5.2 The wound and scarring 4.3.5.3 Restricted clothing choices 4.3.5.4 Reactions from others 4.3.5.5 Exclusive experience of punishing consequences 4.3.5.6 Summary 4.3.6 Summary of Interpretative Functional Analysis results 5. STAGE THREE: EMAIL OR TELEPHONE FOLLOW-UP 6. STAGE FOUR: FEEDBACK QUESTIONNAIRE 6.1 Participants and procedure 6.2 Measure 6.3 Results 7. DISCUSSION 7.1 Forms and frequencies of self-injurious behaviours 7.2 Is self-injury primarily an experientially avoidant behaviour within Aotearoa New Zealand? 7.2.1 The role of cognitions 7.2.2 What about positive reinforcement? 7.3 Strengths of Interpretative Functional Analysis 7.4 Limitations of Interpretative Functional Analysis 7.5 Is participating in a study about self-injury harmful? 7.6 Conclusion Chapter five: Quantifying the functions of self-injury 1. 3. MULTI-FUNCTION MODELS OF SELF-INJURY INTRODUCTION 117 118 118 121 122 123 131 135 139 139 140 141 142 144 145 145 147 148 150 150 150 152 153 154 156 156 157 157 158 158 158 159 161 161 162 163 165 166 166 167 169 170 170 4.2.3.3.4 Consequences 2.1 Procedure g Q 2.4 Open-ended questions 3. QUANTITATIVE RESULTS 3.1 Characteristics of participants’ self-injurious behaviour 3.1.1 Global NSSI episodes 3.1.2 Most recent NSSI episodes 3.2 Is affect regulation the primary function of NSSI? 3.2.1 Self-reported functions of participants’ global NSSI episodes 3.2.2 Self-reported functions of participants’ most recent NSSI episode 3.3 Are intrapersonal functions more highly endorsed then interpersonal? 3.3.1 Cluster analyses 3.3.1.1 Cluster analysis of global NSSI functions 3.3.1.2 Cluster analysis of participants’ most recent NSSI episode 3.3.2 Comparing intrapersonal and interpersonal functions 3.4 Do people report a decrease in negative affect and an increase in positive affect following NSSI? 3.5 Does the content of people’s cognitions change following NSSI? 3.6 Summary of quantitative findings 4. OPEN-ENDED RESPONSES 4.1 Antecedents of NSSI 4.2 Thematic Analysis of the consequences of NSSI 4.2.1 Self becomes transgressor 4.2.1.1 Concealing transgressions from others 4.2.1.2 Being judged by others for transgressions 4.2.1.3 Transgressions cause other to suffer 4.2.2 Self becomes helper 4.2.2.1 Regulating emotions 4.2.2.2 Accessing support and/or treatment 4.2.2.3 The physical wound 4.3 Summary of findings from the open-ended responses 5. DISCUSSION 183 183 183 184 186 188 188 190 192 193 193 195 196 198 201 202 203 203 205 206 208 210 211 211 212 214 215 216 216 4.2.3.3.4 Consequences 4.2.3.3.4 Consequences 4.2.3.3.5 Escape or access: Functions of the target behaviour p g 4.2.3.4 Steps in the Interpretative Functional Analysis 4.3.1 Intrapersonal escape/avoidance 4.3.1.1 Establishing operations 4.3.1.2 Discriminative stimuli Chapter five: Quantifying the functions of self-injury 1. INTRODUCTION 170 170 ix 2. METHOD 2. METHOD 2.1 Procedure 2.1.1 Recruitment strategy 2.1.2 Inclusion/exclusion criteria 2.1.3 General ethical considerations 2.1.4 Ethical issues specific to Internet Mediated Research 2.1.4.1 Meeting the inclusion criteria 2.1.4.2 Exiting the survey before being debriefed 2.1.4.3 Protecting anonymity 2.2 Participants 2.2.1 Flow of participants through the study 2.2.2 Demographics and descriptive characteristics of the sample 2.3 Measures 2.3.1 The Deliberate Self-harm Inventory 2.3.2 Inventory of Statements about Self-injury 2.3.3 Positive and Negative Affect Schedule 2.3.4 Revised Automatic Thoughts Questionnaire 2.4 Open-ended questions 3. QUANTITATIVE RESULTS 3.1 Characteristics of participants’ self-injurious behaviour 3.1.1 Global NSSI episodes 3.1.2 Most recent NSSI episodes 3.2 Is affect regulation the primary function of NSSI? 3.2.1 Self-reported functions of participants’ global NSSI episodes 3.2.2 Self-reported functions of participants’ most recent NSSI episode 3.3 Are intrapersonal functions more highly endorsed then interpersonal? 3.3.1 Cluster analyses 3.3.1.1 Cluster analysis of global NSSI functions 3.3.1.2 Cluster analysis of participants’ most recent NSSI episode 3.3.2 Comparing intrapersonal and interpersonal functions 3.4 Do people report a decrease in negative affect and an increase in positive affect following NSSI? 3.5 Does the content of people’s cognitions change following NSSI? 3.6 Summary of quantitative findings 4. OPEN-ENDED RESPONSES 4.1 Antecedents of NSSI 4.2 Thematic Analysis of the consequences of NSSI 4.2.1 Self becomes transgressor 4.2.1.1 Concealing transgressions from others 4.2.1.2 Being judged by others for transgressions 4.2.1.3 Transgressions cause other to suffer 4.2.2 Self becomes helper 4.2.2.1 Regulating emotions 4.2.2.2 Accessing support and/or treatment 4.2.2.3 The physical wound 4.3 Summary of findings from the open-ended responses 5. DISCUSSION 171 171 171 173 173 173 174 174 175 175 175 177 179 179 179 181 182 183 183 183 184 186 188 188 190 192 193 193 195 196 198 201 202 203 203 205 206 208 210 211 211 212 214 215 216 216 2.1 Procedure 2.1.1 Recruitment strategy 2.1.2 Inclusion/exclusion criteria 2.1.3 General ethical considerations 2.1.4 Ethical issues specific to Internet Mediated Research 2.1.4.1 Meeting the inclusion criteria 2.1.4.2 Exiting the survey before being debriefed 2.1.4.3 Protecting anonymity 2.2 Participants 2.2.1 Flow of participants through the study 2.2.2 Demographics and descriptive characteristics of the sample 2.3 Measures 2.3.1 The Deliberate Self-harm Inventory 2.3.2 Inventory of Statements about Self-injury 2.3.3 Positive and Negative Affect Schedule 2.3.4 Revised Automatic Thoughts Questionnaire 2.4 Open-ended questions 3. QUANTITATIVE RESULTS x 5.1 Affect regulation and self-punishment are the primary single functions of NSSI 5.2 Intrapersonal functions are more highly endorsed than interpersonal functions 5.3 Negative affect decreased and positive affect increased following NSSI 5.4 Negative cognitions decreased following NSSI 5.5 Strengths 5.6 Limitations 5.7 Conclusion Chapter six: Comparing the intrapersonal experiences and coping styles of people with and without a history of NSSI 1. INTRODUCTION 2. METHOD 2.1 Participants 2.2 Procedure 2.3 Measures 2.3.1 The Deliberate Self-harm Inventory 2.3.2 The Acceptance and Action Questionnaire 2.3.3 The Positive and Negative Affect Schedule 2.3.4 The White Bear Suppression Inventory 2.3.5 The Depression Anxiety Stress Scale 2.3.6 The Automatic Thoughts Questionnaire – short version 2.3.7 The Brief COPE 3. RESULTS 3.1 Prevalence and frequencies of self-injury types 3.2 Do people who have self-injured experience more negative emotions and thoughts? 3.3 Do people who have self-injured have a greater tendency towards avoidant coping? 3.4 Is NSSI predicted by negative emotions and thoughts? 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? 4. DISCUSSION 4.1 Experience of negative emotions and thoughts 4.2 Tendencies towards avoidant coping 4.3 Do negative emotions and thoughts cause self-injury? 4.4 Specific types of avoidance may underlie self-injury 4.5 Strengths 4.6 Limitations 4.7 Conclusions Chapter seven: General discussion 216 218 219 220 221 222 222 223 223 226 226 228 229 229 229 230 230 231 231 232 233 233 235 241 244 248 251 251 252 253 254 255 255 255 257 x 5.1 Affect regulation and self-punishment are the primary single functions of NSSI 5.2 Intrapersonal functions are more highly endorsed than interpersonal functions 5.3 Negative affect decreased and positive affect increased following NSSI 5.4 Negative cognitions decreased following NSSI 5.5 Strengths 5.6 Limitations 5.7 Conclusion Chapter six: Comparing the intrapersonal experiences and coping styles of people with and without a history of NSSI 1. INTRODUCTION 2. METHOD 2.1 Participants 2.2 Procedure 2.3 Measures 2.3.1 The Deliberate Self-harm Inventory 2.3.2 The Acceptance and Action Questionnaire 2.3.3 The Positive and Negative Affect Schedule 2.3.4 The White Bear Suppression Inventory 2.3.5 The Depression Anxiety Stress Scale 2.3.6 The Automatic Thoughts Questionnaire – short version 2.3.7 The Brief COPE 3. RESULTS 3.1 Prevalence and frequencies of self-injury types 3.2 Do people who have self-injured experience more negative emotions and thoughts? 3.3 Do people who have self-injured have a greater tendency towards avoidant coping? 3. QUANTITATIVE RESULTS CONCLUSION References Appendices (on CD attached to back cover) Appendix A: Information sheet Appendix B: Screening survey Appendix C: Support organisations Appendix D: Interview questions Appendix E: Consent to contact clinician Appendix F: Consent form Appendix G: Evaluation of research participation Appendix H: Online survey, including information and debriefing sheets Appendix I: Coding scheme Appendix J: Debriefing information (for email) Appendix K: Online survey (Times 1 and 2) Appendix L: Options for support 260 263 265 266 269 269 270 271 273 273 274 275 276 278 2. THE SELF-INJURY MINDSET 3. IS IT USEFUL TO CONCEPTUALISE NSSI AS A COPING STRATEGY? 4. SHOULD COPING STRATEGIES BE CLASSIFIED AS DISORDERS? 5. CLINICAL IMPLICATIONS 6. WHERE TO FROM HERE? 6.1 More sophisticated methods are necessary 6.2 How do emotions and cognitions interact to precipitate NSSI? 6.3 Further research on the EAM is warranted 6.4 What are the similarities and differences between NSSI and other forms of experiential avoidance? 6.5 How do functions of self-injury change over time? 6.6 What does NSSI communicate to others? 6.7 Diverse samples are needed 260 263 265 266 269 269 270 271 273 273 274 275 3. QUANTITATIVE RESULTS 3.4 Is NSSI predicted by negative emotions and thoughts? 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? 4. DISCUSSION 4.1 Experience of negative emotions and thoughts 4.2 Tendencies towards avoidant coping 4.3 Do negative emotions and thoughts cause self-injury? 4.4 Specific types of avoidance may underlie self-injury 4.5 Strengths 4.6 Limitations 4.7 Conclusions 216 218 219 220 221 222 222 223 223 226 226 228 229 229 229 230 230 231 231 232 233 233 235 241 244 248 251 251 252 253 254 255 255 255 p g y p p y 1. INTRODUCTION 2. METHOD 2.1 Participants 2.2 Procedure 2.3 Measures 2.3.1 The Deliberate Self-harm Inventory 2.3.2 The Acceptance and Action Questionnaire 2.3.3 The Positive and Negative Affect Schedule 2.3.4 The White Bear Suppression Inventory 2.3.5 The Depression Anxiety Stress Scale 2.3.6 The Automatic Thoughts Questionnaire – short version 2.3.7 The Brief COPE 3. RESULTS 3.1 Prevalence and frequencies of self-injury types 3.2 Do people who have self-injured experience more negative emotions and thoughts? 3.3 Do people who have self-injured have a greater tendency towards avoidant coping? 3.4 Is NSSI predicted by negative emotions and thoughts? 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? 4. DISCUSSION 4.1 Experience of negative emotions and thoughts 4.2 Tendencies towards avoidant coping 4.3 Do negative emotions and thoughts cause self-injury? 4.4 Specific types of avoidance may underlie self-injury 4.5 Strengths 4.6 Limitations 4.7 Conclusions Chapter seven: General discussion 1. INTRODUCTION 223 226 226 228 229 229 229 230 230 231 231 232 233 233 235 241 244 248 251 251 252 253 254 255 255 255 257 257 xi 2. THE SELF-INJURY MINDSET 3. IS IT USEFUL TO CONCEPTUALISE NSSI AS A COPING STRATEGY? 4. SHOULD COPING STRATEGIES BE CLASSIFIED AS DISORDERS? 5. CLINICAL IMPLICATIONS 6. WHERE TO FROM HERE? 6.1 More sophisticated methods are necessary 6.2 How do emotions and cognitions interact to precipitate NSSI? 6.3 Further research on the EAM is warranted 6.4 What are the similarities and differences between NSSI and other forms of experiential avoidance? 6.5 How do functions of self-injury change over time? 6.6 What does NSSI communicate to others? 6.7 Diverse samples are needed 7. References 278 Appendix A: Information sheet Appendix B: Screening survey Appendix C: Support organisations Appendix D: Interview questions Appendix E: Consent to contact clinician Appendix F: Consent form Appendix G: Evaluation of research participation Appendix H: Online survey, including information and debriefing sheets Appendix I: Coding scheme Appendix J: Debriefing information (for email) Appendix K: Online survey (Times 1 and 2) Appendix L: Options for support xii LIST OF TABLES LIST OF TABLES Table 1. Contrasting definitions and referents of the three most commonly used terms for self-injurious behaviours 11 Table 2. Frequencies and recency of different types of NSSI 104 Table 3. Number of participants (N = 23) who endorsed each statement 160 Table 4. Frequencies of different types of NSSI 185 Table 5. Frequencies of different types of NSSI for participants’ most recent episode 187 Table 6. Descriptive statistics and reliability coefficients for the ISAS in reference to participants’ global and most recent episodes of NSSI 189 Table 7. Agglomeration schedule, Ward’s method and squared Euclidean distance for global NSSI functions 193 Table 8. Agglomeration schedule, Ward’s method and squared Euclidean distance for functions of participants’ most recent NSSI episode 195 Table 9. Descriptive statistics and reliability coefficients for the PANAS-X subscale responses before and after participants’ most recent episode of NSSI 199 Table 10. Ranks for PANAS-X subscales before and after participants’ most recent episode of NSSI 201 Table 11. Antecedent event categories with percentage endorsement and qualitative examples 204 Table 12. Frequencies of NSSI types reported at Times 1 and 2 234 Table 13. NSSI recency at Times 1 and 2 235 Table 14. Time 1 reliability coefficients, descriptive statistics, and ANOVAs for the No NSSI versus Lifetime NSSI groups 237 Table 15. Two binary logistic regression models for NSSI versus No NSSI 245 xiii LIST OF FIGURES Figure 1. The elaborated FFM. 77 Figure 2. The Experiential Avoidance Model. 81 Figure 3. Functional schematic of Melanie’s most recent episode of NSSI. 120 Figure 4. Functions fulfilled by each participant’s most recent episode of NSSI. 121 Figure 5. Flow of participants through the online survey. 176 Figure 6. Dendrogram using Ward linkage for functions of participants’ global episodes of NSSI. 194 Figure 7. Dendrogram using Ward linkage for functions of participants’ most recent NSSI episode. 196 Figure 8. Thematic Map. 206 Figure 9. Flow of participants through the study at Times 1 and 2. 227 Figure 10. Cross-lagged panel model of NSSI and global negative affect. 245 Figure 11. Cross-lagged panel model of NSSI and negative automatic thoughts. 246 Figure 12. Cross-lagged panel model of NSSI and hostility. 246 Figure 13. Cross-lagged panel model of NSSI and guilt. 246 Figure 14. Cross-lagged panel model of NSSI and fear. 247 Figure 15. Cross-lagged panel model of NSSI and sadness. 247 Figure 16. LIST OF TABLES Unstandardised regression coefficients for the relationship between negative affect and NSSI which is not mediated by experiential avoidance. 249 Figure 17. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is partially mediated by thought suppression. 249 Figure 18. Unstandardised regression coefficients for the relationship between fear and NSSI which is not mediated by experiential avoidance. 249 xiv Figure 19. Unstandardised regression coefficients for the relationship between fear and NSSI which is partially mediated by thought suppression. 249 Figure 20. Unstandardised regression coefficients for the relationship between hostility and NSSI which is not mediated by experiential avoidance. 249 Figure 21. Unstandardised regression coefficients for the relationship between hostility and NSSI which is partially mediated by thought suppression. 249 Figure 22. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by experiential avoidance. 250 Figure 23. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by thought suppression. 250 Figure 24. Unstandardised regression coefficients for the relationship between sadness and NSSI which is not mediated by experiential avoidance. 250 Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. 250 Figure 26. Unstandardised regression coefficients for the relationship between automatic thoughts and NSSI which is not mediated by experiential avoidance. 250 Figure 27. Unstandardised regression coefficients for the relationship between automatic thoughts and NSSI which is partially mediated by thought suppression. 250 Figure 28. NSSI within an adaptive coping hierarchy. 264 264 1 1 THESIS OVERVIEW Answering the question of whether Non-Suicidal Self-Injury (NSSI) functions primarily as a experientially avoidant behaviour within Aotearoa New Zealand first necessitated an extensive review of the extant literature about how NSSI is defined, how it functions, and how it is conceptualised within behavioural models of self- injury. This review comprises the first three chapters of my thesis. In Chapter 1, I consider how definitions of self-injurious behaviours are socio-culturally bound, focusing in particular on the ways in which what counts as self-injury within Western cultures has been shaped by both consumer and professional perspectives. I present the evidence in support of NSSI being distinguished from suicide attempts, and critique suggestions that NSSI should be classified as a syndrome, rather than symptom, of psychopathology. To conclude the chapter, I define the parameters of NSSI for the purpose of my thesis. LIST OF TABLES My second chapter concentrates on why people self-injure. I review the current evidence base about risk factors and correlates of NSSI, including individual, psychological, and environmental characteristics. Following this detailed examination of the factors that are associated with, or predict, self-injury, I delineate the most commonly reported intrapersonal (i.e., emotional, cognitive, and physiological) and interpersonal (i.e., support-seeking and avoidance) reasons for self-injury. Literature detailing the self-reported antecedents and consequences of NSSI is also reviewed, along with laboratory studies about why people self-injure. Building on the evidence that I present in Chapter 2, I then move on to discuss functional models of NSSI in Chapter 3. Individual reasons, antecedents, and consequences of self-injury are typically grouped into an array of single function models. I review the literature in support of the eight most common single-function models and discuss the limitations of such conceptualisations of NSSI, before outlining and critiquing two multi-function models—the Four Functions Model (Nock, 2008; Nock & Prinstein, 2004, 2005) and the Experiential Avoidance Model 2 (EAM; Chapman, Gratz, & Brown, 2006)—according to well-established epistemic values. I close the chapter with a summary of the empirical studies that I conducted for the purpose of this thesis. Chapter 4 details the first empirical study I conducted to test whether NSSI functions primarily as an experientially avoidant behaviour within Aotearoa New Zealand. This interview study consisted of four stages: (1) screening potential participants for self-injurious behaviours, (2) interviewing participants about the antecedents and consequences of their most recent episode of NSSI, (3) following up with participants subsequent to the interviews, and (4) distributing a research evaluation questionnaire to interview participants. The methods and results of these four stages are presented and discussed in Chapter 4. My fifth chapter describes the findings of a nation-wide survey I conducted to quantitatively test the assumptions of the EAM in a larger sample of people living in Aotearoa New Zealand. More specifically, I examined whether affect regulation is the primary function of NSSI, whether intrapersonal functions are more highly endorsed than interpersonal functions, and whether the valence of people’s emotions and the content of their cognitions changes following NSSI. A series of open-ended questions, contained within the survey, about the antecedents and consequences of participants’ most recent episode of NSSI are also qualitatively analysed and discussed. What is non-suicidal self-injury? ‚We express our distress through bodily idioms that are both peculiar to distinctive cultural worlds and constrained by our shared human condition.‛ (Kleinman, 1988, p. xiii) LIST OF TABLES In Chapter 6, the final empirical chapter of my thesis, I compare the intrapersonal experiences and coping styles of people who have self-injured with those who have never engaged in self-injurious behaviour across two time-points. I present and discuss a series of analyses conducted to test the following hypotheses: (1) people with a history of NSSI will experience more negative emotions and thoughts, and evidence a greater tendency towards avoidant coping than people without a history of NSSI, (2) NSSI will be predicted by negative emotions and thoughts, and (3) avoidance will mediate the relationships between negative intrapersonal experiences and NSSI. 3 I conclude my thesis in Chapter 7 by discussing the most pertinent findings from my three studies and considering how my research relates to the broader conceptual issues outlined in Chapter 1, such as whether NSSI should be classified as a mental disorder. Finally, before outlining how future research can build on the strengths and limitations of my own studies, I reflect on the clinical implications of my research. 4 1. INTRODUCTION ‚I am a cutter.‛ This deceptively simple phrase is how one of my participants described himself at the start of our interview. Sitting across from me with rolled-up sleeves to expose his heavily scarred forearms, I wondered how and why cutting had become a defining aspect of his identity. What did those scars mean to him and what did they communicate to others in his community, given that self-injury exists firmly outside of the boundaries of what is deemed to be socially acceptable behaviour within Aotearoa New Zealand? Understanding what factors lead to Non-Suicidal Self-Injury (NSSI), why self- injurious behaviours become entrenched over time, and how people make sense of self-injury is complicated because NSSI is a multi-faceted behaviour, that ‚cannot be understood without reference to biological, psychological, social, and cultural components‛ (Favazza & Rosenthal, 1993, p. 138). As a Clinical Psychologist in training, my focus in this thesis is on clarifying the psychological factors that trigger, reinforce, and maintain self-injurious behaviours in populations from Aotearoa New Zealand. Although I touch on biological factors in my discussion of the psychology of self-injury, they are, by and large, beyond the scope of this thesis. Social and cultural norms, however, while not explicitly investigated in my studies do warrant an in-depth discussion at this stage of my thesis because these norms determine what counts as NSSI and, as a result, which behaviours I examine in this research. Every person who self-injures is situated in a particular socio- cultural context, which influences the meaning of self-injury to that person and others in their community. It is therefore imperative, before I begin to delve into the theoretical and empirical research that constitutes my thesis, to consciously reflect on 5 5 how the socio-cultural norms within Aotearoa New Zealand and other Western countries have shaped the way in which the self-injurious behaviours under investigation are defined. Before focusing on how self-injurious behaviours are currently conceptualised in Western societies, I briefly discuss the importance of recognising that definitions of self-injury are social constructs, which shift across time and between cultures. What counts as self-injury within contemporary, Western socio-cultural contexts has been shaped by both professionals and mental health consumers. Two definitional questions, in particular, have been extensively debated in the literature on self- injurious behaviours: (1) Can self-injury be distinguished from suicidal behaviours? 1. INTRODUCTION (2) Should self-injury be categorised as a symptom of mental illness or a separate clinical syndrome? (Cresswell, 2005). After discussing the evidence that self-injury can be differentiated from suicide attempts and the rationale behind classifying NSSI as a syndrome rather than symptom, I conclude this chapter by outlining the definition of self-injurious behaviour used in this thesis. 2. SELF-INJURY DEFINITIONS ARE SOCIO-CULTURALLY BOUND Two ways of exploring how current socio-cultural contexts shape our understanding of NSSI are to reflect on how this understanding has shifted over time and to compare our Western conceptualisations of self-injury with those of other cultures. I concur with Watters (2010) who argues that, ‚Symptoms of mental illnesses are the lightning in the zeitgeist, the product of culture and belief in specific times and specific places‛ (p. 3). Exploring historical and cross-cultural perspectives on NSSI, even though it is not always conceptualised as a symptom of psychopathology, can help us to illuminate the underlying assumptions and values that currently dominate our thinking about self-injurious behaviours (Watters, 2010). 2.1 Historical perspectives 2.1 Historical perspectives Browsing through journal articles on self-injury, it quickly becomes apparent that our knowledge about NSSI in Western cultures has evolved significantly within 6 past decades. For example, it is hard to fathom a journal editor today publishing the following strikingly pejorative and reductive description of women who self-injure: In summary, the cutter is an attractive, intelligent, unmarried young woman, who is either i l f id f il ddi d d bl l f ll past decades. For example, it is hard to fathom a journal editor today publishing the following strikingly pejorative and reductive description of women who self-injure: In summary, the cutter is an attractive, intelligent, unmarried young woman, who is either promiscuous or overly afraid of sex, easily addicted, and unable to relate successfully to others. She is an older one in a group of siblings with a cold, domineering mother and a withdrawn, passive, hypercritical father. She slashes her wrists indiscriminately and repeatedly at the slightest provocation, but she does not commit suicide. She feels relief with the commission of her act. (Graff & Mallin, 1967, p. 38) This extract is objectionable for many reasons: the use of blaming, invalidating statements (‚slightest provocation‛), the blatant contradictions (‚promiscuous‛ versus frigid), the characterisation of parents as villains, the use of stigmatising language such as ‚cutter‛ and ‚slashes‛, and the unnecessary attention to physical features (‚attractive‛), intelligence, and marital status. Furthermore, the overly confident, sweeping assertions made in this description are astounding considering that this research was based on only 20 female patients; the authors excluded the one male because they ‚felt he was atypical‛ (Graff & Mallin, 1967, p. 36). Of course, the socio-cultural context in which such historical accounts were produced was vastly different to the environment we currently inhabit. For a start, Psychoanalysis was the dominant therapeutic modality; understandings of what self- injury was and who self-injured were largely governed by this theoretical framework (Shaw, 2002). In contrast, the contemporary emphasis on evidence-based treatments (i.e., Dialectical Behavioural Therapy [DBT]; Linehan, 1993a) has shaped current conceptualisations of NSSI to focus on behavioural functions (Shaw, 2002). Second, research on NSSI began proliferating from the mid-1980s, a trend that has since continued unabated (Nock, 2010; Shaw, 2002). With the increase in publications on self-injury, the myth of the typical self-injurer (Hodgson, 2004) has been dispelled. 2.1 Historical perspectives Current perspectives on NSSI acknowledge the complexity of self- injurious behaviours, which are carried out by both males and females (Andover, Primack, Gibb, & Pepper, 2010; Claes, Houben, Vandereycken, Bijttebier, & Muehlenkamp, 2010), manifest across diverse cultures (Brausch & Gutierrez, 2010), 7 7 and associated with an array of risk factors and psychopathological correlates (Fliege, Lee, Grimm, & Klapp, 2009; Gratz, 2003; Nock, 2009). Third, the mid-1980s witnessed the advent of the psychiatric survivor movement whose members actively challenged clinical constructions of self- injurious behaviours. The role of mental health consumers in defining what NSSI is (and what it is not) will be discussed in more detail later in this chapter, but it is worth noting here that consumer testimonies about self-injury have exerted considerable influence in the past three decades on both practitioners’ and the general public’s perspectives of NSSI (Cresswell, 2005). Finally, recent technological advances have not only altered the way in which both lay and academic information about self-injury is disseminated, but also the quality and quantity of that information. In particular, the Internet facilitates access to support groups for people who self-injure, along with stories, graphic video clips, and clinical information about NSSI, all of which can contribute to the normalising or ‚narrative reinforcement‛ of these behaviours within certain communities (Whitlock, Lader, & Conterio, 2007, p. 1139). Undoubtedly, this interactive, online environment, which simply did not exist in the past, has influenced the way in which self-injury is understood on an individual and collective level. 2.2 Cross-cultural perspectives Until recently, psychological or psychiatric research with people from non- Western cultures who self-injure was virtually non-existent. Twentieth century academic investigations of NSSI almost exclusively document self-injurious behaviours among middle-class, white women (Shaw, 2002); Favazza’s seminal work on the interplay between culture and self-injury, which was first published in 1987, is one of the major exceptions. Examining the relationships between what Favazza (2011) terms culturally sanctioned body modification and pathological self-injury, brings into clear focus the role of culture in determining not only what types of self- injury are carried out, but also what meaning is ascribed to these behaviours and how that meaning is communicated to others. 8 8 Both socially acceptable and unacceptable self-injurious behaviours are posited to ‚serve an identical purpose, namely, an attempt to correct or prevent a pathological, destabilizing condition that threatens the community, the individual, or both‛ (Favazza, 1996, p. 222). Self-injury is a preventative or remedial activity that allows individuals and communities to create order out of perceived or impending chaos and, as such, examples of ritualistic, religious, and mythic self-injury abound in both Western and Non-Western cultural practices and narratives (see Favazza, 2011 for a detailed review). Given the importance of culture in demarcating the limits of socially acceptable self-injury, the rigorous application of current Western classification systems to Non-Western cultures would inevitably pathologise culturally appropriate self-injurious behaviours. For example, Australian Aboriginal peoples traditionally cut themselves when in mourning (Farrelly & Francis, 2009; Sheldon, 2001). Such ‚sorry cuts‛ are not only socially accepted but may be expected in Aboriginal communities as an expression of grief (Sheldon, 2001, p. 440). These behaviours form a marked contrast to socially acceptable grieving practices within Western cultures. Certainly, it is instructive to contemplate how the treatment of Westerners who cut themselves would change if these behaviours were reframed to become socially acceptable ‚distress cuts‛. While I am not advocating that this should be the case, it is possible that any self-stigma (Ben-Zeev, Young, & Corrigan, 2010) engendered by a history of self-injurious behaviours, which could in turn fuel subsequent episodes, may be ameliorated if these behaviours were consistently viewed as adaptive responses to distress. Indeed, such a reframing is what consumer activists have attempted to achieve (Pembroke, 1996a). 1 Cresswell (2005) uses the word ‚official‛ as a synonym for psychiatric. 3. WHAT COUNTS AS SELF-INJURY WITHIN WESTERN CULTURES? 3. WHAT COUNTS AS SELF-INJURY WITHIN WESTERN CULTURES? It is clear that socio-cultural norms play a fundamental role in how NSSI is defined; however, a shared socio-cultural context does not necessarily imply consensus. What counts as NSSI ultimately depends on who is doing the defining: 9 Acts of self-injury can hold specific meanings for particular individuals or sub- groups who may choose to accept, revise, or challenge hegemonic social and cultural understandings. In seeking to unpack the complexities and contradictions inherent in how self- injurious behaviours are defined and classified within Western cultures, I stumbled upon the burgeoning study of personal epistemology. Broadly speaking, personal epistemology is concerned with how people define, construct, and evaluate knowledge in order to make sense of the world around them (Hofer, 2002). In seeking to unpack the complexities and contradictions inherent in how self- injurious behaviours are defined and classified within Western cultures, I stumbled upon the burgeoning study of personal epistemology. Broadly speaking, personal epistemology is concerned with how people define, construct, and evaluate knowledge in order to make sense of the world around them (Hofer, 2002). Although the field of personal epistemology is not without its own controversies around what are highly abstract and complex concepts (Schommer-Aikins, 2002), my critique, by necessity, is simply informed by this field and does not seek to delve into or resolve these debates. Personal epistemologies are extremely relevant to the question of what counts as NSSI within Western cultures; how researchers, clinicians, and mental health consumers define and categorise self-injury depends on their ways of knowing and what forms of knowledge they privilege. The contrast between empirical knowledge, favoured by professionals, and experiential knowledge, favoured by consumers, is highlighted by Cresswell (2005): ‚‘Official’ knowledge stresses scientific classification, professional expertise, and statistical evidence: ‘Survivor’ knowledge, by contrast, emphasises individual experience, the traumas of the life-course, and the personal testimony of the survivor as itself expert data‛ 1 (p. 1668). By analysing the accounts of self-harm survivors published in Self-harm: Perspectives from personal experience, Cresswell demonstrates how some people actively resist and reject psychiatric hegemony through producing visceral testimonies about survival through self-injury. These testimonies function as a form of political practice designed to challenge and remediate professional conceptualisations of self-injury (Cresswell, 2005). 10 Given that many of these testimonies are directed at bio-medical approaches to NSSI, survivor activists are not necessarily challenging all professional depictions of self-injury. 2 To ensure consistency throughout this thesis, I generally refer to all non-suicidal self-injurious behaviours as NSSI even if this is not the term originally used by the author(s). For example, if authors use the term deliberate self-harm but their definition is consistent with that of non-suicidal self-injury, I refer to the behaviours as NSSI (with the exception of direct quotations). When authors have not distinguished between behaviours on the basis of suicidal intent, I refer to this as self-harm. 3. WHAT COUNTS AS SELF-INJURY WITHIN WESTERN CULTURES? Furthermore, many of those who self-injure do not choose to become activists or even to align themselves with this movement: ‚People vary in the resources available to them to resist or rework the cultural meanings of illness‛ (Kleinman, 1988, p. 26). However, those that do choose to challenge ‘official’ knowledge production are able to locate themselves within the system as the creators, rather than simply the recipients, of knowledge (Clinchy, 2002) and thus begin to wield the power to influence collective understandings of NSSI. For the remainder of this chapter, I will examine how the interplay between professional and consumer epistemologies have shaped the current understanding of NSSI within Western cultures, and how discussions about ways to define and classify self- injury have informed the operationalisation of NSSI for the purpose of this thesis. 3.1 Definitional debates 3.1 Definitional debates Cutting, self-mutilation, self-wounding, self-abuse, self-injury, deliberate self- harm (DSH), and self-inflicted violence are just a selection of the terms that have been used to denote self-injurious behaviours within academic literature.2 Most writers acknowledge the lack of consensus on how self-inflicted, harmful behaviours should be defined and commonly present their own definitions. As is evident in Table 1, this limits comparability of NSSI research findings because these definitions typically draw on idiosyncratic combinations of referents, including: self-inflicted, deliberate/purposeful, direct, suicidal intent, lethality, severity, tissue damage, function, and social acceptability. This inconsistent use of referents has resulted in the same term (e.g., NSSI) being defined in multiple ways, while different terms (e.g., NSSI and DSH) are given This inconsistent use of referents has resulted in the same term (e.g., NSSI) being defined in multiple ways, while different terms (e.g., NSSI and DSH) are given Table 1 Contrasting definitions and referents of the three most commonly used terms for self-injurious behaviours Referents Definition Self-inflicted Deliberate/ Purposeful Direct Suicidal intent Lethality Severity Tissue Damage Function Social acceptability Self-injury ‚Self-injury is intentional, self-effected, low-lethality bodily harm of a socially unacceptable nature, performed to reduce psychological distress‛ (Walsh, 2006, p. 4).      ‚Non-suicidal self-injury (NSSI) is defined as the deliberate destruction of one’s body tissue without suicidal intent and for purposes not socially sanctioned‛ (Klonsky & Glenn, 2009, p. 215).      ‚Nonsuicidal self-injury refers to purposeful, non-life-threatening self-inflicted injuries without suicidal intent that aim to alleviate emotional distress‛ (Kokaliari & Berzoff, 2008, p. 259).      Self-harm ‚Deliberate self-harm may be defined as the deliberate, direct destruction or alteration of body tissue, without conscious suicidal intent but resulting in injury severe enough for tissue damage to occur‛ (Gratz, 2003, p. 192).      ‚Deliberate self-harm includes any intentional act of self-injury or self-poisoning (overdose), irrespective of the apparent motivation or intention‛ (Hawton & Rodham, 2006, p. 11).   ‚Self-harm can be defined as socially unacceptable, intentional alteration or destruction of body tissue without conscious suicidal intent‛ (Croyle & Waltz, 2007, p. 332).     Self-mutilation ‚Self-mutilation refers to a complex group of behaviours in which there is deliberate destruction or alteration of body tissue without conscious suicidal intent‛ (Favazza, 1989, p. 113). 3.1 Definitional debates    ‚Self-mutilative behavior (SMB) refers to the direct and deliberate destruction of one’s own body tissue without suicidal intent‛ (Nock & Prinstein, 2005, p. 140).      ‚A theoretical definition of self-mutilation is the intentional act of tissue destruction with the purpose of shifting overwhelming emotional pain to a more acceptable physical pain‛ (Hicks & Hinck, 2008, p. 412).    Note. This table contains only a small selection of terms, with their accompanying definitions, and is not intended as a representative sample of self-injury terminology. It is modelled on the table presented by Claes and Vanderycken (2007) in their comparison of the different classification systems used to define self-injurious behaviour. Definition f ‚Deliberate self-harm may be defined as the deliberate, direct destruction or alteration of body tissue, without conscious suicidal intent but resulting in injury severe enough for tissue damage to occur‛ (Gratz, 2003, p. 192). ‚Deliberate self-harm may be defined as the deliberate, direct destruction or alteration of body tissue, without conscious suicidal intent but resulting in injury severe enough for tissue damage to occur‛ (Gratz, 2003, p. 192).  ‚Deliberate self-harm includes any intentional act of self-injury or self-poisoning (overdose), irrespective of the apparent motivation or intention‛ (Hawton & Rodham, 2006, p. 11). ‚Deliberate self-harm includes any intentional act of self-injury or self-poisoning (overdose), irrespective of the apparent motivation or intention‛ (Hawton & Rodham, 2006, p. 11). ‚Self-harm can be defined as socially unacceptable, intentional alteration or destruction of body tissue without conscious suicidal intent‛ (Croyle & Waltz, 2007, p. 332). Self-mutilation ‚Self-mutilation refers to a complex group of behaviours in which there is deliberate destruction or alteration of body tissue without conscious suicidal intent‛ (Favazza, 1989, p. 113). ‚Self-mutilation refers to a complex group of behaviours in which there is deliberate destruction or alteration of body tissue without conscious suicidal intent‛ (Favazza, 1989, p. 113).   ‚Self-mutilative behavior (SMB) refers to the direct and deliberate destruction of one’s own body tissue without suicidal intent‛ (Nock & Prinstein, 2005, p. 140).     ‚Self-mutilative behavior (SMB) refers to the direct and deliberate destruction of one’s own body tissue without suicidal intent‛ (Nock & Prinstein, 2005, p. 140). ‚A theoretical definition of self-mutilation is the intentional act of tissue destruction with the purpose o ‚A theoretical definition of self-mutilation is the intentional act of tissue destruction with the purpose of shifting overwhelming emotional pain to a more acceptable physical pain‛ (Hicks & Hinck, 2008, p. 412).    Note. This table contains only a small selection of terms, with their accompanying definitions, and is not intended as a representative sample of self-injury terminology. It is modelled on the table presented by Claes and Vanderycken (2007) in their comparison of the different classification systems used to define self-injurious behaviour. 11 12 the same or similar definitions. For example, Gratz’s (2003) definition of DSH ‚as the deliberate, direct destruction or alteration of body tissue, without conscious suicidal intent but resulting in injury severe enough for tissue damage to occur‛ (p. 192) essentially describes the same behaviours as Klonsky and Glenn’s (2009) definition of NSSI as ‚the deliberate destruction of one’s body tissue without suicidal intent and for purposes not socially sanctioned‛ (p. 215). It is hardly surprising that, given the disparate use of terminology and accompanying referents, varying levels of agreement exist between professionals and mental health consumers as to the empirical and experiential validity of self- injury definitions. Use of certain referents in defining self-injury is undisputed; for example, few researchers, clinicians, or consumers would argue against self-injury being purposeful and self-inflicted. Including the word deliberate, however, has been criticised because it implies ‚premeditation and wilfulness‛ (Pembroke, 1996b, p. 2) and insinuates that the person who self-injures is to blame for their behaviour (Taylor, 2003). Another referent that is seldom disputed in definitions or studies of non- suicidal self-injurious behaviours is direct. Self-mutilation A distinction is typically drawn between direct, self-inflicted, purposeful behaviours where the damage is immediate (e.g., cutting) and indirect behaviours where the damage accrues over time (e.g., eating disorders) (Walsh, 2006). Aside from the temporal qualities of the damage, Walsh (2006) asserts that another difference between indirect and direct self-injury is that people who engage in indirect self-injury seldom do so with the explicit aim of hurting themselves. For example, someone with an eating disorder is more likely to report being motivated to restrict food in order to lose weight (Walsh, 2006). In contrast, people who engage in direct self-injury do consciously aim to hurt themselves (Walsh, 2006). Within the category of purposeful, direct self-injury, the most controversial referent has been suicidal intent because it is an ephemeral construct that is often extremely difficult to quantify (Freedenthal, 2007). One solution is to view self- 13 injurious behaviours on a continuum with the following three anchors: NSSI, ambivalent or ambiguous suicide attempt, and unambiguous suicide attempt (Linehan, Comtois, Brown, Heard, & Wagner, 2006). Although a theoretically sensible approach, it can be difficult on a practical level to determine where individual acts of self-injury fall on the continuum. For instance, if a person burns the back of their hand with a cigarette, it is unproblematic to conclude that this behaviour is a type of NSSI, given that cigarette burns cannot cause death and it would be extremely unlikely for anyone to believe that they could. In comparison, overdosing on prescribed medication or illegal drugs is an ambiguous behaviour because it can result in death and is frequently used as a means to attempt or complete suicide. The latest available suicide statistics from Aotearoa New Zealand show that 11% of people who completed suicide in 2008 poisoned themselves with solids or liquids, compared with only 2% who cut or pierced themselves (Ministry of Health, 2010).3 Other referents (e.g., lethality, tissue damage, function) included in self-injury or self-harm definitions can help to clarify, and support, the parameters of suicidal and non-suicidal behaviours, although this is not always the case. In particular, it can be difficult to classify self-poisoning as suicidal or non-suicidal on the basis of tissue damage and lethality. Tissue damage can result from self-poisoning, but the extent of such damage, if it does occur, can be hard to determine. 3 All of the percentages reported in this chapter have been rounded to zero decimal places. Self-mutilation Likewise, lethality on its own is not always an accurate indicator of intent as some people may hurt themselves with the intent to die but unwittingly use methods that are not sufficiently lethal, whereas others may accidentally complete suicide when they were only intending to self-injure (Brown, Henriques, Sosdjan, & Beck, 2004; Freedenthal, 2007). Given the difficulties associated with clarifying suicidal intent specifically in cases of self-poisoning, overdosing and the ingestion of toxic substances are more 14 typically included in studies where the researchers have not discriminated between harmful behaviours on the basis of suicidal intent. In these studies, self-harm is used as an umbrella term to encompass both self-poisoning and self-injury (e.g., Hawton & Rodham, 2006). However, once again, this is not always the case. Exceptions to this trend include Gratz’s (2003) definition of DSH discussed above, and Nixon, Cloutier, and Jansson’s (2008) use of the term non-suicidal self- harm as a superordinate of self-injury (e.g., cutting), overdosing on medication, using drugs or alcohol, and swallowing non-ingestible items or substances. Behavioural measures of NSSI also vary as to whether or not self-poisoning is included; for example, self-poisoning is excluded from the Deliberate Self-harm Inventory (DSHI; Gratz, 2001), while swallowing chemicals, but not drug overdoses, is included in the Inventory of Statements about Self-injury (ISAS; Klonsky & Olino, 2008). Verifying the absence of suicidal intent by assessing suicidality indicators, such as lethality and extent of tissue damage (Walsh, 2006), thus clearly has the potential to be misleading. In light of these difficulties, the experiential and empirical evidence that supports distinguishing between behaviours on the basis of suicidal intent warrants further discussion given that I am investigating non-suicidal self- injurious behaviours in this thesis. In the following section, I will briefly review the literature that demonstrates NSSI is a discrete set of behaviours to suicide attempts, before examining whether given this distinction it is useful to classify NSSI as either a symptom or syndrome of psychopathology. 3.2 Differentiating non-suicidal self-injury from suicide attempts 3.2 Differentiating non-suicidal self-injury from suicide attempts Although the increase of robust empirical research on the differences between non-suicidal and suicidal self-injury is fairly recent, distinguishing between these behaviours is certainly not a novel endeavour. Writing in 1935, Menninger argued that self-injury was a strategy used to avert suicide completion, which ‚represents a victory<of the life instinct over the death instinct‛ (p. 466). This corresponds with reports from mental health consumers that self-injury provides respite from overwhelming thoughts and emotions, thus allowing them to continue functioning 15 (Alexander & Clare, 2004; Harris, 2000; Himber, 1994), and Favazza and Conterio’s (1988) contention that self-injury is a form of self-help. (Alexander & Clare, 2004; Harris, 2000; Himber, 1994), and Favazza and Conterio’s (1988) contention that self-injury is a form of self-help. Consumer perspectives articulated through personal narratives, research participation, and consultation appear to have informed professional perspectives over time to the point where professional and consumer beliefs about the importance of defining self-injury according to intent have become more aligned. Certainly, there is now substantial experiential and empirical evidence to support distinguishing between NSSI and suicide attempts. 3.2.1 Consumer perspectives Self-injury as self-preservation is powerfully articulated by Kettlewell (1999) in her memoir, Skin Game: Somewhere over the course of that winter I started thinking about killing myself, though not so much because I wanted to be dead, precisely, as because I yearned for resolution, for escape from the scratching distress of now<I needed to kill something in me, this awful feeling like worms tunnelling along my nerves. So when I discovered the razor blade, cutting, if you’ll believe me, was my gesture of hope. That first time, when I was twelve, was like some kind of miracle, a revelation. The blade slipped easily, painlessly through my skin, like a hot knife through butter. As swift and pure as a stroke of lightning, it wrought an absolute and pristine division between before and after. All the chaos, the sound and fury, the uncertainty and confusion and despair—all of it evaporated in an instant, and I was for that moment grounded, coherent, whole. Here is the irreducible self. I drew the line in the sand, marked my body as mine, its flesh and its blood under my command. (p. 57) Accessing hope and avoiding suicide through self-injury has similarly been Accessing hope and avoiding suicide through self-injury has similarly been umented by other women who have written about the fundamental role that self documented by other women who have written about the fundamental role that self- i j i b h i h l d i i th i i l injurious behaviours have played in securing their survival: injurious behaviours have played in securing their survival: I don’t cut myself in an attempt to die! It’s not the same thing! The feelings behind my scars are totally different...I look at these lines and shapes as battle scars—necessary sacrifices for the greater good...I really believe had I not cut, I would have died. The feelings I had while doing this were so intense that they would have overpowered me. In my mind it was a choice. Cut or die. It’s that easy. (Vega, 2007, p. 141) Self harm is a survival strategy and frequently represents the least possible damage an individual can get away with. It is an exercise in extreme restraint. Self harm is a survival strategy and frequently represents the least possible damage an individual can get away with. It is an exercise in extreme restraint. Self harm is about self-worth, self-preservation, lack of choices, coping with the uncopeable, speaking the unspeakable. Self harm gives a physical face to pain that might otherwise extinguish life. If I had not self-injured, I wouldn’t be here today. (Pembroke, 1998, p. 20) Self harm is about self-worth, self-preservation, lack of choices, coping with the uncopeable, speaking the unspeakable. Self harm gives a physical face to pain that might otherwise extinguish life. If I had not self-injured, I wouldn’t be here today. (Pembroke, 1998, p. 20) 16 A harm minimisation philosophy is evident within these extracts in that self-injury, when compared to suicide, is presented as the less damaging, and therefore more desirable, option. In recognition of the paradox that self-injury can sustain life but also cause accidental death, the National Self-harm Network (NSHN) in the United Kingdom published a book entitled: Cutting the risk: Self-harm, self-care & risk reduction. This book provides explicit instructions about how people should injure themselves to prevent long-term damage or unintended death. When introducing the book, Pembroke (2000) writes that, ‚Professionals frequently equate recovery with the cessation of self-harm, but that’s simplistically shallow and unrealistic. If we do less damage and feel better about ourselves, take greater care of ourselves, then that’s a success‛ (p. 7). Directly challenging professionals who define self-injury as a form of suicidal behaviour, the NSHN frames it as ‚a valid method of survival, until survival is possible by other means‛ (p. 6). 3.2.2 Professional perspectives Recent empirical findings support the experiential evidence (that self-injury is not synonymous with attempted suicide) presented by mental health consumers, such as Pembroke (1998), Kettlewell (1999), and Vega (2007). Researchers have identified that while non-suicidal and suicidal self-injurious behaviours do share certain features, there are sufficient, significant differences to warrant distinguishing between these behaviours (Brausch & Gutierrez, 2010; Whitlock & Knox, 2007; Wichstrøm, 2009). Studies investigating the relationship between suicidal behaviours and NSSI routinely demonstrate that not all people who self-injure report past suicidal ideation or attempts (Plener, Libal, Keller, Fegert, & Muehlenkamp, 2009; Whitlock, Eckenrode, & Silverman, 2006). In one study, 66% of university students who had self-injured did not report any past suicidal ideation or attempts (Whitlock et al., 2006), while in another study, 25% of adolescents had self-injured but only 7% had attempted suicide (Plener et al., 2009). 17 These prevalence rates, however, differ between community and psychiatric samples. Among a group of adolescent inpatients who had self-injured in the previous year, 70% also reported having attempted suicide (Nock, Joiner, Gordon, Lloyd-Richardson, & Prinstein, 2006). Although this rate is much higher than reported in community samples, there were still a substantial minority of adolescents (30%) who had self-injured but had never attempted suicide. Furthermore, people with a history of self-injury and suicide attempts typically report that they have self-injured significantly more times than they have attempted suicide (Walsh, 2006). Recent ecological momentary assessment research where adolescents and young adults were invited to complete questions, multiple times a day, about any self-destructive thoughts or behaviours they experienced over a two-week period, showed that out of the 1,262 episodes reported, 27% were categorised as NSSI thoughts, 8% as NSSI behaviour, and 2% as suicidal ideation (Nock, Prinstein, & Sterba, 2009). Additionally, while 87% of the participants self- injured without suicidal intent at least once during the course of the study, a minority of 33% reported suicidal ideation, and none reported any suicide attempts. Among people who self-injure, non-suicidal self-injurious thoughts and behaviours are not only more prevalent than suicide ideation and attempts, but also are associated with, and predicted by, discrete factors (Wichstrøm, 2009). Prior NSSI and becoming sexually active at younger age have been identified as specific risk factors for NSSI, while conduct problems and suicidal thoughts have been identified as specific risk factors for suicide attempts (Wichstrøm, 2009). 3.2.2 Professional perspectives Protective factors specific to NSSI and suicide attempts were satisfaction with social support and parental care respectively (Wichstrøm, 2009). Studies have also identified that suicidal and non-suicidal self-injury serve different functions (Brown, Comtois & Linehan, 2002; Himber, 1994; Polk & Liss, 2009), lending further support to distinguishing between these behaviours. Within- and between-person analyses showed that women diagnosed with Borderline Personality Disorder (BPD) more often endorsed using NSSI, as opposed to suicide 18 attempts, to express anger, reduce dissociation, and distract themselves (Brown et al., 2002). In comparison, suicide attempts were more often endorsed as a way to improve the lives of others. Self-punishment evidenced a more complicated relationship with NSSI and suicide attempts (Brown et al., 2002). While self- punishment was more likely to be endorsed in the context of NSSI than suicide attempts between persons, this difference was not identified within persons, suggesting that women with BPD who injure themselves both with and without suicidal intent are likely to do so as a form of self-punishment (Brown et al., 2002). Although the evidence to date supports the argument that NSSI and suicide attempts are phenomenologically different behaviours, researchers have also found a number of similarities between these behaviours (Brausch & Gutierrez, 2010; Muehlenkamp & Gutierrez, 2007; Wichstrøm, 2009). For example, Brown and colleagues (2002) found that particular functions (e.g., emotion relief, interpersonal influence) were similarly endorsed for both NSSI and suicide attempts. Certain risk factors have also been identified as common to adolescents with history of both NSSI and suicide attempts, and adolescents with a history of NSSI only, but these shared factors tend to vary in intensity between the two groups. Adolescents who have self-injured and attempted suicide report fewer reasons for living, higher rates of suicidal ideation and depressive symptoms, and judge themselves more harshly than adolescents who have only self-injured (Brausch & Gutierrez, 2010; Dougherty et al., 2009; Muehlenkamp & Gutierrez, 2007). However, adolescents who have attempted suicide and/or self-injured are at significantly greater risk of suicide completion than adolescents who have never engaged in self- injurious behaviours (Brausch & Gutierrez, 2010; Muehlenkamp & Gutierrez, 2007). 3.3 Should self-injury be classified as a symptom or a syndrome? While non-suicidal and suicidal self-injurious behaviours share certain features, it is clear that they can, and should, be viewed as distinct behaviours. 3.2.2 Professional perspectives Several researchers who support this distinction have debated whether NSSI should be classified as a symptom of psychopathology or a psychopathological syndrome 19 (Favazza & Rosenthal, 1993; Muehlenkamp, 2005; Pattison & Kahan, 1983). The evolution of this classification debate is especially pertinent in light of the proposed addition of Non-Suicidal Self-Injury Disorder to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5; American Psychiatric Association, 2010). Of course, as the following discussion will emphasise, both personal epistemologies and socio-cultural norms once again come into play when defining what counts as a symptom of disorder versus a disorder in and of itself. It is therefore timely to reflect on the propriety and utility of including a self-injury syndrome in the DSM-5, and the impact that such an inclusion may have on non- Western cultures. 3.3.1 Self-injury as a symptom Traditionally, NSSI among typically developing populations has been conceptualised within academic discourse as a symptom of mental disturbance or disorder, most notably that of BPD. Unfortunately because self-injury is a DSM-IV- TR diagnostic criterion for BPD (American Psychiatric Association, 2000), the relationship between self-injurious behaviour and BPD can easily become tautological; self-injurious behaviour is taken as evidence of a BPD diagnosis, and the presence of a BPD diagnosis is then used to explain future episodes of NSSI (Cipani & Schock, 2007). Reducing self-injury to the status of a BPD symptom has been criticised by Babiker and Arnold (1997) who declare their professional understanding of self- injury to be ‚at odds‛ (p. 11) with the structuralist epistemology that informs psychiatric research and practice. Self-injury is perceived as one symptom in a ‚pathology package‛ (p. 14), which requires immediate intervention and eradication. In contrast, they view self-injury as an embodied signifier of distress, thus emphasising the sociological and communicative aspects of self-injurious behaviour. Conceptualising self-injury as a sign of suffering, rather than psychopathology, corresponds with how many people who self-injure articulate the 20 reasons behind their behaviours (Kettlewell, 1999; Pembroke, 1996a; Vega, 2007). The juxtaposition between professional and consumer understandings of NSSI is a reminder that: ‚The meanings of symptoms are standardized ‚truths‛ in a local cultural system, inasmuch as the groups’ categories are projected onto the world, then called natural because they are found there‛ (Kleinman, 1988, p. 10). When consumers call into question professional conceptualisations of NSSI as indicative of pathology they are questioning these ‚truths‛. Recent research similarly refutes the primacy of self-injury as a symptom of BPD finding that not all people who self-injure meet the criteria for a mental health disorder, and those who do meet the criteria for one or more disorders are a diagnostically heterogeneous group (Hintikka et al., 2009; Nock et al., 2006). Given this heterogeneity, self-injurious behaviours are now more routinely conceptualised within academic discourse as a maladaptive coping mechanism, rather than a symptom of a specific disorder. Through emphasising the coping function of self-injury, professional perspectives have become more aligned with those of consumer activists, although one important difference remains. Consumer activists tend to dispute the use of the qualifier, maladaptive, preferring instead to endorse self-injury as a legitimate and adaptive strategy for managing overwhelming, and often life-threatening, distress (Pembroke, 1996a). 3.3.1 Self-injury as a symptom The utility of self-injury thus lies in the functions that these behaviours fulfil, which will be discussed in more detail in Chapters 2 and 3. Certainly from a functional perspective, any behaviour can be understood as an adaptive response to the environment (Sturmey, Ward-Horner, Marroquin, & Doran, 2007a), thus making the use of the word maladaptive a misnomer within this paradigm. Through emphasising the coping function of self-injury, professional perspectives have become more aligned with those of consumer activists, although one important difference remains. Consumer activists tend to dispute the use of the qualifier, maladaptive, preferring instead to endorse self-injury as a legitimate and adaptive strategy for managing overwhelming, and often life-threatening, distress (Pembroke, 1996a). The utility of self-injury thus lies in the functions that these behaviours fulfil, which will be discussed in more detail in Chapters 2 and 3. Certainly from a functional perspective, any behaviour can be understood as an adaptive response to the environment (Sturmey, Ward-Horner, Marroquin, & Doran, 2007a), thus making the use of the word maladaptive a misnomer within this paradigm. One way in which consumers challenge the labelling of NSSI as maladaptive is by critiquing the similarities and differences between NSSI and socially acceptable forms of self-injury: 21 Socially acceptable forms of self-harm include; excessive smoking, drinking, exercise, liposuction, bikini-line waxing, high heels and body piercing<The socially acceptable range of self-harm clearly does not include; self-cutting, burning, smashing bones and pouring toxic substances over or into our bodies<Some forms of self-harm don’t have the social seal of approval. It is denied as an expression of distress. It goes against the pre-occupation with maintaining ‘beauty’ and achieving some perceived image of ‘perfection’. (Pembroke, 1996b, pp. 2-3) An examination of the parallels between socially acceptable and unacceptable self- injury (Turp, 2003) serves to destabilise the legitimacy of dominant academic and practitioner conceptualisations of NSSI by focusing on the role of social and cultural norms in defining where the boundaries of psychopathology lie. 3.3.2 Self-injury as a syndrome Building on the argument that self-injury is a maladaptive coping mechanism, several academics at different times have advocated for non-suicidal self-injurious behaviours to be classified as a unique clinical syndrome. Proposed diagnostic labels include: deliberate self-harm syndrome (Pattison & Kahan, 1983), repetitive self- mutilation syndrome (Favazza & Rosenthal, 1993), and deliberate self-injury syndrome (Muehlenkamp, 2005). 3.3.1 Self-injury as a symptom The possible inclusion of NSSI disorder in the fifth edition of the DSM (American Psychiatric Association, 2010), due to be published in 2013, has revived this nosological debate. In the following sections, I discuss the proposed diagnostic criteria for a self-injury syndrome or disorder, whether classifying NSSI within a psychiatric taxonomy is useful or necessary, and finally whether such a classification would impact negatively on non-Western cultures. 3.3.2.1 Proposed diagnostic criteria In 1983, Pattison and Kahan presented the first comprehensive attempt at describing a deliberate self-harm syndrome, characterised by direct, low lethality self-harmful behaviours. They suggested that the syndrome, which typically begins in late adolescence and comprises of multiple self-injury episodes over a number of years, should be included in the fourth edition of the DSM as an impulse control disorder. Drawing on 56 published case studies, they identified the following six psychological symptoms: an irresistible urge to self-injure, feeling overwhelmed and 22 unable to cope, heightened negative emotions, an inability to problem-solve due to cognitive constriction, feeling relieved after self-injury, and depression. Informed by the work of Pattison and Kahan (1983), Favazza and Rosenthal (1993) similarly proposed that self-injury should be included in the DSM as an impulse disorder, although they used the label, repetitive self-mutilation syndrome. Their suggested diagnostic criteria predominantly focused on the trajectory of self- injury: (A) a preoccupation with thoughts of self-injury, (B) a persistent inability to resist the urge to self-injure, (C) an escalation of tension immediately prior to self- injury, and (D) a feeling of relief or gratification during self-injury. Criterion E differentiated NSSI from injuries carried out with conscious suicidal intent or in the context of psychotic experiences or intellectual disabilities. Furthermore, Favazza and Rosenthal advised that self-injury be considered a symptom of disorder until it becomes ‚an overwhelming preoccupation‛ (p. 136), at which stage it should be elevated to the status of a syndrome. More recently, Muehlenkamp (2005) advocated for the inclusion of a Deliberate Self-Injury Syndrome in the DSM. This syndrome bears close resemblance to previous incarnations (Favazza & Rosenthal, 1993; Pattison & Kahan, 1983), but expands on some of the earlier criteria. For example, Muehlenkamp’s description of the emotional precipitants of NSSI are more detailed: ‚Preceding the act of self- injury, there is a psychological experience of increasing tension, anger, anxiety, dysphoria, or general distress, which the person feels he or she cannot escape from or control‛ (p. 333). 3.3.1 Self-injury as a symptom As mentioned earlier, the debate about whether self-injury should be classified as a syndrome has been revived because of the impending publication of the DSM-5. It has been proposed that a diagnosis of NSSI be included in the fifth edition under the category ‘Other Disorders’ (American Psychiatric Association, 2010). Criterion A of the self-injury disorder specifies the location (i.e., on the body surface, thus excluding overdoses, ingestion of toxic substances or objects), recency (i.e., within the past 12 months), number of episodes (i.e., injured on at least 5 days), 23 and severity (i.e., bleeding, bruising, or pain is probable) of the self-injurious behaviour. Lack of suicidal intent is determined through self-report or inferred from the regular use of non-lethal means. To qualify for the diagnosis, people also need to show evidence of at least two of the four precipitating factors described in Criterion B: (1) negative emotions or cognitions, (2) a preoccupation with self-injury, (3) recurrent urges to self-injure, and (4) anticipation that the self-injury will serve a useful purpose, such as emotional relief. Criterion C stipulates that the ‚behaviour and its consequences cause clinically significant distress or impairment in interpersonal, academic, or other important areas of functioning‛, while Criterion D differentiates NSSI from other disorders that include self-injurious behaviours. 3.3.2.2 Is taxonomising self-injury within the DSM useful or necessary? Those in favour of a self-injury diagnosis argue that classification is useful to ensure a clear, consistent definition of self-injurious behaviours, which in turn will facilitate much-needed research on the etiology, maintenance, and treatment of NSSI (Muehlenkamp, 2005). As a separate diagnosis, NSSI would no longer be conflated with BPD or suicidal behaviours (Muehlenkamp, 2005; Pattison & Kahan, 1983). Finally, the clinical utility of a self-injury diagnosis for people without any other mental health disorders has been highlighted (Muehlenkamp, 2005). Academic opposition to the creation of a self-injury syndrome has centred on the limited research available on the causes and course of self-injurious behaviours, the link between self-injury and suicide, and the evidence of high psychiatric comorbidity in people who injure themselves on purpose (Muehlenkamp, 2005). Given the now well-established differences between self-injury and suicide attempts discussed above and the fact that comorbidity does not preclude the existence or diagnosis of other disorders (Muehlenkamp, 2005), I will briefly speculate about the possible consequences of creating a new diagnosis in the absence of sufficient research. 3.3.1 Self-injury as a symptom 24 The paucity of longitudinal research into how self-injurious behaviours manifest and change over time (Muehlenkamp, 2005; Prinstein, 2008) is problematic as it could result in the unnecessary pathologising of what may be predominantly adolescence-limited behaviours (cf. Moffitt, 1993). One unintended consequence (Wykes & Callard, 2010) of a self-injury diagnosis could be that young people who experiment with self-injury are ‘encouraged’ to adopt the identity of a self-injurer through being diagnosed as such. Furthermore, an NSSI diagnosis could constrain our developing understanding of how to treat and ultimately prevent more chronic self-injurious behaviour (Wykes & Callard, 2010). Of course, the dilemma is that there is likely insufficient research to warrant the creation of a self-injury diagnosis, but without a fixed definition of NSSI that would accompany a diagnosis, it is more difficult to develop a robust evidence base about self-injury (Muehlenkamp, 2005). The proposal to classify self-injury within the structuralist bounds of the DSM also runs counter to the increasingly functionalist approach taken by self-injury researchers. The importance of functionalism in understanding why people self- injure will be discussed in more detail in Chapter 4, but it is useful to note here that one of the key differences between structuralists and functionalists is that the former maintain the causes of behaviour lie within people, whereas the latter maintain the causes of behaviour lie within the environment (Sturmey et al., 2007a). Diagnoses therefore can be counter-productive in dealing with problem behaviours, particularly if they prevent a thorough assessment of the contingencies maintaining the behaviour (Cipani & Schock, 2007). This can lead to the tautology described earlier in this chapter where certain behaviours are interpreted as evidence of a disorder, and the disorder is then used to justify the presence of the behaviours (Cipani & Schock, 2007). The resulting danger is that practitioners fail to appreciate, or remain cognisant of, the influence of social and cultural norms on definitions and manifestations of self-injury. It is imperative to consider the impact that a self-injury diagnosis may have on people who self-injure and qualify for the diagnosis, as well as those who self- 25 injure but do not qualify for the diagnosis. While it is likely that some consumers will find a self-injury diagnosis validating, others may feel stigmatised by being labelled with NSSI disorder. 3.3.1 Self-injury as a symptom Conversely, those people who do not meet the threshold required for a diagnosis may be denied access to mental health support or feel that their distress has been minimised. 3.3.2.3 How would a NSSI disorder impact on non-Western cultures? Although the DSM is a classification system based on Western conceptualisations of mental distress and disorder, it is increasingly being used around the world to guide diagnosis and treatment (Watters, 2010; Wykes & Callard, 2010). Given that socio-cultural norms determine what meanings are ascribed to self- injurious behaviours, it is important to consider how the proposed addition of NSSI disorder to the DSM-5 (American Psychiatric Association, 2010) will shape non- Western cultures’ understanding and treatment of these behaviours. In Crazy like us: The globalization of the American psyche, Watters (2010) argues that American constructions of mental illness are not only rapidly changing the way in which psychological distress is perceived in non-Western societies, but also the way in which this distress is experienced. He contends that mental health practitioners who promote the use of DSM criteria in countries that do not subscribe to Western models of health and wellbeing unintentionally become vectors for the disorders they are attempting to treat. To support his argument, Watters (2010) refers to the work of Shorter (1987) who maintains that each culture has a ‚symptom pool‛ (p. 69) that changes over time; people communicate psychological distress through a restricted set of symptoms that are drawn from this pool. Particular behaviours become legitimised as culturally appropriate signifiers of disorder through their inclusion in the symptom pool (Shorter, 1987). Given the global power exerted by the DSM, the so- called ‚bible‛ of mental disorders (Watters, 2010, p. 3), a NSSI diagnosis may influence the way in which other cultures manifest distress and inadvertently increase the prevalence of self-injurious behaviours. 26 Why do people self-injure? ‚I know why I self-injure. I do it at times of extreme emotion: anger, self-hatred, stress, grief and guilt. I do it to punish myself. When I feel I am losing control, I reach for a razor and prove to myself that I can, at least, have control over my body.‛ (Ross, 1996, p. 13) CHAPTER TWO CHAPTER TWO 4. DEFINING SELF-INJURY FOR THE PURPOSE OF THIS THESIS This chapter has highlighted the importance of considering how both socio- cultural norms and personal epistemologies shape our understanding of what counts as non-suicidal self-injurious behaviours. As a trainee Clinical Psychologist, I value structuralist, functionalist, and experiential ways of knowing, and believe that all of these epistemologies can, and should, inform research and practice. By using a mixed-methods design (interviews and surveys) in this thesis, I have attempted to blend these three approaches and balance both experiential and empirical knowledge. The following definition of self-injury, used in this thesis, is a combination of the definitions presented in Table 1 and is informed by the debates discussed earlier in this chapter: NSSI is the purposeful, direct, and self-inflicted destruction or alteration of one’s body tissue without suicidal intent and for reasons not socially sanctioned. It is distinct from major or stereotypic self-injury conducted in the context of psychotic or developmental disorders respectively (Favazza, 2011). Furthermore, given the difficulties in clarifying suicidal intent in cases of self- poisoning (see pp. 13-14), overdosing and the ingestion of toxic substances are also excluded from my definition of NSSI. Finally, my understanding of self-injury is consistent with current clinical guidelines (National Institute for Clinical Excellence, 2004; Penrose-Wall, Farris, & Berkery, 2005); self-injury is a behaviour, not a disorder, which fulfils an array of different functions. In the following two chapters, I review what leads people to injure themselves on purpose in the absence of suicidal intent and why they may become reliant on these behaviours to cope with distress, before describing the three studies I conducted to investigate whether self-injury functions primarily as an experientially avoidant behaviour within Aotearoa New Zealand. 27 4 It should be noted that my review about why people are motivated to self-injure is similarly structured to those presented by other authors (e.g., Klonsky, 2007). 1. INTRODUCTION Publications on self-injury have more than tripled within the last decade (Nock, 2010), which has contributed to an increased awareness of the incidence and prevalence of these behaviours, especially among young people (Ross, Heath, & Toste, 2009). The burgeoning evidence base addressing self-injurious behaviours can be divided into two broad domains: studies that investigate the risk factors and correlates of NSSI (e.g., Gratz, Conrad, & Roemer, 2002; Hankin & Abela, 2011; Wichstrøm, 2009), and studies that investigate why people self-injure (e.g., Klonsky & Glenn, 2009; Lloyd-Richardson, Perrine, Dierker, & Kelley, 2007; Nock & Prinstein, 2004). Both streams of research are vital to developing a comprehensive understanding of what factors increase the likelihood that people will self-injure, and, once they begin self-injuring, how their self-injurious behaviours are reinforced and maintained over time. The studies for this thesis fall into the latter domain; my research is concerned with why people living in Aotearoa New Zealand self-injure and seeks to further clarify the behavioural functions of NSSI. In particular, I am interested in whether self-injury, carried out within the context of Aotearoa New Zealand, can be understood primarily as a form of experiential avoidance, a behavioural process that reflects people’s unwillingness to tolerate aversive, intrapersonal experiences (Hayes, Wilson, Gifford, Follette, & Strosahl, 1996). Extant research suggests that affect regulation is the primary function of NSSI (e.g., Klonsky, 2007; Klonsky & Glenn, 2009; Nock & Prinstein, 2004), but the broader, overarching concept of experiential avoidance, which includes but is not limited to affect regulation, has 28 seldom been examined in the international literature and has never been empirically investigated within Aotearoa New Zealand. Although I am not focussing on the risk factors and correlates of NSSI, it would be remiss to omit a discussion of these characteristics because they provide the context, through specifying who is more likely to self-injure, in which to situate my empirical work for this thesis. With this in mind, I review the research conducted on the individual, psychological, and environmental factors that predict, or are associated with, self-injury, before focusing in more detail on what we currently know about why people are motivated to hurt themselves on purpose.4 3. RISK FACTORS AND CORRELATES OF SELF-INJURY The equifinality of NSSI is evident in the wide range of individual, psychological, and environmental characteristics that are associated with self- injurious behaviours. Individual factors that have been implicated in the development of NSSI include gender, ethnicity, and sexuality. Psychological factors, such as mental health disorders, suicidality, temperament, and alexithymia are also thought to play an aetiological role in self-injury, along with environmental characteristics, including experiences of maltreatment during childhood and peer victimisation. 2. HOW PREVALENT IS SELF-INJURY? Before discussing self-injury correlates, risk factors, and motivations, it is essential to briefly summarise how many people self-injure and when they begin engaging in these behaviours. Unfortunately, precise prevalence rates of NSSI in clinical and community populations are unknown; figures tend to vary considerably across studies due to inconsistent operational definitions and modes of measurement (Ross et al., 2009). Evidence from population-based surveys suggests that 2% to 6% of adults have self-injured without suicidal intent in their lifetimes (Bebbington et al., 2010; Briere & Gil, 1998; Klonsky, 2011), although the rate of lifetime NSSI was as high as 17% in a random sample of university students (Whitlock et al., 2006). The identical prevalence rate of 17% was reported in a population-based study of adolescents and young adults (Nixon et al., 2008). Much higher prevalence rates of 36% to 63% have been observed among clinical populations (Claes, Vandereycken, & Vertommen, 2007; Swenson, Spirito, Dyl, Kittler, & Hunt, 2008; Weismoore & Esposito-Smythers, 2010). On average, people begin self-injuring between the ages of 11 to 15 (Hankin & Abela, 2011; Nixon, Cloutier, & Aggarwal, 2002; Yates, Carlson, & Egeland, 2008). 29 3.1.1 Gender Non-suicidal self-injury traditionally has been considered to be a gendered phenomenon, with more females than males engaging in NSSI, until recent evidence from community-based studies suggested that this gender ratio may be a sampling artefact. Much of the earlier research on self-injury focused on clinical populations, which were comprised of a disproportionate number of women (Whitlock et al., 2006). Within these populations, people diagnosed with BPD have been of specific interest to researchers because self-injury is one of the DSM-IV-TR criteria for the disorder (American Psychiatric Association, 2000). Since women are purportedly three times more likely to be diagnosed with BPD than men (Skodol & Bender, 2003), the research focus on self-injury within BPD populations has arguably supported a sampling bias in the study of NSSI. Preliminary evidence has also shown that there are gender differences in the forms of NSSI used by females and males, with females being more likely to cut themselves and males being more likely to burn themselves (Andover et al., 2010; Whitlock et al., 2006). Studies that have focussed exclusively on particular forms of self-injury (e.g., cutting) thus may have drawn premature conclusions about gender differences in NSSI, as a result of gendered preferences for certain forms of self- injury. 30 Although it is possible that being female is not necessarily a risk factor for NSSI, the evidence to support gender equivalency in NSSI prevalence rates is far from conclusive. Several cross-sectional studies of community-based adolescents and young adults have reported that females are significantly more likely to self- injure than males (Hoff & Muehlenkamp, 2009; Nixon et al., 2008; Plener et al., 2009; Ross & Heath, 2002), while others have failed to find any significant gender differences in NSSI prevalence (Andover et al., 2010; Claes, Houben, et al., 2010; Gratz et al., 2002; Muehlenkamp & Gutierrez, 2004). In a prospective study with Norwegian adolescents conducted over five years, females were twice as likely to self-injure as males and being female was a risk factor for NSSI (Wichstrøm, 2009). One of the reasons for these conflicting results could be due to the age group being assessed for NSSI, as the ratio of female to male self-injury may vary according to stages in the life cycle (Hawton & Harriss, 2008). 3.1.1 Gender Data collected on hospital presentations for DSH over 10 years showed that overall, females were one and a half times more likely than males to present to hospital following an act of DSH, but this general ratio masked sizeable differences between age groups. Females were eight times more likely to self-harm than males in the 10-14 year old age group and approximately three times more likely to self-harm in the 15-19 year old age group. This female to male ratio in DSH rates then decreased considerably from the age of 20 until age 50, when males engaged in higher rates of DSH than women (Hawton & Harriss, 2008). Although caution is required when extrapolating these results to gender differences in NSSI—this study relied exclusively on hospital admissions for self- harm and did not distinguish between different types of self-harm on the basis of intent—it is possible that a similar, age-related trend may be observable in female to male ratios of NSSI. An archival study of self-reported, lifetime prevalence rates of NSSI among secondary school students over 5 years showed that while there were no gender differences in NSSI rates for the first three years of secondary school, self- injury was more prevalent amongst females than males during the last two years of 31 school (Muehlenkamp, Williams, Gutierrez, & Claes, 2009). Population-based research on the prevalence of NSSI throughout the life cycle is needed to determine whether self-injurious behaviours are actually more common among females than males. 3.1.2 Ethnicity Sampling biases have also precluded a thorough investigation of the role of ethnicity in the development of NSSI. Historically, self-injury has been conceptualised as a largely Caucasian phenomenon, but this conclusion is based on a body of literature where the overwhelming majority of participants are white (Gratz, 2006; Shaw, 2002). The dearth of evidence into the impact of ethnicity on developmental trajectories of NSSI is especially problematic considering that, as I discussed in Chapter 1, what counts as pathological self-injury is culturally bound (Favazza, 2011). The few studies that have recruited ethnically diverse samples have presented conflicting results as to whether Caucasians are more likely than other ethnicities to engage in NSSI. No significant differences in the lifetime prevalence of NSSI were found in an ethnically diverse sample (i.e., 35% Caucasian, 37% African American, 16% multi-ethnic etc.) of secondary school students (Brausch & Gutierrez, 2010). However, in an inpatient sample with approximately even numbers of Caucasian and African American adolescents and a small percentage of other ethnicities (e.g., Hispanic, Native American), young people from ethnic minorities were less likely to have engaged in NSSI, but more likely to have attempted suicide (Boxer, 2010). In Aotearoa New Zealand, young Māori people are two to three times more likely to complete suicide than non-Māori youth (Beautrais & Fergusson, 2006), but the difference in NSSI prevalence between ethnicities is unclear. More Māori (25%) than Pākehā (19%) youth reported DSH in a recent national survey of health and wellbeing among young New Zealanders (Fortune et al., 2010), but the survey question referred to engaging in self-harm or any actions that may have resulted in 32 injury or death, rendering it impossible to separate out non-suicidal from suicidal self-injury. Given the importance of culture in delineating the taxonomical boundaries of NSSI, studies conducted by Māori researchers with tangata whaiora5 and community-based Māori populations are essential to determine the extent of NSSI among Māori people. Utilising questions about self-injury, which are developed within a Western paradigm of NSSI, to determine how many Māori self-injure and the factors that place Māori at risk of self-injuring, makes it less likely that an in- depth understanding of this phenomenon among Māori will result. Furthermore, significant cross-cultural differences may be overlooked because Western-based questions about NSSI do not take into account contextual factors that are specific to Māori, or other indigenous peoples and minority groups. 5 Tangata whaiora is the Māori term for mental health consumers. 3.1.2 Ethnicity For example, it has been argued that the higher suicide rate among Māori is a result of the enduring consequences of colonisation, such as the loss of cultural identity and connectedness to Māori culture (Lawson-Te Aho & Liu, 2010). Any research that examines NSSI among Māori needs to take the complex dynamics of post- colonialism into account (Wilson, 1999). Aotearoa New Zealand is also home to a number of other ethnic groups, including Pacific peoples and Asians, whose experiences of NSSI are yet to be examined. Unique socio-cultural factors will undoubtedly affect NSSI rates in these communities. For instance, a recent review found that South Asian women living in the United Kingdom are significantly more likely to self-harm than Caucasian women, but the authors did not discriminate between suicidal and non-suicidal self- harm (Husain, Waheed, & Husain, 2006). Factors implicated in the high rates of DSH among these women included racism, domestic violence, forced marriage, and the pressure to abide by their families’ or communities’ beliefs about izzat, defined as Aotearoa New Zealand is also home to a number of other ethnic groups, including Pacific peoples and Asians, whose experiences of NSSI are yet to be examined. Unique socio-cultural factors will undoubtedly affect NSSI rates in these communities. For instance, a recent review found that South Asian women living in the United Kingdom are significantly more likely to self-harm than Caucasian women, but the authors did not discriminate between suicidal and non-suicidal self- harm (Husain, Waheed, & Husain, 2006). Factors implicated in the high rates of DSH among these women included racism, domestic violence, forced marriage, and the pressure to abide by their families’ or communities’ beliefs about izzat, defined as 33 ‚family or personal honour/respect, or as status and prestige in the eyes of the community‛ (Chew-Graham, Bashir, Chantler, Burman, & Batsleer, 2002, p. 342). 3.1.3 Sexuality Other individual characteristics that have been associated with higher rates of NSSI are sexual orientation and same-sex attraction. In one study, university students who were uncertain about their sexual orientation or who identified as bisexual were more likely to report self-injury than heterosexual students (Whitlock et al., 2006). Concerns about sexual orientation were also associated with DSH in a community sample of adolescents, but given that the definition of DSH included suicide attempts, the specific role of NSSI in this association is unclear (O’Connor, Rasmussen, Miles, & Hawton, 2009). 3.1.2 Ethnicity In another study that examined the relationship between NSSI and sexual orientation, adolescents who identified as homosexual or bisexual were significantly more likely to have self-injured than heterosexual adolescents (Deliberto & Nock, 2008). Within Aotearoa New Zealand, the impact of sexual attraction on self-harm has been examined in two research studies. Same-sex attraction or being attracted to both sexes placed male and female secondary school students at significantly higher risk of engaging in self-harm behaviours (Lucassen et al., 2011). Adult New Zealanders who reported same-sex attraction were similarly found to be at risk of self-harm (Skegg, Nada-Raja, Dickson, Paul, & Williams, 2003). Unfortunately NSSI was conflated with DSH in both of these studies making it impossible to generalise these results to people who self-injure without suicidal intent. Further research is needed before it can be conclusively determined whether non-heterosexual orientation and/or attraction are risk factors for NSSI. Such research should take advantage of increasingly sophisticated understandings of sexuality. For instance, in their longitudinal study of adolescent health, Savin- Williams and Ream (2007) assert that sexual orientation should be measured as a dimensional, rather than categorical, construct and define sexuality on the basis of attraction, behaviour, and identity. 34 3.2.1 Mental health disorders Although NSSI is listed as a symptom of BPD in the DSM-IV-TR (American Psychiatric Association, 2000), it has been associated with a range of other mental health conditions, including disordered eating (Ross et al., 2009; Whitlock et al., 2006), depression, and anxiety disorders (Hintikka et al., 2009; Hoff & Muehlenkamp, 2009). In a Finnish study, adolescent girls who cut themselves were more than three times as likely to have a mental health diagnosis than age-matched controls (Hintikka et al., 2009), although given that the study design was cross- sectional, it is not possible to determine whether the high rate of mental disorders in this sample was a causal factor in the development of NSSI. Furthermore, since the type of NSSI was limited to cutting, such high rates of mental disorders may not be observed among people who use other forms of self-injury. Although NSSI is listed as a symptom of BPD in the DSM-IV-TR (American Psychiatric Association, 2000), it has been associated with a range of other mental health conditions, including disordered eating (Ross et al., 2009; Whitlock et al., 2006), depression, and anxiety disorders (Hintikka et al., 2009; Hoff & Preliminary support for the suggestion that different forms of NSSI may be associated with different types of mental disorders has been provided by Andover, Pepper, Ryabchenko, Orrico, and Gibb (2005). University students who had cut themselves reported more anxiety than those who used other forms of self-injury, but the rates of depressive symptoms were similar in both groups. Further analyses by Andover and colleagues identified that although the students who had self- injured were significantly more likely to report symptoms of depression and anxiety compared to a control group, these differences were no longer significant after controlling for BPD symptoms even though none of their participants met the threshold for a diagnosis of BPD. The nature of the relationships between mental health disorders and NSSI, however, will most likely differ according to the severity of psychopathology experienced by participants. For example, in a prospective research study conducted over 10 years, Zanarini, Laudate, Frankenburg, Reich, and Fitzmaurice (2011) found that major depression was a significant predictor of NSSI in people diagnosed with BPD. Additional studies are needed to tease out the contribution of various forms of 35 psychopathology to NSSI and how comorbid disorders interact to influence the manifestation of self-injurious behaviour. 3.2.1 Mental health disorders It is also probable that any interactions between mental disorders and NSSI will change over time both within and between individuals. In a study of secondary school students using archival data gathered over five years, Muehlenkamp et al. (2009) found that as females’ rates of NSSI increased over time, their symptoms of depression decreased. Given that NSSI is thought to function primarily as an affect regulation strategy (Klonsky, 2009), one possible explanation for this pattern is that self-injury was increasingly used to reduce depressive symptoms. It is unclear why this pattern was not observed in males, but Muehlenkamp et al. (2009) hypothesise their ability to identify statistically significant changes may have been impeded by the small number of male participants. It is important to keep in mind that although a range of mental health disorders have been identified as correlates or predictors of NSSI, the experience of psychopathology is not a prerequisite for engaging in self-injurious behaviours. In one study, 21% of the adolescents who had cut themselves did not meet the criteria for an Axis-I disorder as measured by the Structured Clinical Interview for DSM-IV- TR (Hintikka et al., 2009). However, the authors acknowledge that these adolescents may meet the criteria for the Axis-II disorder of BPD later in life. 3.2.2 Suicidality Owing to the historical conflation of non-suicidal and suicidal self-injury, which I discussed in-depth in Chapter 1 (see pp. 14-18), research on the role of suicidality (i.e., suicidal ideation and attempts) in NSSI is limited. In Chapter 1, I cited experiential and empirical evidence in support of a distinction between NSSI and suicide attempts. In the current section, I review this empirical evidence in more detail to further unpack the complex relationship between non-suicidal and suicidal self-injury. Repetitive DSH, irrespective of suicidal intent, has been identified as a suicide risk factor for both females and males (Zahl & Hawton, 2004), but the unique 36 contribution of NSSI to suicide completion is unclear. To address this, researchers have started to compare people who have self-injured or attempted suicide, with those who have self-injured and attempted suicide, in order to identify correlates and risk factors specific to each group. In particular, the impact of suicidal ideation and past suicide attempts on NSSI has been examined. 3.2.1 Mental health disorders A comparison of a group of secondary school students who had self-injured to a group who had attempted suicide found no difference in suicidal ideation between the two groups (Muehlenkamp & Gutierrez, 2004). However, when secondary school students who had self-injured and attempted suicide were compared to those who had only engaged in self-injury, those with a history of NSSI and suicide attempt(s) reported significantly higher levels of suicidal ideation than those who with a history of NSSI only (Brausch & Gutierrez, 2010; Muehlenkamp & Gutierrez, 2007). Slightly different results were obtained in a sample of adolescent outpatients (Jacobson, Muehlenkamp, Miller, & Turner, 2008). In this study, when adolescents with a history of NSSI only were compared to adolescents who had engaged in NSSI plus attempted suicide, and adolescents who had only attempted suicide, those who had only engaged in self-injury had significantly lower rates of suicidal ideation. The group of adolescents who had self-injured and attempted suicide, and the group who had only attempted suicide but had not self-injured, did not differ significantly from each other on suicidal ideation. Evidence in support of a relationship between NSSI and suicide attempts has been mixed. No association between NSSI episodes and suicide attempts was found in a group of inpatient adolescents (Nock et al., 2006), but in a group of adult inpatients, those with a history of NSSI were more likely to have attempted suicide than those without a history of NSSI (Andover & Gibb, 2010). Moreover, Andover and Gibb (2010) found that suicide attempts were positively associated with NSSI frequency. Although there was no difference between current suicidal ideation versus NSSI frequency and history in predicting suicide attempts, the number of 37 times people had self-injured was a stronger predictor of suicide attempts than hopelessness, depression, and BPD symptoms. Additionally, the presence of an NSSI history was a significant predictor of attempted suicide regardless of how many times people had self-injured. The predictive value of NSSI has been examined not only for suicide attempts and ideation, but also for plans and gestures (Whitlock & Knox, 2007). Among people who reported NSSI and suicidality, NSSI strongly predicted all forms of suicidality (i.e., ideation, plans, gestures, and attempts). However, these results rely on the assumption that NSSI precedes or occurs simultaneously with suicidality (Whitlock & Knox, 2007), which may not be the case. 3.2.1 Mental health disorders Evidence from a prospective study of Norwegian adolescents showed that while previous suicide attempts predicted future NSSI, previous NSSI did not predict future suicide attempts, suggesting that NSSI is not a risk factor for suicide attempts but, rather, prior suicide attempts place adolescents at risk of engaging in NSSI (Wichstrøm, 2009). Given that self-injuring without suicidal intent does appear to place some people at risk of attempting suicide, it is important to consider why this may be the case. Joiner (2005) argues that one way people acquire the ability to complete suicide is through practising self-injury until these behaviours become habitual and unthreatening. Cross-sectional studies that have identified a positive association between the frequency of NSSI and suicide attempts (Andover & Gibb, 2010; Whitlock & Knox, 2007) provide preliminary support for Joiner’s theory, but such support is tempered by research that has failed to find evidence of this association (Nock et al., 2006). If people do become habituated to the prospect of suicide through self-injury, what prompts the shift from one to the other? One possibility is that when people who use self-injury as a coping strategy surpass a particular threshold of distress, which cannot be alleviated by other means (including self-injury), they may attempt suicide (Whitlock & Knox, 2007). More specifically, the connection between suicidal and non-suicidal self-injury may lie in the shared, unresolved distress that motivates 38 these behaviours (Whitlock et al., 2006). Prospective, longitudinal research is clearly needed to test Joiner’s (2005) habituation hypothesis. 3.2.3 Temperament Specific temperament traits, such as impulsivity and emotional reactivity, have been identified as individual risk factors for NSSI. As mentioned earlier, several researchers have suggested that NSSI be classified as an impulse control disorder (Favazza & Rosenthal, 1993; Pattison & Kahan, 1983), but findings about the relationship between NSSI and impulsivity have been mixed. Utilising child and parent reports of temperament dimensions, Baetens, Claes, Willem, Muehlenkamp, and Bijttebier (2011) found that lack of effortful control (i.e., a decreased capacity for attention and behaviour regulation) was one of the most robust predictors of NSSI in a group of secondary school students. However, others maintain that there is insufficient evidence to support the role of impulsivity in NSSI, in part, because it is a heterogeneous construct that has been operationalised and measured differently across studies, resulting in conflicting findings (Glenn & Klonsky, 2010a). For example, Herpertz (1995) categorised NSSI episodes as impulsive if people evidenced the following three criteria: a lack of premeditation and consideration of consequences, acting on the decision to self- injure within minutes, and an urge to self-injure, whereas other studies have assessed the association between impulsivity and self-injury through assorted validated measures (e.g., Jutengren, Kerr, & Stattin, 2011; MacLaren & Best, 2010). Two studies that utilised multiple methods (e.g., self-report and laboratory- based tasks) to measure impulsivity sought to clarify whether people who self-injure are more impulsive than those who do not self-injure (Glenn & Klonsky, 2010a; Janis & Nock, 2009). Both studies found the same pattern of results: people who had self- injured reported greater impulsivity than people who did not have a history of NSSI, but these differences were not evident in performance-based measures of impulsivity. 39 One possible explanation for these inconsistencies is that only particular negative emotions trigger impulsivity in people who self-injure; such emotions would therefore need to be induced in a laboratory setting to capture the relationship between NSSI and impulsivity (Glenn & Klonsky, 2010a; Janis & Nock, 2009). It is also essential to examine this relationship over time in order to disentangle whether impulsivity is a cause or effect of NSSI (Janis & Nock, 2009). Another temperamental risk factor for self-injury that is garnering increased attention from researchers is whether people are predisposed to experience, and react to, emotions in specific ways (Nock, Wedig, Holmberg, & Hooley, 2008). 3.2.4 Alexithymia 3.2.4 Alexithymia Researchers are not only interested in the way in which people who self- injure react to emotions, but also how they understand and articulate these emotions. Alexithymia, which has been associated with NSSI (Paivio & McCulloch, 2004; Zlotnick et al., 1996), is a term that encapsulates a set of difficulties with identifying and discriminating between various emotions, and with verbally communicating those emotions (Kooiman, Spinhoven, & Trijsburg, 2002). Little is known about whether alexithymia is causally related to self-injury as the research conducted to date has been cross-sectional. Certainly, more work is needed on whether specific groups of people who self-injure are more likely to exhibit alexithymic tendencies, how the different components of alexithymia contribute to the manifestation of self-injury, and whether alexithymia is a causal factor for NSSI. For instance, Oyefeso, Brown, Chiang, and Clancy (2008) found that people addicted to opiates who had self-injured reported significantly more difficulty identifying their emotions than people addicted to opiates who had not self-injured. However, these two groups did not differ on their self-reported ability to describe how they were feeling. There is also preliminary evidence to suggest that people who have difficulty identifying and expressing their emotions may turn to self- injury following traumatic experiences. In a cross-sectional study with female university students, alexithymia fully mediated the relationship between child maltreatment (i.e. physical and emotional abuse and neglect) and self-injurious behaviours (Paivio & McCulloch, 2004). 3.2.3 Temperament This focus is congruent with the conceptualisation of NSSI as an emotion regulation strategy and draws on research conducted with people diagnosed with BPD (Rosenthal et al., 2008). Emotional vulnerability has been proposed as a core mechanism underlying the development of BPD; people who are emotionally vulnerable are characterised by ‚high sensitivity to emotional stimuli, emotional intensity, and slow return to emotional baseline‛ (Linehan, 1993b, p. 43). Emotion reactivity is comparable to emotion vulnerability in that it has been theorised to include three components: (1) how emotionally sensitive people are to stimuli, (2) how intensely they experience emotions, and (3) how long emotions persist for following their initiation (Nock et al., 2008). Due to the absence of a comprehensive, self-report measure of emotion reactivity, Nock et al. (2008) recently developed the Emotion Reactivity Scale to assess emotional sensitivity, arousal/intensity, and persistence. Preliminary evidence demonstrated the validity and reliability of the scale, and people who had recently self-injured reported themselves as significantly more emotionally reactive compared to people who had not self-injured. Moreover, the relationship between psychopathology and NSSI was mediated by emotion reactivity. Unfortunately, the cross-sectional nature of the data precluded any definitive conclusions about whether emotion reactivity drives people with mental health disorders to engage in NSSI (Nock et al., 2008). 40 3.3.1 Child maltreatment The experience of child maltreatment has been proposed as a causal factor in the aetiology of NSSI (Yates, 2004), yet the evidence to support this contention remains conflicted. A group of university students who had been abused were significantly more likely to have self-injured than those without an abuse history (Muehlenkamp, Kerr, Bradley, & Adams Larsen, 2010), but neither childhood 41 physical or sexual abuse was associated with NSSI in a sample of adolescent inpatients (Weismoore & Esposito-Smythers, 2010). However, in another adolescent sample, self-injuring without suicidal intent in the previous 12 months was significantly related to physical neglect, emotional abuse, and sexual abuse (Glassman, Weierich, Hooley, Deliberto, & Nock, 2007). Within Aotearoa New Zealand, childhood sexual abuse has been identified as a risk factor for self-injury among depressed adults (Joyce et al., 2006). The type of maltreatment is one factor which may influence the nature of the relationship between NSSI and abuse history. Indeed, Yates et al. (2008) reported that community-based adults who had been sexually abused as children were almost 10 times as likely to engage in recurrent self-injury (three or more episodes) but those who had been physically abused were more than seven times as likely to engage in intermittent self-injury (one to two episodes). The finding that the type of abuse experienced may impact on the frequency of NSSI was supported in a study by Whitlock et al. (2006). In their sample of university students, single versus repeated incidents of NSSI were differentially associated with particular types of abuse. When compared to students who had never self-injured, those who had self- injured once were more likely to report past emotional abuse, while those who had self-injured repeatedly were more likely to report past emotional and sexual abuse. Child sexual abuse (CSA) has arguably received the most attention in relation to NSSI, potentially as a result of the high prevalence of both CSA and self-injury in populations with BPD (Lieb, Zanarini, Schmahl, Linehan, & Bohus, 2004). Indeed, CSA was a significant predictor of NSSI in people with BPD in a longitudinal 10-year study (Zanarini et al., 2011). However, meta-analytic and review studies have not supported such definitive results. A recent meta-analysis of 43 studies demonstrated a small relationship between CSA and self-injury, with CSA explaining at most 5% of the variance in NSSI aetiology (Klonsky & Moyer, 2008). 3.3.1 Child maltreatment Furthermore, this relationship became more tenuous when other psychological and environmental risk factors (e.g., 42 dissociation, family functioning) were controlled for. The authors concluded that (1) CSA is either a proxy risk factor for NSSI in that the relationship between CSA and NSSI exists because of shared psychological risk factors or (2) CSA may mediate the relationship between other risk factors and NSSI. This conclusion is partially supported by a review that identified CSA as a non-specific risk factor for engaging in self-harm irrespective of suicidal intent (Maniglio, 2011). In light of the conflicting evidence regarding the contribution of different types of childhood maltreatment to the development of NSSI, it is important for researchers to examine factors that may mediate or moderate the relationships between types of maltreatment and self-injury to shed light on these relationships. For example, when Weierich and Nock (2008) examined the impact of CSA and physical and emotional abuse/neglect on adolescent self-injury, they found that NSSI was only significantly related to CSA. Further analyses revealed that this relationship was fully mediated by Post-traumatic Stress Disorder (PTSD) symptom clusters of avoidance/numbing and re-experiencing. Shenk, Noll, and Cassarly (2010) similarly found that posttraumatic stress symptoms fully mediated the relationship between child abuse and NSSI among female adolescents. 3.3.2 Peer victimisation Another factor that has been implicated in the development of NSSI among young people is bullying, but it has not been extensively investigated. Research from Aotearoa New Zealand showed that reports of bullying were greater among secondary school students who had self-harmed than those who had not self- harmed, but did this study did not distinguish between NSSI and DSH (Garisch & Wilson, 2010). Peer victimisation was identified as a risk factor for NSSI in a Swedish study where secondary school students were interviewed at two time points, 12 months apart (Jutengren et al., 2011), but did not predict NSSI among a community group of adolescents in the United States (Heilbron & Prinstein, 2010). Indeed, the relationship between different types of peer victimisation and NSSI appears complex. An investigation of peer-nominated overt (e.g., hitting) and 43 relational (e.g., exclusion, spreading rumours) victimisation in community-based adolescents showed that female victims of overt bullying were less likely to report having self-injured, while male victims of overt bullying were more likely to report having self-injured (Heilbron & Prinstein, 2010). In a sample of female, adolescent inpatients, the relationship between peer relational difficulties (i.e., overt victimisation, relational victimisation, and negative friendship interactions) and NSSI was mediated by emotional dysregulation (Adrian, Zeman, Erdley, Lisa, & Sim, 2011). Once again, further research is needed to replicate and clarify these findings. relational (e.g., exclusion, spreading rumours) victimisation in community-based adolescents showed that female victims of overt bullying were less likely to report having self-injured, while male victims of overt bullying were more likely to report having self-injured (Heilbron & Prinstein, 2010). In a sample of female, adolescent inpatients, the relationship between peer relational difficulties (i.e., overt relational (e.g., exclusion, spreading rumours) victimisation in community-based adolescents showed that female victims of overt bullying were less likely to report having self-injured, while male victims of overt bullying were more likely to report having self-injured (Heilbron & Prinstein, 2010). In a sample of female, adolescent inpatients, the relationship between peer relational difficulties (i.e., overt 3.4 Summary and limitations Given the diversity of the individual, psychological, and environmental correlates and risk factors for self-injurious behaviour, it is worth investigating whether there are particular typologies of NSSI. In two recent studies with university students, researchers have attempted to clarify whether people who self- injure can be divided into clinically distinct subgroups (Klonsky & Olino, 2008; Whitlock, Muehlenkamp, & Eckenrode, 2008). Utilising latent class analyses, both studies provided evidence for distinct typologies of NSSI, which were based on factors such as the form, frequency, contextual features, and functions of the self- injurious behaviour. Although identifying clinically relevant distinctions between subgroups of people who self-injure is a promising start to unpacking the complexity and heterogeneity of NSSI, there are still many unresolved questions that need to be addressed. When considered in isolation, the majority of the risk factors that have been empirically tested do not only contribute to the aetiology of NSSI, but are predictive of numerous psychopathological outcomes (Nock, 2010). Little research has been conducted on the interaction of multiple risk factors to determine how these interactions influence the development and maintenance of NSSI (Gratz, 2003). Furthermore, Fliege et al. (2009) have disputed whether many of the characteristics identified in the literature as predictors of NSSI are, in fact, risk 44 factors for self-injury. After reviewing 59 studies on NSSI, they concluded that many of the risk factors cannot be characterised as such because NSSI is typically investigated cross-sectionally rather than longitudinally, there is insufficient evidence that the risk factors were present before the self-injurious behaviour began, and none of the studies prospectively investigated new incidents of NSSI. Certainly, Glenn and Klonsky (2011) identified that several well-established correlates of NSSI failed to prospectively predict self-injurious behaviours. In sum, identifying the contribution of varied risk factors and correlates to the development of NSSI is essential to create effective prevention programmes, and much more work is needed in this area. However, even if a solid, comprehensive evidence base about these factors did exist, this information would still be of limited treatment utility because the factors that are implicated in the development of problem behaviours do not necessarily maintain those behaviours (Cipani & Schock, 2007). In other words, knowing who is more likely to self-injure does not provide the answer to why people self-injure. 3.4 Summary and limitations To understand why people engage in NSSI, it is essential to examine how these behaviours are reinforced and maintained over time by identifying the reasons or motivations people give for their self-injury, and the antecedents and consequences of these behaviours. 4. SELF-REPORTED REASONS AND MOTIVATIONS FOR SELF-INJURY 4. SELF-REPORTED REASONS AND MOTIVATIONS FOR SELF-INJURY Reasons for NSSI can be divided into two broad categories: those that are motivated by intrapersonal factors and those that are motivated by interpersonal factors (Yates, 2004). Intrapersonal reasons are driven by a person’s desire to alter their internal state, while interpersonal reasons are driven by a person’s desire to communicate with others in their environment (Yates, 2004). In the following section, I review the most commonly reported intrapersonal and interpersonal reasons for self-injury, across a range of populations, before discussing the limitations of these studies. It should be noted that I have followed the precedent set by other researchers in using the words ‘reason’ and ‘motivation’ interchangeably to refer to the purpose of the self-injurious behaviour (Klonsky, 2007). 45 Huang (2009) as follows: The basic idea is that during emotional experience (‘‘how do I feel?’’) and emotion perception (‘‘is the rat afraid?’’; ‘‘is my friend angry?’’; ‘‘is my dog guilty?), representations of internal sensations from the body (experienced as affect) and external sensations from the world are made meaningful via the process of categorization (just as visual sensation are transformed into sight). This categorization uses emotion knowledge that has been learned via prior experience. Together, different recipes (the combination and weighting of these three sources of information—sensations from the world, sensations from the body, and prior experience) create the variety of mental states that represent your own feelings of your experience or someone else’s behavior named with emotion words. (p. 431) Although quantitative explorations of the reasons and motivations for NSSI cannot do justice to this complexity, they can provide insight into people’s internal experiences and how these experiences contribute to self-injury. Although quantitative explorations of the reasons and motivations for NSSI cannot do justice to this complexity, they can provide insight into people’s internal experiences and how these experiences contribute to self-injury. experiences and how these experiences contribute to self-injury. 4.1 Intrapersonal Reasons The most common self-reported intrapersonal reasons for NSSI reflect a need to decrease, eliminate, or escape from unwanted negative emotions (e.g., anger), cognitions (e.g., suicidal ideation, traumatic memories), affect states (e.g., dissociation), and physiological conditions (e.g., tension). Additionally, many people report self-injuring to punish themselves which, in all likelihood, stems from the experience of negative emotions (Chapman et al., 2006) or cognitions as will be discussed later in this chapter. Given my approach of inspecting individual items where possible, I have divided the reasons reported in the literature according to whether they fall under the categories of emotions/affect, cognitions, or physiological states. Of course, it should be noted that these intrapersonal events are all intricately interconnected and, as a result, they overlap experientially and conceptually. The complex interplay of experiences that results in named emotion is explicated by Barrett, Gendron, and Huang (2009) as follows: 6 To maintain consistency, all the percentages in this chapter have been reported without decimal places. Where percentages were reported with one or more decimal places in the original source, I have rounded these figures to zero decimal places. 4.1.1 Emotional reasons Emotional motivations for self-injury predominate in measures designed to assess why people self-injure, and can be conceptualised along two intersecting continua of arousal (i.e., bodily activation) and valence (i.e., pleasantness) (Barrett, 1998). The conceptualisation of NSSI as a strategy for regulating aversive emotions 46 or mood states has led researchers to primarily focus on the role of negatively, rather than positively, valenced emotions in the maintenance of self-injury. 4.1.1.1 Specific emotions A number of specific emotions, such as anger, frustration, loneliness, and excitement have been investigated in studies of why people self-injure; these emotions have been differentially endorsed as reasons for NSSI across diverse populations of study participants. Reasons for self-injury that describe the elimination or expression of anger and frustration, both high arousal, negative emotions, have been reported by a majority of the participants in several studies (Brown et al., 2002; Laye-Gindhu & Schonert-Reichl, 2005; Nixon et al., 2002). It is typically unclear whether the anger and frustration experienced by participants is directed at themselves or other people as studies seldom differentiate between self- and other-directed anger. One of the few studies that did make this distinction found that 63% of a community group of adolescents reported self-injuring because they were angry with themselves, whereas only 39% reported self-injuring because they were angry with others (e.g., parents, friends) (Laye-Gindhu & Schonert-Reichl, 2005). 6 Low arousal, negative emotions that have been identified as reasons for NSSI in both community and psychiatric populations include feeling empty, lonely, and helpless (Favazza & Conterio, 1989; Nixon et al., 2002; Oyefeso et al., 2008; Swannell, Martin, Scott, Gibbons, & Gifford, 2008). Like high arousal, negative emotions, the particular low arousal, negative emotions measured, and the extent to which they are endorsed, depends on what questionnaire is used and the population sampled. One emotion that has been examined in several NSSI studies is loneliness. In the first large-scale survey study to examine the motivations for self-injury, 47% of an all female sample reported injuring themselves ‚to feel less lonely‛ (Favazza & 6 To maintain consistency, all the percentages in this chapter have been reported without decimal places. Where percentages were reported with one or more decimal places in the original source, I have rounded these figures to zero decimal places. 47 Conterio, 1989, p. 286). 4.1.1 Emotional reasons In contrast, 76% of adolescent inpatients reported injuring themselves to ‚reduce a feeling of being utterly alone‛ (Swannell et al., 2008, p. 101) and 63% of community-based adolescents reported injuring themselves because they ‚felt all alone‛ (Laye-Ghindhu & Schonert-Reichl, 2005, p. 452). Few positive emotions have been investigated as reasons for NSSI and when they are included in questionnaires, the results tend to be variable. For example, self- injuring for excitement was endorsed by 53% of the adolescent inpatients in one study (Swannell et al., 2008), but by only 7% of the adolescent inpatients in another study (Nixon et al., 2002). This large discrepancy is surprising considering the items were worded very similarly and both study samples consisted of adolescent inpatients. The instructions on how to complete the questions may have differed between measures, but these instructions were not reported. Concluding whether excitement motivates adolescents to self-injure is impossible from these studies. It is equally as difficult to determine whether excitement is a common motivation for NSSI in adult inpatient or community populations because other studies that have examined self-injuring for excitement Concluding whether excitement motivates adolescents to self-injure is impossible from these studies. It is equally as difficult to determine whether excitement is a common motivation for NSSI in adult inpatient or community populations because other studies that have examined self-injuring for excitement did not report how many of their participants endorsed this reason (Kumar, Pepe, & Steer, 2004; Osuch, Noll, & Putnam, 1999). It is likely that self-injuring for excitement is motivated by boredom, which may be more common among adolescent and adult inpatients in restricted environments, but further research is necessary to determine whether this is the case. 4.1.1.2 Specific affect states The two affect states that are most commonly investigated in studies about the reasons and motivations for self-injury are depression and dissociation. Over 80% of the community-based and inpatient adolescent participants in two separate studies endorsed self-injuring because they were depressed or trying to cope with depressed mood, making this the most commonly endorsed motivation for NSSI in these samples (Laye-Gindhu & Schonert-Reichl, 2005; Nixon et al., 2002). A lower 48 rate of endorsement (58%) for the item, ‚to feel less depressed‛ (p. 286), was found among women in Favazza and Conterio’s (1989) study. rate of endorsement (58%) for the item, ‚to feel less depressed‛ (p. 286), was found among women in Favazza and Conterio’s (1989) study. In general, items about using NSSI to manage dissociation do not appear to rate as highly as depression items. Among two adult samples, 38% to 57% of people reported self-injuring to end dissociative states (Briere & Gil, 1998, Favazza & Conterio, 1989). Similar results were found in a group of hospitalised adolescents where just under half reported self-injuring ‚to stop feeling numb or out of touch with reality‛ (Nixon et al., 2002, p. 1337). In contrast, 87% of the inpatient adolescents in Swannell et al.’s (2008) study endorsed self-injuring to ‚decrease an empty feeling‛ (p. 101) making this the second most highly endorsed reason for NSSI in this sample. Non-suicidal self-injury has not only been reported as an anti-dissociation strategy, but also has occasionally been examined as a means of inducing dissociation. A substantial proportion of adolescents (30%-82%) have reported injuring themselves to induce feelings of numbness, which provided some respite from their overwhelming emotions (Laye-Ghindhu & Schonert-Reichl, 2005; Swannell et al., 2008). Apart from these two studies, self-injuring to induce, rather than reduce, dissociation does not appear to have been widely investigated. 4.1.1.3 General emotional experiences Some studies included items that lacked specificity and referred to general, aversive emotional or affective experiences, such as ‚to stop bad feelings‛ (Lloyd- Richardson et al., 2007, p. 1189), ‚to cope with emotional pain‛ (Oyefeso et al., 2008, p. 230), and ‚to get rid of intolerable emotions‛ (Klonsky, 2009, p. 263). Although these items were endorsed by a majority of the participants in these studies, it is impossible to isolate the specific emotions they refer to. 4.1.1.2 Specific affect states Along with emotional reasons for NSSI, particular cognitions, especially to do with suicidal ideation or traumatic memories, have been identified by numerous 49 people as motivations for their self-injurious behaviour. Self-injury, as a strategy to prevent suicidal ideation or attempts, has been reported by 41% to 74% of community-based and inpatient adolescents (Laye-Gindhu & Schonert-Reichl, 2005; Nixon et al., 2002; Swannell et al., 2008). Certainly, endorsing self-injury as survival is consistent with the consumer perspectives presented in Chapter 1 (see pp. 15-16). However, in studies where participants have been asked to rank their reasons for NSSI, averting suicidal thoughts has not been rated very highly. Only 7% of women with BPD rated ‚to prevent me from acting on suicidal feelings‛ as one of their top three reasons for NSSI (Shearer, 1994, p. 525), while 6% of university students rated ‚to avoid the impulse to attempt suicide‛ as a primary reason for self- injury and 18% rated it as a secondary reason (Klonsky, 2009, p. 264). A number of people also report using self-injury as a way of distracting themselves from traumatic memories. In a clinical sample, in which over 90% of the participants had been sexually abused, 58% reported self-injuring as a ‚distraction from memories‛ and 39% to ‚stop flashbacks‛ (Briere & Gil, 1998, p. 615). Using self- injury to avoid or distract oneself from negative memories has also been reported by more than half of two inpatient adolescent samples (Nixon et al., 2002; Swannell et al., 2008). A lower rate was found among a group of women diagnosed with BPD—only 15% endorsed self-injuring to ‚keep bad memories away‛ (Shearer, 1994, p. 525)— but caution is required when interpreting these results as the women who participated in this study were asked to rank their top three reasons for NSSI. Consequently, the results may underestimate the number of women with BPD who self-injure to avoid or escape negative memories. Unfortunately, detailed information about the content of the memories or flashbacks is not reported in any of these studies. 4.1.2.2 General cognitive states 4.1.3 Physiological reasons 4.1.3.1 Releasing pressure or tension 4.1.2.2 General cognitive states As with the general affect items, some researchers have included vague items about the cognitive motivations for NSSI in their questionnaires. For example, 72% 50 of the women in Favazza and Conterio’s (1989) sample reported self-injuring ‚to control their mind when it is racing‛ (p. 286), but the content of these thoughts is unclear. Likewise, 55% of a group of secondary school students recalled self-injuring to get their mind off their problems and 20% to prevent themselves from ‚thinking bad thoughts‛ (Laye-Gindhu & Schonert-Reichl, 2005, p. 452). Once again, these items are too general to provide sufficient information about the specific thoughts or problems that motivated these people to injure themselves. 4.1.3.1 Releasing pressure or tension Items that refer to physiological reasons for self-injury often describe NSSI as a means through which to release tension. It should be noted that the release of tension is closely linked with self-injuring to cope with negative emotions. Indeed, it is probable that experiences of tension are triggered by intense emotions (Chapman et al., 2006). Furthermore, a number of questionnaire items actually specify the release of emotional tension. Eighty-five percent of a group of university students reported that their primary motivation for self-injuring was ‚to release emotional pressure that builds up inside me‛, while the remaining 15% reported it as their secondary motivation for engaging in NSSI (Klonsky, 2009, p. 263). In Briere and Gil’s (1998) study, 77% of the participants reportedly self-injured to ‚release pent-up feelings‛ (p. 615). Measures used in other studies have not specified the release of emotional tension per se, but have rather referred to the elimination of tension in general. In two studies, one involving women diagnosed with BPD (Kleindienst et al., 2008) and one involving adult psychiatric inpatients (Herpertz, 1995), tension release was the most frequently reported motivation for NSSI. Similarly high rates of endorsement (45%-74%) for the utility of NSSI as a tension release or relaxation strategy have been found in other studies (Favazza & Conterio, 1989; Laye-Ghindhu & Schonert-Reichl, 2005; Nixon et al., 2002). 51 4.1.3.2 Inducing stimulation 4.1.3.2 Inducing stimulation 4.1.3.2 Inducing stimulation Aside from using self-injury to release tension, people have also reported self- injuring to produce stimulation, an experience which is often likened to a drug high (Kleindienst et al., 2008; Oyefeso et al., 2008; Swannell et al., 2008). For example, 47% of a clinical sample of women with BPD endorsed using NSSI to ‚achieve a kick or high‛ (Kleindienst et al., 2008, p. 233) and 66% of a clinical sample of adolescents endorsed using NSSI ‚to experience a high that feels like a drug high‛ (Swannell et al., 2008, p. 101). Inducing stimulation through self-injury can be thought of as comparable to self-injuring for excitement, which was discussed earlier. Another motivation for self-injury that is rarely investigated and, when it is examined, seldom endorsed is using self-injury to induce sexual arousal. In a sample of women with BPD, only 5% reported self-injuring ‚to provide a sense of physical release that feels much like sexual release‛(Shearer, 1994, p. 525), while 12% of another clinical sample purportedly self-injured to experience ‚sexual arousal or pleasure‛ (Briere & Gil, 1998, p. 615). The role of pain as a stimulus has also been explicitly investigated in some of the measures of the reasons and motivations for self-injury. However, in these items, physical pain is presented as a means through which people can gain control over, or distract themselves from, unpleasant emotions or affect states. For example, 92% of adolescent inpatients reported self-injuring because physical pain distracted them from their emotional pain (Swannell et al., 2008). Other items that have been endorsed by more than half of the participants in two separate studies include using self-injury to ‚produce a pain I can control‛ (Klonsky, 2009, p. 263) and ‚to feel something, even if it was pain‛ (Lloyd-Richardson et al., 2007, p. 1189). In all of these items, the primary motivation for NSSI appears to be the control or regulation of affect, which is made possible through the distraction provided by experiencing pain. 52 4.1.4 Self-punishment 4.1.4 Self-punishment Self-punishment is often one of the most highly endorsed reasons for self- injury. In three adult clinical samples, self-punishment received the highest level of endorsement of all the reasons provided for NSSI (Briere & Gil, 1998; Brown et al., 2002; Shearer, 1994). However, it is a construct that defies simple classification within the emotional, cognitive, or physiological categories discussed thus far because it could feasibly fit within all three of these categories. Some studies have demonstrated that the desire to punish oneself through self-injury can be triggered by positive and negative experiences or affect states. For example, 50% of hospitalised adolescents reported self-injuring to ‚punish self for being bad/bad thoughts‛ and 26% reported self-injuring to ‚punish self for feeling good‛ (Nixon et al., 2002, p. 1337). In another study of adolescent inpatients, a majority endorsed the motivations to ‚punish myself for being bad‛ (84%), to ‚punish myself for positive feelings/experiences‛ (68%), and to ‚punish myself for telling secrets‛ (53%) (Swannell et al., 2008, p. 101). Self-punishment was also primarily driven by negative self-evaluations in a sample of women with BPD; 49% endorsed using self-injury ‚to punish myself for being ‚bad‛ in some way (e.g., angry, selfish, etc.)‛ (Shearer, 1994, p. 525). In Oyefeso and colleagues’ (2008) study, 68% of the participants used NSSI to express their self-hatred, while Laye-Gindhu and Schonert-Reichl (2005) found that more than 60% of the adolescents in their study were motivated to self-injure because they did not like themselves and felt like failures. It is hardly surprising that people report injuring themselves as punishment for both positive and negative experiences, given that NSSI is associated with low self-esteem (Brausch & Gutierrez, 2010; Claes, Houben, et al., 2010). If the desire to self-punish is driven by low self-worth, it is plausible that people would blame themselves for any negative experiences they have and feel undeserving of positive experiences. In such cases, both negative and positive experiences may lead to feelings of guilt and shame which, in turn, could trigger self-injurious behaviour. It 53 is thus clear from the examples discussed that self-injury can be used as a form of punishment in the context of negative emotions and cognitions about oneself, and for experiencing positive emotions. 4.2 Interpersonal Reasons Interpersonal reasons for self-injury, which reflect support-seeking or avoidance, have not been investigated as thoroughly in the literature as intrapersonal reasons for self-injury, and when they have been included in measures assessing why people self-injure, they are seldom endorsed as highly as intrapersonal reasons. Concerns about perpetuating damaging stereotypes about self-injury as a purely attention-seeking behaviour may have encouraged researchers to avoid investigating intrapersonal reasons for NSSI (Nock, 2008). However, some people do claim to use self-injury to communicate with those around them or to exert control over their interpersonal environment (Nock, 2008; Yates, 2004) and, as a result, it is important to examine these motivations. 4.2.2 Avoidance Even less commonly investigated interpersonal reasons involve using NSSI to avoid having to complete tasks, fulfil responsibilities, or face the consequences of particular actions. More than 50% of a group of secondary school students reported self-injuring ‚to avoid school, work, or other activities‛ (Lloyd-Richardson et al., 2007, p. 1189). However, in another sample of secondary school students, only 16% endorsed self-injuring because they wanted to avoid certain tasks (Laye-Gindhu & Schonert-Reichl, 2005). Further research is necessary to determine whether avoiding responsibilities is a common motivation for NSSI across different study populations. 4.2.1 Support-seeking As is common with all of the research investigating motivations for NSSI that I have presented thus far, the number of people who report using self-injury as a way of eliciting attention and support from others varies between studies. In one adult clinical sample, 40% of the participants endorsed self-injuring to ‚get attention, ask for help‛ but only 16% reported injuring themselves to get attention from their therapist and 9% to receive medical attention (Briere & Gil, 1998, p. 615). The discrepancy in these ratings may indicate that NSSI is used more frequently to elicit support and attention from family members or friends, rather than clinicians. Indeed, over 50% of a sample of secondary school students were motivated to injure themselves to receive more attention from their friends or parents (Lloyd- Richardson et al., 2007). Similarly, 41% of community-based adolescents reported injuring themselves so that others would notice them (Laye-Gindhu & Schonert- Reichl, 2005). Much lower rates of attention-seeking were reported in an adolescent inpatient population; only 10% recalled self-injuring to ‚get care or attention from 54 others‛ (Nixon et al., 2002, p. 1337). Among a clinical group of women diagnosed with BPD, 17% reported using self-injury as an indirect way of obtaining care and support from others because they felt unable to request this support verbally (Shearer, 1994). The need for support and care often seems to occur in the context of distressing, overwhelming emotions. Over 70% of one group of adolescent inpatients reported injuring themselves to demonstrate to others how angry and hurt they felt (Swannell et al., 2008), whereas approximately one third of two different samples of secondary school students were motivated to hurt themselves to show others how desperate they were (Laye-Gindhu & Schonert-Reichl, 2005; Lloyd-Richardson et al., 2007). Some adolescents have also reported using NSSI as a way to hurt, shock, irritate, or get back at people (Laye-Gindhu & Schonert-Reichl, 2005; Swannell et al., 2008), or to feel more connected to a group of peers (Lloyd-Richardson et al., 2007). 4.2.2 Avoidance 4.3 Limitations The literature on self-reported reasons and motivations for NSSI is limited in several ways. First, researchers investigating why people injure themselves on purpose typically develop questionnaires based on clinical experience and extant publications. As a result, only three self-report measures designed to assess why people self-injure have been used in more than one published study: the Functional Assessment of Self-mutilation (Lloyd-Richardson et al., 2007), the Self-injury 55 Motivation Scale (Osuch et al., 1999), and the Inventory of Statements About Self- injury (Klonsky & Glenn, 2009). The measures used in each study typically include various numbers of items about the same concepts (e.g., managing negative emotions through NSSI), but the different wording used in these items makes it difficult to reliably compare study results. Not only do some items include specific emotions, affect states, or cognitions, while others simply refer in general to negative feelings or thoughts, but the verbs used in each item differ. Some of the verbs contained in the items discussed include: to get rid of, to cope with, to express, to distract, to control, to release, to decrease, to feel, and to produce. These verbs hint at different functional mechanisms underlying the motivations for self-injury and, as such, items that include the same emotion or cognition word but have different verbs cannot be compared directly. For example, someone who endorses self-injuring to get rid of unpleasant feelings may not endorse self-injuring to express unpleasant feelings, as the latter implies a communicative function. Furthermore, each of these questionnaires measures the reasons for NSSI in different ways. In some studies, participants rated their reasons for NSSI on likert scales (e.g., Kleindienst et al., 2008; Laye-Gindhu & Schonert-Reichl, 2005); in others, they identified their primary and secondary motivations (Klonsky, 2009), ranked their top three motivations for self-injury (Shearer, 1994), or simply checked yes or no to identify which motivations were relevant to their experience of NSSI (Oyefeso et al., 2008). These diverse levels of measurement make it difficult to compare results across studies. Second, the length of time since the participants’ most recent episode of NSSI and their completion of the study measures varies widely both within and between studies. In Favazza and Conterio’s (1989) study, 64% of the participants had injured themselves in the month prior to the study whereas only 13% of the participants in Oyefeso’s et al. (2008) study had self-injured in the previous 12 months. 4.3 Limitations Moreover, many authors do not report how much time has elapsed between people’s most 56 recent self-injurious behaviour and their study participation, or even if that information was collected (e.g., Herpertz, 1995; Osuch et al., 1999; Swannell et al., 2008). One study did specify that participants had to have injured themselves within the previous two months to be included in the research (Brown et al., 2002), while another only included participants who had self-injured every month for the six months prior to the study (Nixon et al., 2002). Such specifications, however, are rare and the variation in time since last self-injury is a concern given that all of the studies reviewed thus far have relied on retrospective self-report and the accuracy of participants’ memories are likely to decrease as the length of time since their last episode of NSSI increases (Tourangeau, 2000). Finally, it is possible that the self-reported reasons for NSSI do not accurately reflect people’s motivations for injuring themselves. Rather, people may be motivated to provide socially desirable responses (Nock, 2008) or their reasons may be post-hoc attributions to help them to make sense of their self-injury (Yates, 2004). The way in which people retrospectively appraise and justify their self-injurious behaviour, however, may be as clinically relevant as any factors that are truly motivating that behaviour: If people recall successfully escaping aversive affect states or accessing support through self-injuring, they are more likely to use NSSI in the future when they feel similarly distressed and overwhelmed (Chapman & Dixon-Gordon, 2007; Kamphuis, Ruyling, & Reijntjies, 2007). 4 4 S 4.4 Summary Several researchers therefore have retrospectively or prospectively examined specific emotional and physiological antecedents and 58 consequences of self-injurious behaviour; cognitive antecedents and consequences have been largely neglected. 4.4 Summary In spite of the limitations associated with self-report studies of the reasons and motivations for self-injury, it is reasonable to conclude from this literature that self-injury is used primarily to alleviate or escape from aversive intrapersonal experiences, including emotions, cognitions, and physiological states (Klonsky, 2007; Nock, 2008). Although interpersonal reasons have been examined in fewer studies, where they have been included they are seldom as highly endorsed as intrapersonal reasons for NSSI. However, a substantial number of people still report using NSSI to 57 communicate with, or exert influence over, others and, as such, interpersonal reasons for NSSI cannot be dismissed as irrelevant. Rather, it is apparent that people injure themselves for multiple intrapersonal and interpersonal reasons. In studies where the actual number of reasons for NSSI has been reported, rates range from a median of four among drug dependent adults (Oyefeso et al., 2008) to an average of eight among adolescent inpatients (Nixon et al., 2002). However, these figures apply to participants’ general experiences of self- injury rather than to specific episodes. Only one study, to date, has examined the motives given by participants for a single episode of NSSI: Women with BPD identified an average of 10 reasons for their most recent self-injury (Brown et al., 2002). This study demonstrates that multiple reasons or motivations also underlie individual episodes of self-injury, at least amongst a sample of women with BPD. Further research is necessary to determine whether other populations, such as inpatient or community-based adolescents, would similarly report multiple motivations for single episodes of NSSI. 5. SELF-REPORTED ANTECEDENTS AND CONSEQUENCES OF SELF-INJURY The literature on self-reported reasons for NSSI demonstrates that people are primarily motivated to hurt themselves to reduce, eliminate, or gain control over aversive intrapersonal experiences. In particular, there appears to be strongest support within these studies for reasons to do with negative emotions or affect states. Unfortunately, this research only suggests, but does not explicitly investigate, the emotional antecedents for NSSI, and fails to provide evidence that these emotions actually do change following acts of self-injury. Rather the process of change is typically implied in the way in which items are worded. For example, endorsing a reason, such as ‘I self-injure to relieve negative emotions’, assumes rather than directly asks whether negative emotions are present before and are eliminated after self-injuring. 5.1 Retrospective Studies Antecedents and consequences of NSSI are usually identified by asking research participants to indicate which affect or physiological states they commonly experience before and after NSSI. People routinely report decreased negative affect and increased positive affect following their engagement in self-injurious behaviour (Chapman & Dixon-Gordon, 2007; Kamphuis et al., 2007; Kemperman, Russ, & Shearin, 1997; Kleindienst et al., 2008). In one study, women recruited from a self- injury support group completed a measure of negative and positive mood states, in relation to how they felt immediately before, after, and one day following episodes of NSSI (Kamphuis et al., 2007). Anger, depression, fatigue, and tension scores decreased significantly following self-injurious behaviour, while vigour (e.g., energetic, alert) scores showed a significant increase. Significantly more women reported a decrease in tension than any other internal state, leading the authors to conclude that NSSI functions primarily as a tension reduction strategy. Similar results have been obtained in other studies. More than 90% of a sample of women diagnosed with BPD reported that the tension and pressure they felt prior to injuring themselves was significantly reduced after NSSI, while their ratings of positive affect items (e.g., ‚relaxed‛, ‚euphoria‛) were significantly higher after NSSI (Kleindienst et al., 2008). Analyses of change scores (i.e., rates of change from before to after NSSI) for university students showed that the high arousal, negative affect state—overwhelmed—decreased the most while low arousal, positive affect states—relief, calmness, satisfaction, and relaxation—increased the most (Klonsky, 2009). However, not all people experience a decrease in negative emotion following NSSI. Almost half of a group of female inmates reported that the main emotional consequence of their most recent episode of NSSI was negative (e.g., sadness, guilt) (Chapman & Dixon-Gordon, 2007). Other researchers have similarly found that self- 59 conscious emotions, such as shame and guilt, are reported to increase following engagement in self-injurious behaviour (Kemperman et al., 1997; Laye-Gindhu & Schonert-Reichl, 2005). It thus appears that while NSSI is an adaptive strategy to reduce overwhelming tension and certain aversive emotions, the act of self-injuring can also evoke other negative emotions (e.g., guilt). Given that the experience of aversive emotions precipitates NSSI, negative feelings consequent to self-injury have the potential to trigger future self-injurious behaviour, leading to a vicious cycle. 5.2 Prospective Studies The application of prospective methodologies, such as ecological momentary assessment (EMA), to the study of NSSI is a recent empirical development. To date, only two studies have used EMA to examine the antecedents and consequences of NSSI. In one of these studies, women diagnosed with Bulimia Nervosa used palmtop computers to report their emotions, stressors, and any self-injurious behaviours or Bulimic symptoms at six, semi-random times a day for two weeks (Muehlenkamp et al., 2009). They were also asked to complete an additional set of questions each time they engaged in one or more of a set of pre-identified behaviours, including self- injury. Analyses of the NSSI episodes showed a significant increase in negative affect and decrease in positive affect in the lead-up to the self-injurious behaviour. While positive affect increased significantly following NSSI, there was no significant reduction in negative affect. The authors speculate that the lack of a significant decrease in negative affect may be an artefact of their small sample size and cannot be generalised to people without Bulimia who self-injure (Muehlenkamp et al., 2009). Given a larger sample, it may have been useful to examine the trajectories of particular affect states that precede and follow NSSI episodes. By analysing global positive and negative affect scores, significant decreases in specific negative emotions or affect states may have been overlooked. As previously discussed, evidence from retrospective self-report studies of the emotional antecedents and consequences of NSSI suggests that some 60 negative emotions (e.g., anger) typically decrease following self-injury while other emotions (e.g., shame) may increase. Ecological momentary assessment was also utilised by Nock and colleagues (2009) to investigate the antecedents and consequences of self-injurious thoughts and behaviours. Over two weeks, participants completed a set of questions twice a day and following any self-injurious thoughts or behaviours. Analyses showed that experiencing self- and other-directed anger, self-hatred, rejection, and numbness predicted NSSI episodes, while contrary to expectations, feeling sad or worthless decreased the likelihood of self-injury. 5.3 Summary and limitations As was evident in studies of self-reported motivations for NSSI, one of the major limitations of the literature on the antecedents and consequences of self-injury is once again the failure of researchers to use standardised measures. Only two studies used a validated measure of emotion words (Kamphuis et al., 2007; major limitations of the literature on the antecedents and consequences of self-injury is once again the failure of researchers to use standardised measures. Only two studies used a validated measure of emotion words (Kamphuis et al., 2007; Muehlenkamp et al., 2009); all others simply examined a range of emotions and affect states, presumably drawn from the NSSI literature. For example, Chapman and Dixon-Gordon (2007) report that they used a ‚standard list of nine emotions‛ (p. 546) but it is unclear where this list is derived from or why the emotions are considered standard. This use of disparate emotions and affect states makes it difficult to compare results across studies. Muehlenkamp et al., 2009); all others simply examined a range of emotions and affect states, presumably drawn from the NSSI literature. For example, Chapman and Dixon-Gordon (2007) report that they used a ‚standard list of nine emotions‛ (p. 546) but it is unclear where this list is derived from or why the emotions are considered standard. This use of disparate emotions and affect states makes it difficult to compare results across studies. Although prospective investigations of the antecedents and consequences of NSSI involve real-time assessments of emotional states and are thus an improvement on retrospective studies, they nonetheless rely on participants being able to identify and accurately report on their emotional experience (Muehlenkamp et al., 2009) There is also the potential for such studies to have an iatrogenic effect in that people may be more likely to think about, and engage in, self-injury because they are participating in a daily study about NSSI. 61 6. LABORATORY STUDIES While phenomenological research of NSSI provides useful information about how the process of self-injury is perceived by those who engage in these behaviours, one of the drawbacks of most of these studies is their reliance on the single method of retrospective self-report. To address this limitation, some researchers have used self-injury proxies to try to simulate the psychophysiological experience of NSSI within a laboratory setting, while concurrently assessing self-reported affect states. While the external validity of such proxies is debatable (Klonsky, 2007), these studies nonetheless provide further evidence in support of the hypotheses that self-injury serves to reduce negative affect states and aversive psychophysiological arousal. Both the cold pressor test (Russ et al., 1992) and guided imagery (Haines, Williams, Brain, & Wilson, 1995; Welch, Linehan, Sylvers, Chittams, & Rizvi, 2008) have been used to simulate NSSI within laboratory settings. In one study, participants completed the cold pressor test by immersing their left hands into a 10°C cold-water bath for four minutes, once a day, for three consecutive days. Analyses of averaged mood ratings before and after the cold pressor test showed that women who experienced no pain during NSSI reported significantly lower depression, anxiety, anger, and confusion scores, and significantly higher vigour scores after administration of the test (Russ et al., 1992). In contrast, women who experienced pain during NSSI did not report any significant differences in emotion. The only significant result for the control group was reduced anxiety. Both the cold pressor test (Russ et al., 1992) and guided imagery (Haines, Williams, Brain, & Wilson, 1995; Welch, Linehan, Sylvers, Chittams, & Rizvi, 2008) have been used to simulate NSSI within laboratory settings. In one study, These results lend support to the notion that NSSI functions as an affect regulation strategy although it is unclear why this was not the case for women who experienced pain during self-injury. It is possible that the cold pressor test was an inadequate self-injury proxy for these women, who may rely on reaching a certain threshold of pain during NSSI before they experience an alteration in mood (Russ et al., 1992). Guided imagery has also been used in attempts to reproduce, within a controlled laboratory environment, the psychophysiological arousal patterns 62 associated with NSSI, thus allowing researchers to examine whether NSSI serves a tension reduction function (Haines et al., 1995; Welch et al., 2008). 6. LABORATORY STUDIES In one study, individualised self-injury scripts were developed for men based on their descriptions of their most recent or salient episode of NSSI (Haines et al., 1995). Each script contained four discrete phases: setting the context, the lead-up to the event, the actual event, and the consequences of the event. The men were instructed to visualise the events described in each script while their levels of psychophysiological arousal (e.g., heart rate, skin conductance response) were recorded. Psychophysiological arousal was found to increase in the lead-up to the recalled NSSI episode, decrease during the description of the actual self-injury, and remain low subsequent to the episode. This same pattern of arousal was not observed for scripts detailing neutral, aggressive, or accidental injury events. Self-reported ratings of negative emotions were also significantly lower in the final stage of the self-injury scripts when participants visualised the consequences of their self-injurious behaviour (Haines et al., 1995). Welch and colleagues (2008) replicated the guided imagery procedure conducted by Haines et al. (1995) with a sample of people diagnosed with BPD. Similarly to Haines et al. (1995), participants’ negative emotion and urge to self- injure ratings, along with their skin conductance response rates, were significantly lower following the imagined NSSI episode, thus supporting the conceptualisation of NSSI as a negatively reinforced, emotion regulation strategy (Welch et al., 2008). Laboratory-based studies lend support to the arguments that NSSI serves as both an emotion regulation and tension reduction strategy, but the external validity of self-injury proxies remain questionable (Klonsky, 2007). It is evident from the research reviewed thus far that NSSI is a complex, multi-determined behaviour (Suyemoto, 1998), making it very difficult to reliably recreate the experience of self- injury and any accompanying contextual features within an experimental context. 63 7. CONCLUSION 7. CONCLUSION People endorse both intrapersonal and interpersonal motivations for NSSI, but it appears that intrapersonal reasons, particularly those pertaining to emotion regulation, are reported most frequently across a range of study populations. People endorse both intrapersonal and interpersonal motivations for NSSI, but it appears that intrapersonal reasons, particularly those pertaining to emotion regulation, are reported most frequently across a range of study populations. Indeed, conceptualising NSSI as an emotion regulation strategy is so pervasive that some authors even include this function within their definitions of the behaviour (e.g., Walsh, 2006). However, it is premature to define NSSI solely in terms of emotion regulation because of the limited research that has been conducted both on the role of cognitions in self-injurious behaviours and interpersonal reasons for NSSI. Although cognitive precipitants for NSSI (e.g., suicidal ideation, traumatic memories) are not as highly or consistently endorsed as emotional ones, the impact of certain types of cognitions, such as negative automatic thoughts, has not been investigated. Of course as I discussed earlier, the experience of cognitions and emotions are intricately linked (Barrett et al., 2009). Indeed, it is probable that any cognitive motivations for self-injury are underpinned by the experience of aversive affect (Chapman et al., 2006). However, it is still necessary to analyse the extent to which different negative thoughts precipitate engagement in self-injury. Furthermore, it would be useful to determine whether particular thoughts evoke negative emotional responses that people with a history of self-injury find difficult to manage, or whether the experience of particular emotions triggers intrusive, negative thoughts. It is likely that a threshold is reached through the combination of sufficiently intense negative emotions, thoughts, and physiological arousal. Interpersonal factors have been similarly neglected in studies of why people self-injure (Klonsky, 2007). As a result, caution is required before drawing definitive conclusions about the limited role of interpersonal factors in NSSI. Studies that do examine interpersonal reasons for self-injury need to take into account that people may not endorse these reasons for fear they will be perceived as manipulative or attention-seeking, although social desirability is unlikely to influence responses 64 when questionnaires are completed anonymously (Klonsky, 2007; Nock, 2008). People also may lack insight into interpersonal motivations for their self-injurious behaviour or may not completely understand why they self-injure (Klonsky, 2007). 7. CONCLUSION It is also possible that intrapersonal reasons for NSSI are more salient and therefore easier to recall than interpersonal ones. Furthermore, while the evidence to date suggests the most common interpersonal motivation for NSSI is communicating one’s distress to others to receive attention and support, avoidance has seldom been investigated. However, future studies may find that a strict delineation between intrapersonal and interpersonal factors is not warranted. For instance, it has been suggested that certain interpersonal reasons for NSSI may indirectly serve an affect regulation function (Nock, 2008). People who are unable to effectively regulate their own emotions may look to others for help in managing their emotional well-being; NSSI is the strategy they use to communicate that such help is needed (Nock, 2008). Extant research on why people are motivated to hurt themselves is also limited in that the majority of studies have relied on retrospective self-reports from convenience samples of predominantly Caucasian women. These results may not be generalisable to other populations. For example, studies involving secondary school and university students have demonstrated that some motivations for self-injuring differ according to gender (Klonsky & Glenn, 2009; Laye-Ghindhu & Schonert- Reichl, 2005; Lloyd-Richardson et al., 2007). In one study, males were more likely than females to report injuring themselves to provoke anger in others, whereas females were more likely than males to report injuring themselves as a form of self- punishment (Lloyd-Richardson et al., 2007). In the next chapter, I will discuss how the literature on the reasons for NSSI, and the antecedents and consequences of these behaviours, has been used to inform the development of both single- and multi-function models of self-injury. These models provide frameworks for organising the diverse reasons people give for self- 65 injuring and have been instrumental in further developing our understanding of why people choose to hurt themselves on purpose. injuring and have been instrumental in further developing our understanding of why people choose to hurt themselves on purpose. 66 CHAPTER THREE 1. INTRODUCTION Successful treatment of self-injurious behaviours necessitates a sophisticated understanding of how these behaviours are reinforced and maintained over time. Various functional models of NSSI, each drawing on different psychological theories, have been proposed to account for why people injure themselves on purpose (Klonsky, 2007; Messer & Fremouw, 2008; Suyemoto, 1998; Yates, 2004). Evidence for these models was originally derived from case studies and clinical experience (e.g., Menninger, 1935); more recently, the phenomenological and psychophysiological studies reviewed in Chapter 2 have been used to support the empirical and clinical validity of functional models of self-injurious behaviour. In many cases, the development of what I shall call single-function models has simply been a matter of reframing people’s self-reported motivations for self- injury into specific NSSI models. Single-function models therefore can be conceptualised as being one step up from the reasons and motivations for NSSI that I reviewed in the previous chapter, in that they provide functional categories into which related reasons for self-injury can be grouped; in other words, the single- function models subsume individual reasons, motivations, antecedents, and consequences. For example, emotional reasons, antecedents, or consequences can be grouped within the overarching functional model of affect regulation; people who report self-injuring to release anger or manage feelings of depression are understood to self-injure to regulate negative affect. Individual single-function models, however, cannot adequately account for why people are motivated to hurt themselves on purpose because NSSI, as an overdetermined behaviour, fulfils multiple functions at the same time (Klonsky & Muehlenkamp, 2007; Suyemoto, 1998). To understand why any one person is 67 engaging in NSSI, single-function models thus need to be considered concurrently. Without an overarching theoretical framework to connect these models, any attempt to integrate them is bound to be haphazard and, potentially, of limited clinical utility. Furthermore, I would argue that since little is known about the underlying mechanisms of these models, it is difficult to arrive at a parsimonious solution about which models should be grouped together. Fortunately, several theory-driven, rather than data-driven, models have been proposed more recently, which I will refer to as multi-function models because they subsume individual single-function models. 1. INTRODUCTION Some of these models have focused on specific populations, such as people with BPD (Crowell, Beauchaine, & Linehan, 2009) or those with a history of trauma (Yates, 2004), or particular forms of self- injury, such as cutting (Yip, 2005), and thus have limited applicability for my thesis because I am interested in how NSSI functions across different populations and multiple forms of self-injury. Two multi-function models, however, that are applicable across populations and forms of NSSI are the Four Functions Model (FFM; Nock, 2008; Nock & Prinstein, 2004, 2005) and the Experiential Avoidance Model (EAM; Chapman et al., 2006). These models have been proposed in an attempt to draw together the miscellaneous single-function theories of self-injury and, ultimately, to clarify the functions of NSSI. Unlike the disparate and often under-theorised single-function models, both the FFM and the EAM define function in accordance with social learning and behavioural perspectives (Chapman et al., 2006; Nock, 2008) and, as such, identifying the functions of self-injury requires simultaneously analysing the antecedents and consequences that cause or maintain self-injurious behaviours (Nock, 2008). In this way, functions of NSSI are conceptualised as temporal processes, rather than as categories of particular reasons or motivations. Defining the functions of self-injury in terms of the features which control the behaviour is thought to be fundamental to developing sophisticated treatment and prevention 68 programmes that have the capacity to respond to individual characteristics (Nock, 2008; Nock & Prinstein, 2004, 2005). In this chapter, I briefly describe the basic premise of each of the single- function models, the evidence in support of the hypothesised functions, and the proposed causal mechanisms underlying these functions. I then examine the assumptions and empirical evidence in support of the EAM and FFM, before evaluating these two models within an epistemic values framework. 2. SINGLE-FUNCTION MODELS OF SELF-INJURY According to recent reviews, the following eight single-function models have been repeatedly discussed within the literature on self-injurious behaviours: affect regulation, anti-dissociation, anti-suicide, self-punishment, sexual, interpersonal boundaries, interpersonal influence, and sensation-seeking (Klonsky, 2007; Messer & Fremouw, 2008; Suyemoto, 1998). Although not an exhaustive list, I have chosen to concentrate my discussion on these single-function models because they have regularly featured in the NSSI literature as explanations for why people self-injure. 2.1 Affect regulation 7 In developing the ISAS, Klonsky and Glenn (2009) aimed to include all the functions identified within the NSSI literature, as well as additional functions identified by researchers, clinicians, and 2.1 Affect regulation The affect regulation model proposes that people self-injure to reduce or eliminate intense, negative emotions or affective arousal (Chapman et al., 2006; Klonsky, 2007). Evidence in support of this model is primarily derived from the self- report studies of reasons for NSSI and the laboratory studies that I reviewed in Chapter 2. These studies have typically found that self-injury functions as an adaptive strategy to manage aversive affect and arousal, making affect regulation the most empirically supported, single-function model of NSSI (Klonsky, 2007). Only one measure—the Inventory of Statements About Self-injury (ISAS)— has been developed specifically to assess the single-function models of NSSI by grouping empirically supported reasons and motivations for NSSI into 13 functional subscales (Klonsky & Glenn, 2009). 7 Using the ISAS with a group of university 7 In developing the ISAS, Klonsky and Glenn (2009) aimed to include all the functions identified within the NSSI literature, as well as additional functions identified by researchers, clinicians, and 69 students, Klonsky and Glenn (2009) found that affect regulation was the most highly endorsed function for self-injury. Despite the compelling evidence from these studies that NSSI functions as an affect regulation strategy, there is limited understanding of how self-injuring actually enables people to regulate their emotional experiences. Both psychological and biological explanations have been proposed to account for this effect (Klonsky, 2007). Psychological explanations have focused on the sense of control that may be gained through expressing overwhelming emotions (Suyemoto, 1998) or engaging in self-care (Klonsky, 2007). People who struggle to cope with psychological distress may be more adept at attending to their physical wounds, thus providing them with a sense of mastery over their distress (Klonsky, 2007). Biological explanations have focused on pain reduction and mood elevation following the release of endogenous opiods (see Sher & Stanley, 2008 for a review), or the role of physical stimulation in triggering an attentional shift (Niedtfeld et al., 2010). In a brain imaging study, Niedtfeld and colleagues (2010) identified that thermal stimulation (warmth or painful heat), experienced in the context of hyperarousal elicited by negative images, was followed by reduced limbic activity in both controls and participants diagnosed with BPD. mental health consumers. This comprehensive approach resulted in the inclusion of 13 functions in the ISAS. 2.1 Affect regulation They hypothesise that sensory stimulation causes an attentional shift and although both groups demonstrated this shift, it may be that the intense affective arousal experienced by people with BPD when distressed necessitates that they use more painful methods of self-stimulation (e.g., cutting) in order to sufficiently shift their attention (Niedtfeld et al., 2010). The hypothesis that self-injuring causes an attentional shift is consistent with reports from people that the physical sensation of NSSI distracts them from the emotional distress they are experiencing (Himber, 1994; Polk & Liss, 2009). Additionally, it is possible that seeing blood prompts a shift of attention from aversive, internal experiences to the site of the physical injury (Glenn & Klonsky, mental health consumers. This comprehensive approach resulted in the inclusion of 13 functions in the ISAS. 70 2010b), which may be particularly useful for people who do not feel pain during NSSI. Caring for the wounds might then serve to maintain this focus on the physical body, rather than on emotional distress. Indeed, the sight of blood has been identified as an important component of the ritual of self-injury for some people as it is associated with calmness and tension relief (Glenn & Klonsky, 2010b). Although the mechanism through which blood exerts this effect is unknown, it is possible that seeing blood either results in heart rate deceleration or heightened activation of the sympathetic nervous system, which is then followed by an intense parasympathetic reaction because there is no immediate danger (Glenn & Klonsky, 2010b). Both of these physiological processes—heart rate deceleration and parasympathetic rebound—would result in the suppression of negative emotions (Glenn & Klonsky, 2010b). 2.2 Self-punishment Self-injury is thought to function as self-punishment by providing a means through which people can denigrate themselves or communicate how angry they are at themselves (Klonsky, 2007). Self-punishment was the second most highly endorsed function of NSSI, after affect regulation, in a population of university students (Klonsky & Glenn, 2009) and, as I demonstrated in Chapter 2 (see pp. 52- 53), self-punishment is one of the most commonly reported reasons for NSSI. More specifically, studies where people have explicitly reported self-injuring to punish themselves (e.g., Nixon et al., 2002; Shearer, 1994), to regulate self-directed anger (e.g., Laye-Gindhu & Schonert-Reichl, 2005), or to express self-hatred (e.g., Nock et al., 2009; Oyefeso et al., 2008) can all be interpreted as support for the self- punishment model. 2.1 Affect regulation Beyond this phenomenological research, the role of self-punishment in the development and maintenance of NSSI is not well understood. Given that self- injuring to alleviate anger or guilt, or to express self-hatred is emotionally driven, it is possible that self-punishment is a sub-category of the affect regulation model (Chapman et al., 2006). This is seemingly consistent with Klonsky’s (2009) finding 71 that although affect regulation and self-punishment items were the most commonly endorsed motivations for NSSI among university students, affect-regulation motivations were typically reported as primary while self-punishment motivations were reported as secondary. Self-punishment therefore may be one specific type of affect regulation. A number of specific psychological mechanisms have been proposed to underlie the self-punishment function of NSSI. Drawing on self-verification theory (Swann, Hixon, Stein-Seroussi, & Gilbert, 1990) and cognitive dissonance theory (Festinger, 1978), Chapman et al. (2006) propose that when the disconfirmation of core self-beliefs or cognitive dissonance leads to aversive affect, people may self- injure as a form of punishment. Getting the punishment they ‘deserve’ (i.e., self- injury) validates their negative self-beliefs and restores cognitive balance, which in turn reduces aversive affect. In this way, ‚self-injury may be experienced as familiar, ego-syntonic, or self-soothing in the face of distress‛ (Klonsky & Muehlenkamp, 2007, p. 1050). Social learning theory (Bandura, 1977) also provides a possible explanation for why people punish themselves through self-injury (Chapman et al., 2006). Socially conditioned relationships between other-inflicted punishment and subsequent relief may generalise to self-punishment for any perceived or actual transgressions. People may also self-injure as a form of pre-emptive self-punishment to minimise or avert punishment by others. If self-punishment can be subsumed within the affect regulation model, then the physiological mechanisms proposed to underlie affect regulation (e.g., attentional shift as a result of pain or the sight of blood) should be applicable to self- punishment. Furthermore, self-injuring as a form of punishment can result in a sense of satisfaction (Nock, 2010), which may be related to the release of endorphins. 2.3 Anti-dissociation Anti-dissociation, like self-punishment, has also been categorised as a type of affect regulation (Suyemoto, 1998). According to this model, self-injuring terminates 72 depersonalisation or dissociation through the generation of feeling (Klonsky, 2007). As discussed in Chapter 2 (see pp. 2.1 Affect regulation 47-48), empirical evidence in support of the anti- dissociation model has been mixed (Klonsky, 2007); 38% to 87% of adolescents and adults who self-injure report doing so to end dissociation or feelings of emptiness (Briere & Gil, 1998, Favazza & Conterio, 1989; Nixon et al., 2002; Swannell et al., 2008). Once again, it is likely that self-injury fulfils an anti-dissociative function because the physical sensation and/or the sight of blood trigger certain physiological reactions (Klonsky & Muehlenkamp, 2007; Polk & Liss, 2009; Schoppmann, Schröck, Schnepp, & Büscher, 2007). One such reaction may be the orienting response (Pavlov, 1927), which is the increased cortical activity that occurs in response to novel stimulation (Chapman et al., 2006). Although the role of the orienting response in NSSI is yet to be empirically examined, Chapman et al. (2006) propose that an orienting response to physical pain or the sight of blood may be sufficient to end dissociation, although the intensity of this response should decrease over time as people become habituated to experiencing pain or seeing blood when self-injuring. Identifying the mechanisms through which NSSI functions as an anti- dissociation strategy becomes more complicated when taking into account evidence that self-injury not only ends dissociative episodes, but also has been reported to induce dissociation by providing an escape from overwhelming, internal experiences (Himber, 1994; Laye-Ghindhu & Schonert-Reichl, 2005; Swannell et al., 2008). In one study, retrospective, self-reported levels of dissociation peaked during episodes of NSSI in a group of female inpatients diagnosed with BPD, regardless of whether or not they experienced pain while self-injuring (Kemperman et al., 1997). It appears that NSSI fulfils an antithetical function to anti-dissociation in such cases. 2.4 Anti-suicide Drawing on psychoanalytic theory, NSSI is conceptualised within the anti- suicide model as a compromise between the urges to live and die; in other words, self-injury is a form of temporary self-destruction that serves as a substitute for 73 suicide (Firestone & Seiden, 1990; Menninger, 1935; Suyemoto, 1998). This notion of using NSSI to avert suicidal impulses or to manage suicidal ideation, discussed in both Chapters 1 (see pp. 15-16) and 2 (see pp. 48-49), has been supported in a number of self-report studies (Klonsky, 2007). It is probable that the act of physical injury distracts people from ruminating about taking their own life through the attentional shift process described above and/or improves their mood, at least temporarily, perhaps through the release of endorphins, so that suicide is no longer as salient as the best or only option for dealing with their distress. In this way, self-injuring to avert suicide could also be subsumed under the general framework of affect regulation in that the act of injury may decrease intense, negative feelings associated with suicidal urges or cognitions (Klonsky & Muehlenkamp, 2007). 2.5 Sexual The sexual model of self-injury is also informed by psychoanalytic theory and contends that people self-injure for sexual satisfaction, to exert control over their sexual development, or to avoid or punish themselves for sexual behaviours or feelings (Suyemoto, 1998). Although a historically prevalent explanation for why people self-injure, the sexual model has rarely been empirically investigated and, when it has been, it has received negligible support (Briere & Gil, 1998; Kemperman et al., 1997; Shearer, 1994). That this model holds little weight in contemporary understandings of the functions of NSSI is evident in the fact that the ISAS does not include a subscale addressing the purported sexual functions of self-injury (Klonsky & Glenn, 2009) and self-injurious behaviours linked to sexual arousal were explicitly excluded from one study (Polk & Liss, 2007), perhaps because including such motivations would have blurred the distinction between NSSI and sadomasochism. For the few people who do report being sexually gratified through self-injury (e.g., Briere & Gil, 1998), it is likely that this is associated with the release of endorphins. 74 2.6 Sensation-seeking Self-injuring to produce exhilaration or excitement falls within the sensation- seeking model of NSSI and in studies where such reasons for NSSI have been examined, they have typically been endorsed by a low percentage of participants (Klonsky, 2007). Once again, it is likely that any heightened arousal following NSSI, which is sometimes likened to a drug high (Swannell et al., 2008), results from the release of endorphins. It is possible that sensation-seeking lies on the opposite side of the same coin to anti-dissociation, in that the desire to seek positive emotional arousal through self- injury may stem from feelings of dissociation. If sensation-seeking was simply another descriptor for anti-dissociation, both functions should be similarly endorsed. However, this does not appear to be the case as anti-dissociation is more frequently reported than sensation-seeking (Klonsky & Glenn, 2009). 2.7 Interpersonal boundaries The interpersonal boundaries model of self-injury, which has rarely been empirically examined perhaps owing to its psychoanalytic roots, is based on object- relations theory and proposes that people self-injure in order to distinguish themselves from others and to assert their autonomy (Klonsky, 2007). The action of marking the body may engender a sense of control because such marks physically delineate the boundaries between self, other, and the environment (Klonsky, 2007; Klonsky & Muehlenkamp, 2007). 2.8 Interpersonal influence Self-injuring to influence others tends to be perceived as the most contentious single-function model of NSSI in light of pejorative depictions of people who self- injure as attention-seeking or manipulative (Nock, 2008). Although self-injuring to access support or care seems to be fairly common (Klonsky & Muehlenkamp, 2007), interpersonal functions for NSSI are less frequently endorsed than intrapersonal functions (Klonsky & Glenn, 2009; Nock & Prinstein, 2004). However, it is possible that these functions are not reported as often because they are considered to be 75 socially undesirable (Claes, Klonsky, Muehlenkamp, Kuppens, & Vandereycken, 2010). The interpersonal influence model draws on behavioural theory in that the person’s self-injury is thought to signal to others that support is needed, and when support is provided, the self-injurious behaviour is reinforced (Klonsky, 2007). Arguably, using self-injury as support-seeking strategy could be conceptualised as affect regulation by proxy; people who struggle to regulate their own affective arousal and negative emotions may need the help of others to cope (Nock, 2008). Although the causal mechanism underlying this model is interpersonal reinforcement, people who self-injure to influence others may not be cognisant that their behaviour is being reinforced by others’ responses, which would also influence how frequently they endorsed interpersonal functions (Klonsky, 2007). 2.9 Summary and limitations 2.9 Summary and limitations A range of single-function models, which subsume the reasons, antecedents, and consequences of NSSI discussed in detail in Chapter 2, have been proposed to account for why people self-injure in the absence of suicidal intent. On the surface, some of these models appear to have little in common, but once they are considered in more depth, it becomes apparent that many of the models can be incorporated within an affect regulation framework and, in all likelihood, share the same or similar underlying mechanisms. Furthermore, as an overdetermined behaviour (Suyemoto, 1998), self-injury is thought to serve multiple functions at the same time which makes it necessary to integrate the single-function models into a cohesive, clinically useful paradigm. One of the most promising ways to achieve this integration has been through the use of behavioural theory as an overarching framework. Behavioural models successfully capture the commonalities and differences of the diverse single-function models because they define the function of a behaviour by identifying both the antecedents and consequences of that behaviour (Nock & Prinstein, 2004). The result is a paring down of the functions of NSSI into four, simple contingencies: intrapersonal and 76 interpersonal consequences that are either negatively or positively reinforced (Nock, 2010). 3. MULTI-FUNCTION MODELS OF SELF-INJURY The FFM (Nock, 2008; Nock & Prinstein, 2004, 2005) and EAM (Chapman et al., 2006) are two promising behavioural models of NSSI that are garnering varying degrees of empirical support (e.g., Anderson, 2009; Armey & Crowther, 2008; Klonsky & Glenn, 2009; Lloyd-Richardson et al., 2007). In the following section, I will examine the assumptions and research evidence in support of each of these models, before evaluating them according to well-established epistemic values. 3.1.1 Assumptions of the FFM The FFM (Nock, 2008; Nock & Prinstein, 2004, 2005) proposes that self-injury can be categorised according to whether it serves an automatic (i.e., intrapersonal) or social (i.e., interpersonal) purpose, which, in turn, is either positively or negatively reinforced. These two dimensions intersect to form four primary functions of NSSI: automatic negative reinforcement (i.e., to eradicate or escape from negative thoughts or affective states), automatic positive reinforcement (i.e., to induce positive, physiological states), social negative reinforcement (i.e., to escape interpersonal events or demands), and social positive reinforcement (i.e., to get attention or desired objects) (Nock, 2008; Nock & Prinstein, 2004, 2005). The term automatic in the context of the FFM refers to self-imposed reinforcement rather than actions that are carried out without conscious awareness (Nock & Prinstein, 2004, 2005). In a later publication, Nock (2010) revised the FFM terminology to refer instead to automatic consequences as intrapersonal and social consequences as interpersonal. This is consistent with the language used by other NSSI researchers and, as such, the terms intrapersonal and interpersonal are used in this thesis. In light of the limited empirical support for interpersonal functions, Nock (2008) presented an expanded version of the FFM, which includes hypotheses about the potential mechanisms underlying the social functions of NSSI (see Figure 1). The 77 expanded model explains why people may use NSSI as a way to communicate with others. Drawing on theories of animal behaviour and anthropological studies, Nock (2008) suggests that self-injury may be used as a signal to convey strength and fitness or distress. People may begin to self-injure because less costly and less severe communication strategies have not produced desired results. Figure 1. The elaborated FFM. Reproduced from ‚Actions speak louder than words: An elaborated theoretical model of the social functions of self-injury and other harmful behaviours,‛ by M. Nock, 2008, Applied and Preventive Psychology, 12, p. 164. Figure 1. The elaborated FFM. Reproduced from ‚Actions speak louder than words: An elaborated theoretical model of the social functions of self-injury and other harmful behaviours,‛ by M. Nock, 2008, Applied and Preventive Psychology, 12, p. 164. For example, if a person seeks comfort from others and comfort is not provided, they may resort to NSSI as a way to gain attention and validation (i.e., self-injury is a signal of distress). If this strategy is successful, they may be more likely to use it in the future when similarly distressed. 3.1.1 Assumptions of the FFM Likewise, someone may use self-injury to avoid or escape from fulfilling interpersonal obligations when less extreme behaviours (e.g., acting out) have failed. Alternatively, people may injure themselves to prevent victimisation by others or to demonstrate their connectedness with a particular social group (i.e., as a signal of strength or fitness). Once again, if 78 self-injury proves to be an effective means of displaying one’s strength or fitness to others, it is likely to be reinforced (Nock, 2008). 3.1.2 Empirical support for the FFM To test the hypothesised four functions of self-injury, adolescent inpatients completed the Functional Assessment of Self-mutilation (FASM; Lloyd, Kelley, & Hope, 1997, cited in Nock & Prinstein, 2004), which includes a list of 22 reasons for engaging in self-injury (e.g., ‚to stop bad feelings‛, ‚to punish yourself‛). To test the hypothesised four functions of self-injury, adolescent inpatients completed the Functional Assessment of Self-mutilation (FASM; Lloyd, Kelley, & Hope, 1997, cited in Nock & Prinstein, 2004), which includes a list of 22 reasons for engaging in self-injury (e.g., ‚to stop bad feelings‛, ‚to punish yourself‛). Participants were able to endorse multiple reasons. The majority of the participants had injured themselves at least once in the past year, while almost half reported injuring themselves more than 10 times. Participants were able to endorse multiple reasons. The majority of the participants had injured themselves at least once in the past year, while almost half reported injuring themselves more than 10 times. After categorising the FASM reasons for self-injury into the four hypothesised functional domains of negatively or positively reinforced intrapersonal and interpersonal consequences, Nock and Prinstein (2004) conducted a factor analysis which confirmed their model’s goodness-of-fit with the data. Further analyses provided evidence that the four functions represented associated, but distinct paradigms. Although this study supported the four function conceptualisation of NSSI, not all functions were equally endorsed (Nock & Prinstein, 2004). Negatively reinforced, intrapersonal scores were significantly higher than the scores for the other three functions, while the scores for the positively reinforced, intrapersonal function were significantly higher than both the negatively and positively reinforced interpersonal functions. Additionally, 24% to 53% of the participants endorsed intrapersonal items, but only 6% to 24% of participants endorsed interpersonal items, suggesting that while adolescent inpatients self-injure for multiple reasons, the primary function of their self-injury is intrapersonal emotion regulation (Nock & Prinstein, 2004). Research with community-based adolescents provided further support for the structural validity of the FFM, although in contrast to Nock and Prinstein’s (2004) findings, interpersonal functions received similar levels of endorsement in this 79 sample as intrapersonal functions (Lloyd-Richardson et al., 2007). Furthermore, the endorsement of the different functions of NSSI varied according to the severity of the adolescents’ self-injurious behaviour. 3.1.2 Empirical support for the FFM Minor NSSI (i.e., hitting, pulling out hair, inserting objects, biting, or excoriation) was only significantly associated with intrapersonal functions, while moderate/severe NSSI (i.e., cutting, carving, burning, self-tattooing, scraping or erasing skin) was significantly associated with both intrapersonal and interpersonal functions. Adolescents within the moderate/severe category also used more types of NSSI and injured themselves more often than those in the minor NSSI category. It thus appears that adolescents who have a more chronic history of NSSI are more likely to endorse multiple functions of self-injury. Furthermore, it is possible that people who are experiencing particular forms of psychopathology may be more likely to endorse certain functions of NSSI. Unfortunately, research efforts to identify clinical correlates of specific functions have yielded discrepant results. For example, past suicide attempts and current suicide ideation were significantly associated with all four functions among community-based adolescents (Lloyd-Richardson et al., 2007), whereas suicide attempts and hopelessness were only associated with intrapersonal, negative reinforcement among adolescent inpatients (Nock & Prinstein, 2005). Depressive symptoms have been associated with both intrapersonal (Hilt, Cha, & Nolen-Hoeksema, 2008; Lloyd-Richardson et al., 2007) and interpersonal reinforcement (Nock & Prinstein, 2005). Although it seems counter-intuitive that depressive symptoms and suicidal ideation would be significantly correlated with social functions, people may be using NSSI as a way to communicate to others that they are depressed and in need of support (i.e., social positive reinforcement) (Nock & Prinstein, 2005), or to avoid obligations that they feel unable to fulfil because of their depressive symptoms. In sum, there is evidence to support four behavioural functions of NSSI, but it appears that intrapersonal consequences, particularly those that are negatively 80 reinforced, are more commonly endorsed than interpersonal functions. However, social desirability (Claes, Klonsky, et al., 2010) or a lack of awareness about how the behaviours are functioning may lead to the underreporting of interpersonal functions (Klonsky, 2007). Further research needs to be conducted to determine whether certain individual risk factors, diagnostic categories, or socio-cultural environments are more commonly associated with specific functions. 3.2.1 Assumptions of the EAM Non-suicidal self-injury is conceptualised within the EAM (Chapman et al Non-suicidal self-injury is conceptualised within the EAM (Chapman et al., 2006) as a form of experiential avoidance, a functional diagnostic dimension defined as: the phenomenon that occurs when a person is unwilling to remain in contact with particular private experiences (e.g., bodily sensations, emotions, thoughts, memories, behavioral predispositions) and takes steps to alter the form or frequency of these events and the contexts that occasion them. (Hayes et al., 1996, p. 1154) the phenomenon that occurs when a person is unwilling to remain in contact with particular private experiences (e.g., bodily sensations, emotions, thoughts, memories, behavioral predispositions) and takes steps to alter the form or frequency of these events and the contexts that occasion them. (Hayes et al., 1996, p. 1154) Avoidance is thus broadly conceptualised by Hayes and colleagues (1996) to include all escape and avoidant behaviours, which serve to change intrapersonal events and the environmental factors that cause such events to occur. Avoidance is thus broadly conceptualised by Hayes and colleagues (1996) to include all escape and avoidant behaviours, which serve to change intrapersonal events and the environmental factors that cause such events to occur. Given that experiential avoidance is a functional diagnostic dimension, Hayes et al. (1996) argue that numerous mental disorders can be reframed as forms of experiential avoidance for particular client subgroups, thus facilitating the integration of diverse theoretical approaches and promoting research on specific behaviours across traditionally demarcated fields. Clinical syndromes such as Obsessive Compulsive Disorder, BPD, and Substance Use can be classified as experiential avoidance because they all involve clients experiencing aversive, intrapersonal events and then using unhelpful coping strategies in an attempt to avoid these experiences (Hayes et al., 1996). In developing the EAM, Chapman and colleagues (2006) have followed Hayes et al.’s (1996) lead by reconceptualising self-injurious behaviour as a form of experiential avoidance. The EAM proposes that people experience an unwanted, 81 negative emotional response (e.g., anger or frustration) to a particular stimulus (e.g., a fight with their partner) that they are unable to tolerate or manage. As a result, they feel an urge to escape from the undesirable state of arousal. To reduce or eliminate the distress that they are feeling, they injure themselves which provides them with a sense of relief. 3.2.1 Assumptions of the EAM However, this relief is temporary, necessitating the continued use of self-injury to regulate their emotions and, as a result, the behaviour becomes an automatic way of responding to negative, emotional arousal (see Figure 2). 2). Figure 2. The Experiential Avoidance Model. Reproduced from ‚Solving the puzzle of deliberate self-harm: The experiential avoidance model,‛ by A.L. Chapman et al., 2006, Behaviour Research and Therapy, 44, p. 373. Figure 2. The Experiential Avoidance Model. Reproduced from ‚Solving the puzzle of deliberate self-harm: The experiential avoidance model,‛ by A.L. Chapman et al., 2006, Behaviour Research and Therapy, 44, p. 373. Figure 2. The Experiential Avoidance Model. Reproduced from ‚Solving the puzzle of deliberate self-harm: The experiential avoidance model,‛ by A.L. Chapman et al., 2006, Behaviour Research and Therapy, 44, p. 373. There are three key assumptions underlying the EAM. First, Chapman, and colleagues (2006) maintain that NSSI functions primarily as a behaviour of emotional avoidance although they do not discount the possibility that individuals may also utilise self-injury to escape from, or avoid, other unwanted internal experiences such as thoughts, memories, and bodily sensations. Second, NSSI is negatively reinforced 82 because the reduction or elimination of unwanted internal states (particularly aversive emotions) is a direct consequence of the self-injurious behaviour. Third, the model suggests that experiential avoidance is a functional response class and, as a result, people who self-injure will also use other experientially avoidant strategies (e.g., thought suppression, substance use). By drawing on diverse psychological literatures to explain (a) why individuals who self-injure may have heightened experiential avoidance response tendencies, (b) what mechanisms could underlie the function of experiential avoidance in people who self-injure and (c) why NSSI persists over time, Chapman et al. (2006) construct a comprehensive, functional model of self-injury. Although they briefly consider how innate temperamental factors, such as impulsivity, may contribute to heightened experiential avoidance response tendencies, they focus predominantly on the way in which people experience and regulate their emotions. More specifically, the model proposes that if people who self-injure experience their emotions very intensely, they may find it difficult to control their heightened arousal levels, which in turn may increase their susceptibility to using NSSI as an experiential avoidance strategy. Alternatively, people who self-injure may not experience emotions more intensely, but rather may be unable to tolerate emotional distress. 3.2.1 Assumptions of the EAM Low distress tolerance may be more extreme when experiencing emotions that typically induce a desire to escape or avoid (e.g., shame). The authors also suggest that emotion regulation skills could be a key factor in determining whether individuals are likely to utilise experientially avoidant behaviours. People who are unable to regulate their emotions effectively, either through the lack of skills or a failure to employ skills when highly aroused, may be at risk of attempting to avoid or escape aversive emotions through the use of NSSI (Chapman et al., 2006). Although these hypotheses—that people may be susceptible to self-injury because they experience emotions more intensely than others, have low distress tolerance, and/or are unable to regulate their emotions effectively—are useful in identifying people at risk of engaging in NSSI, they do not account for why self- 83 injury is a form of avoidance. Three underlying mechanisms are hypothesised to support the theory that NSSI functions primarily as behaviour of emotional avoidance: The act of self-injuring causes the release of endogenous opiates which provide a sense of analgesic relief; the physical pain of self-injury distracts from emotional pain; and/or self-injury is used as a form of punishment for perceived wrongdoing, or to avert actual punishment, and once sufficiently punished, emotional arousal decreases (Chapman et al., 2006). Of course, these are consistent with the proposed mechanisms for the single-function models discussed earlier in this chapter but, as I have already discussed, empirical evidence in support of these hypotheses is lacking (Chapman et al., 2006). If NSSI does function as a form of avoidance, it becomes necessary to determine how the avoidance is maintained over time. According to the EAM, the repetitive nature of NSSI is potentially related to four factors (Chapman et al., 2006). First, it is more likely that aversive emotions will recur with an increased intensity and frequency if a person attempts to avoid or escape from these emotions by engaging in NSSI. Future attempts to regulate these emotions establish a cyclical pattern of repeated self-injury. Second, people who are unwilling to fully experience their aversive emotions may never learn that such emotions can be tolerated and, as a result, may come to see NSSI as the only or best solution for emotional regulation. 3.2.1 Assumptions of the EAM Third, people who adopt a verbal rule equating NSSI with emotional release may never give themselves the opportunity to learn alternative ways of coping or may fail to learn from the negative effects of self-injury. Finally, people may simply habituate to the characteristics and negative consequences of NSSI over time, which makes it less likely that they will cease engaging in the behaviour. 3.2.2 Empirical support for the EAM The three key assumptions of the EAM—that NSSI is primarily an emotionally avoidant behaviour, that it is negatively reinforced, and that people who self-injure will also engage in other avoidant behaviours—have been empirically investigated to varying degrees albeit not within Aotearoa New Zealand. 84 Evidence that NSSI is an emotionally avoidant, negatively reinforced behaviour is primarily drawn from the self-report, laboratory, and ecological momentary assessment studies reviewed in Chapter 2 (see pp. 44-62). Additionally, research on the functions of NSSI discussed earlier in this chapter overwhelmingly supports the primacy of intrapersonal, negative reinforcement (Klonsky & Glenn, 2009; Nock & Prinstein, 2004). The hypothesis that self-injury is a negatively reinforced, avoidant behaviour has also been examined by testing the relationship between the frequency of people’s self-injurious behaviours and their intrapersonal experiences. Aversive self- awareness (i.e., the experience of negative thoughts and feelings about oneself) predicted engagement in NSSI among university students (Armey & Crowther, 2008), providing further support for the experiential avoidance function of self- injury. However, this study concentrated on antecedents that predicted NSSI, rather than on the consequences that may have reinforced and maintained the self- injurious behaviours over time. Studies that have examined whether the frequency of NSSI is related to negatively reinforced consequences have yielded mixed results. Decreases in negatively valenced, high arousal affect states and increases in positively valenced, low arousal affect states following NSSI, predicted lifetime cutting rates among university students, partially supporting the negative reinforcement hypothesis of NSSI (Klonsky, 2009). Among female inpatients with eating disorders, self-injury frequency was similarly related to increases in positive affect subsequent to NSSI, although this relationship did not reach statistical significance (Claes, Klonsky, et al., 2010). Decreases in negative affect and increases in positive affect following self- injury suggest that NSSI may be both negatively and positively reinforced. The final assumption of the EAM, that people who self-injure will engage in other avoidant behaviours (i.e., that NSSI fits within an avoidant response class), has received some support. Undergraduate students with a history of NSSI, who were matched to controls on psychological distress levels, reported significantly higher 85 rates of avoidant coping (Andover, Pepper, & Gibb, 2007). Another study of young adults similarly found significantly higher levels of avoidant coping among people with a history of self-injury when compared to those who had never self-injured (Hasking, Momeni, Swannell, & Chia, 2008). 3.2.2 Empirical support for the EAM Moreover, in the latter study, as the severity of self-injurious behaviours increased, so did avoidant coping, which suggests that self-injury may belong in a response class of avoidant coping strategies. However, it is not clear whether these avoidant coping strategies or behaviours (e.g., thought suppression, substance use) mediate, moderate, or simply co-occur with NSSI. Recently, Najmi, Wegner, and Nock (2007) proposed that people who self-injure and are highly reactive to emotions will try to suppress their negative thoughts as a way of gaining control over their distress. However, unless thought suppression is used to focus on one particular distracter, it typically increases the frequency of the thoughts (Najmi at al., 2007) because ‚the intention to suppress a thought instigates a monitoring process that ironically increases the cognitive accessibility of the unwanted thought‛ (Wegner & Zanakos, 1994, p. 616). Following this paradoxical increase in aversive thoughts and emotions, Najmi and colleagues hypothesised that people will graduate to self-injury because it serves as a focused distracter. They found that the association between emotional reactivity and rates of self- injurious behaviour was partially mediated by thought suppression, which suggests that in some incidents, self-injury may be utilised following an unsuccessful attempt to suppress distressing thoughts (Najmi et al., 2007). However, these cross-sectional results would need to be replicated longitudinally before reaching any definitive conclusions about the temporal sequencing of thought suppression and NSSI. Further research is needed to identify the exact nature of the relationships between NSSI and other forms of avoidance. Further research is needed to identify the exact nature of the relationships between NSSI and other forms of avoidance. Finally, NSSI is associated with a number of clinical syndromes, such as Bulimia and Substance Use Disorders (Klonsky & Muehlenkamp, 2007), that are 86 similarly thought to function as forms of experiential avoidance (Hayes et al., 1996; Heatherton & Baumeister, 1991). This lends support to the hypothesis that NSSI belongs in a functional response class of behaviours primarily aimed at escaping or avoiding negative internal states. 3.3 Evaluating the FFM and the EAM Both the EAM and the FFM rely on operant conditioning principles to explain why people are motivated to injure themselves, and how this behaviour is reinforced and maintained over time. While the FFM includes intrapersonal and interpersonal functions which are either positively or negatively reinforced, the EAM focuses exclusively on the negatively reinforced, intrapersonal function of experiential avoidance. Epistemic values provide an evaluative framework, which can be used to identify the strengths and limitations of each of these models. Although I utilise this framework for my critique, it should be noted that epistemic values are not objective, definitive criteria, but rather subjective, multi-faceted constructs (Rooney, 1992). Nonetheless, they provide a useful starting point for comparing and contrasting theoretical models. Drawing on the work of Kuhn (1977) and McMullin (1983), Howard (1985) identifies and defines six epistemic values: predictive accuracy, internal coherence, external consistency, unifying power, fertility, and simplicity; values which I use to compare the EAM with the FFM. Arguably, the key criterion in determining the validity of a theoretical model is whether that model accurately predicts its hypothesised outcomes (Howard, 1985). To determine whether the EAM and the FFM have adequate predictive accuracy, it is first necessary to revisit the basic assumptions of each model. The FFM proposes that NSSI will serve four primary functions: intrapersonal negative and positive reinforcement, and interpersonal negative and positive reinforcement. In contrast, the EAM predicts that NSSI will serve one primary, intrapersonal function of experiential avoidance. Additionally, the EAM posits that individuals who self- 87 injure to avoid or escape unwanted internal experiences will also use other experientially avoidant coping strategies (e.g., binge eating). Both the EAM and FFM propose, in line with the principles of operant conditioning, that understanding the function of a behaviour involves identifying the antecedents and consequences of that behaviour. Furthermore, a consequence of self-injury would only be considered to reinforce self-injurious behaviour if it increased the likelihood of the person engaging in subsequent episodes of self-injury (Cipani & Schock, 2007). However, establishing a direct, temporal relationship between consequences and future episodes of self-injury is extremely difficult. Someone may experience a particular consequence (e.g., increased attention from loved ones) following an episode of self-injury, but this does not mean that they injured themselves to elicit that consequence or that they will injure themselves to elicit the same consequence in the future (Nock, 2008). 3.3 Evaluating the FFM and the EAM Although the evidence to date supports the contention that there are four functions of NSSI, people routinely endorse intrapersonal, negative reinforcement as primary to the maintenance of their self-injurious behaviour. The EAM thus surpasses the FFM in terms of predictive accuracy as it is far more likely that a person will report engaging in NSSI to escape or avoid negative intrapersonal experiences, than any of the other three hypothesised functions of NSSI. However, the predictive utility of the EAM and the FFM ultimately needs to be determined through prospectively and longitudinally assessing the antecedents and consequences of self-injurious behaviours; a challenging task in light of the necessary ethical constraints that accompany any NSSI research (Prinstein, 2008). It is likely that ecological momentary assessment and longitudinal survey methods will prove to be essential in testing and supporting the reinforcement hypotheses of the FFM and EAM (see Muehlenkamp et al., 2009; Nock et al., 2009). 3.3.2 Internal coherence An internally coherent, theoretical model needs to be logically consistent; that is, all the elements of the model should fit together (Howard, 1985). The EAM 88 provides a comprehensive overview of how the cycle of self-injury is triggered, reinforced, and maintained over time. As such, it reflects its premise that understanding the function of a behaviour requires identifying both the antecedents and consequences of that behaviour. The only potentially incongruent aspect of the EAM is that the experience of relief following NSSI is hypothesised to negatively reinforce self-injury. If relief is viewed as a positive affect state, then experiencing relief after self-injury would positively reinforce the behaviour (Klonsky, 2009). However, if relief is defined as an absence of distress (Watson & Tellegen, 1985), then conceptualising it as a negatively reinforced consequence of NSSI is no longer anomalous (Klonsky, 2009). In contrast to the EAM, the FFM lacks internal coherence because the conceptualisation of the model is inconsistent with the definition of function proposed by Nock and Prinstein (2004). In their paper, they define function as the antecedents and consequences that cause and maintain a behaviour, but they fail to include any antecedents in their model, which focuses exclusively on how self- injurious behaviour is reinforced. 3.3 Evaluating the FFM and the EAM The researchers acknowledge this shortcoming and attempt to address it in a subsequent paper (Nock & Prinstein, 2005), yet instead of investigating specific antecedents, they focus on clinical correlates (e.g., suicide attempts) and contextual features (e.g., experience of pain while self-injuring). The cross-sectional nature of their data prohibits them from demonstrating whether these correlates and contextual factors actually trigger episodes of self-injury. Furthermore, although Nock and Prinstein (2004) hypothesised four primary functions of NSSI, they found significantly more support for the intrapersonal negative reinforcement function over the other three functions. This begs the question whether a four functions model, where each of the functions is viewed as primary to the cause and maintenance of self-injury, is conceptually valid. It may be more prudent to consider the automatic negative reinforcement function as primary, with the other functions as secondary. It should be noted, however, that the intrapersonal and interpersonal functions of NSSI were similarly endorsed in one of 89 the only other confirmatory factor analyses of the FFM (Lloyd-Richardson et al., 2007). 3.3.3 External consistency Theoretical models that are consistent with established theories from the same or similar disciplines meet the criterion of external consistency (Howard, 1985). As previously discussed, well-established behavioural principles of operant conditioning are fundamental to both the EAM and the FFM. The multi-function structure of the FFM is also consistent with the extensive evidence base on stereotypical self-injury in developmentally disabled populations (Nock & Prinstein, 2004, 2005). Furthermore, in his elaboration of the social functions of the FFM, Nock (2008) draws on evidence from both anthropology and theories of animal behaviour (see section 3.1.1). Through arguing that NSSI functions as an experientially avoidant behaviour, Chapman et al. (2006) rely on the burgeoning evidence base that suggests experiential avoidance is the key to understanding a range of psychological disorders. This literature forms the theoretical foundation of the EAM and was discussed in detail earlier in this chapter (see section 3.2.1). Unifying power refers to a theoretical model’s capacity to draw together diverse strands of knowledge into a cohesive whole (Howard, 1985). Both the FFM and EAM draw various single-function models of self-injury into more comprehensive models of NSSI. The FFM is an exhaustive multi-function model in that every single-function model presented in the literature could feasibly be categorised according to whether the purpose for self-injuring reflects an attempt to alter one’s intrapersonal or interpersonal environment, and whether it is positively or negatively reinforced. In contrast, Chapman and colleagues (2006) argue that many of the single- function models of NSSI propose that self-injury enables people to avoid, or escape from, unwanted internal experiences, although the emphasis on what the person 90 who self-injures is trying to escape from or avoid varies between models. Given this commonality, they propose that the overarching construct of experiential avoidance subsumes the hypotheses put forward by many single-function models. Both the FFM and the EAM thus have the capacity to account for the multiple reasons, reviewed in Chapter 2, which people give for self-injuring. 3.3.5 Fertility A model is considered fertile if it generates new directions for future research on the topic (Howard, 1985). Beyond proposing that NSSI serves four functions, Nock and Prinstein (2004) do not provide any hypotheses about why NSSI may serve different functions for different people, how these functions relate to one another, and what mechanisms underlie each of the four functions. 3.3.3 External consistency While Nock (2008) did elaborate on the FFM in a subsequent paper, he focussed exclusively on the interpersonal functions of NSSI and, as a result, the intrapersonal functions remain under-theorised. In comparison, the EAM proposes a gamut of testable hypotheses about why people who self-injure may have heightened experiential avoidance response tendencies, what mechanisms underlie the function of experiential avoidance in people who self-injure, and why NSSI becomes a habitual behaviour (see section 3.2.1). 3.3.6 Simplicity The EAM meets the criteria of simplicity in its depiction of self-injury as a negatively reinforced, cyclical process carried out to avoid or escape from aversive internal states. It could be considered a limitation that the EAM to fails include positive reinforcement contingencies and does not account for any interpersonal functions of NSSI, given the evidence that people do use self-injury to communicate with, and influence, others (e.g., Lloyd-Richardson et al., 2007; Nock & Prinstein, 2004). However, Chapman et al. (2006) acknowledge that NSSI likely serves multiple functions but are explicit in their assertion that the primary function of NSSI is emotional (experiential) avoidance. 91 In comparison, the FFM has a broader scope with its emphasis on four primary functions of NSSI. The four functions of the FFM complement each other to cover a range of contingencies and on the surface the FFM appears to be a simple, falsifiable model. But the difficulty arises when, as discussed earlier, one attempts to determine how the functions relate to one another, whether the model proposes that people who self-injure are likely to endorse all four functions within and/or between episodes, or whether the functions served by self-injury will depend on contextual features and/or psychopathological symptoms. The manner in which the FFM is conceptualised provides no hypotheses for these questions and, as such, it could be thought of as an under-theorised model that is complicated by its over-inclusivity. 4. THE CURRENT THESIS 4. THE CURRENT THESIS Although several single-function models have been proposed for why people self-injure, research has shown that NSSI serves multiple functions and, as such, individual single-function models are unable to account for the full range of NSSI functions. In contrast, multi-function models, particularly those conceptualised within a behavioural paradigm such as the FFM and the EAM, succeed at integrating diverse single-function models to present more comprehensive perspectives on self- injurious behaviour. Both the FFM and the EAM are empirically supported when compared on the basis of adherence to common epistemic values (Howard, 1985), but the EAM is a more internally coherent, fertile, and parsimonious theoretical model of why people hurt themselves on purpose. Additionally, the evidence to date suggests that people primarily injure themselves to regulate their emotional experiences and to punish themselves (Klonsky, 2007), which is consistent with the emphasis placed by Chapman and colleagues (2006) on emotional avoidance. Indeed, as discussed earlier in this chapter, all of the most empirically supported single-function models (e.g., affect regulation, self-punishment, anti-suicide, and anti-dissociation) can be unified under the rubric of experiential avoidance. 92 In light of this evidence, my overarching research question for this thesis is: Does NSSI function primarily as an experientially avoidant behaviour within Aotearoa New Zealand? This is an important topic to investigate because this question has not been asked of populations living in Aotearoa New Zealand. As such, our current understanding of why people in this country self-injure is mostly limited to anecdotal reports and evidence gleaned from international studies, which have focused primarily on testing single- rather than multi-function models. This focus has resulted in particular forms of experiential avoidance, such as affect regulation, being extensively investigated, while other forms of experiential avoidance, such as cognitive avoidance, have been largely neglected. Given that developing a sophisticated understanding of the functions of NSSI is essential to effectively treat these behaviours, it is imperative that we do not simply assume that the functions of self-injury within Aotearoa New Zealand are comparable to those endorsed overseas, but rather that we investigate why people living here self-injure in order to establish a New Zealand-specific evidence base about NSSI. That being said, my general hypothesis is that NSSI will primarily function as a way for people living in Aotearoa New Zealand to escape from or avoid aversive emotional experiences. 4. THE CURRENT THESIS Therefore, I did not expect to find that the functions of my participants’ self-injurious behaviours would differ significantly from those endorsed by people living in other developed Western countries. If the EAM is a valid, explanatory framework for NSSI in Aotearoa New Zealand, this may have important clinical implications. In particular, it may warrant the investigation of Acceptance and Commitment Therapy (ACT) as a treatment for self-injurious behaviours because the central goal of ACT is to decrease experiential avoidance by enhancing psychological flexibility (Hayes, Luoma, Bond, Masuda, & Lillis, 2006). To determine whether NSSI functions primarily as an experientially avoidant behaviour within Aotearoa New Zealand, I designed and conducted three studies. For my first study, I interviewed people who had self-injured without suicidal intent in the previous 12 months to elucidate the temporal process of their most recent 93 episode of self-injury. By asking about the situational, emotional, and cognitive antecedents and consequences of their self-injurious behaviour, I was able to functionally assess each episode of NSSI to determine whether the majority of these episodes fulfilled an experientially avoidant function as predicted by the EAM (Chapman et al., 2006). In my second study, an online survey of people across Aotearoa New Zealand who had self-injured in the past 12 months, I examined three primary hypotheses, all of which were informed by the EAM (Chapman et al., 2006), extant literature on the functions of NSSI (e.g., Klonsky, 2007), and the results from my first study. First, I hypothesised that participants would endorse affect regulation as the primary function of NSSI both in relation to their most recent and general episodes of NSSI. Second, I expected participants to endorse intrapersonal functions of NSSI more strongly than interpersonal functions, both in regards to their most recent and general episodes of NSSI. Third, I hypothesised that participants would report a decrease in negative affect/emotion and an increase in positive affect/emotion following their most recent episode of self-injury. Although any increases in positive affect would oppose the exclusive focus on negative reinforcement in the EAM, the findings from my first study and other empirical evidence to date suggested that this possibility should not be excluded. An Interpretative Functional Analysis of self-injury ‚Behavior analysis is based on a pragmatic philosophy: what is true is what works. Behavior is understood in terms of its function, not its form, and function is always understood in relation to a context.‛ (Hayes & Bissett, 2000, p. 239) 4. THE CURRENT THESIS My final study involved comparing university students, with a history of NSSI, to those who had never self-injured, in order to determine whether people who have self-injured experience higher levels of negative emotions and/or thoughts, and use more avoidant coping strategies in general. Additionally, I was interested in investigating whether the experience of negative emotions and/or thoughts predicts the frequency of NSSI, and, if this is the case, whether these relationships are mediated by people’s propensity to avoid aversive intrapersonal experiences. 94 1. INTRODUCTION Most studies examining why people hurt themselves on purpose have relied exclusively on self-report methodologies, but few of these have qualitatively examined people’s descriptions of their motivations for self-injury. As highlighted in Chapters 2 and 3, phenomenological research on NSSI has predominantly involved participants completing survey measures, many of which have been developed from the extant literature specifically for each study. People are typically presented with a predetermined set of reasons or motivations for NSSI and asked to endorse the items that reflect their experiences of self-injury. Although the uniformity and brevity of surveys are an advantage— quantitative methods are used to examine the functions of NSSI later in this thesis— the information that can be extracted from such measures is limited in scope and depth. Quantitative and qualitative methodologies complement one another, and both are needed to clarify the functions of self-injury; quantitative research provides explanations of phenomena while qualitative research promotes understanding of those phenomena (Hjelmeland & Knizek, 2010). Indeed, one of the research recommendations in the self-harm guidelines developed by the National Institute for Health and Clinical Excellence (2004) is to utilise qualitative designs to investigate why people hurt themselves on purpose. Despite being limited in number, qualitative studies on NSSI to date have addressed diverse topics, including understanding the phenomenological experiences of men who self-injure (Russell, Moss, & Miller, 2010), the role of parents in triggering adolescent self-injury (Kam-shing, Mei-yuk, & Lam, 2003), and the 95 ‚hostile care‛ received by women who seek help for self-injury from Accident and Emergency Departments (Harris, 2000, p. 167). However, elucidating the functions of self-injury has seldom been the focus of this research. Rather, the reasons or motivations for NSSI presented in these studies have emerged from discussions or written accounts of self-injurious behaviours in general. These reasons, however, have been largely consistent with those reported in quantitative studies; for example, self-injury has been described as a way to regulate affect, self-punish, express emotions, and gain control (Harris, 2000; Kam-shing et al., 2003; Rissanen, Kylmä, & Laukkanen, 2008; Russell et al., 2010). Unfortunately, none of these studies—qualitative or quantitative—about why people self-injure have been carried out in Aotearoa New Zealand. 8 There are a few notable exceptions. For examples, see Curtis (2003) and Garisch (2010). 1. INTRODUCTION Furthermore, to my knowledge, in the majority of research on self-injurious behaviours within populations from Aotearoa New Zealand, these behaviours have been operationalised as DSH not NSSI (see for example Fortune, 2006; Garisch & Wilson, 2010; Hatcher, Sharon, & Collins, 2009; Skegg et al., 2003). Non-suicidal self-injury among New Zealanders thus remains a largely unexplored phenomenon.8 The striking absence of both qualitative and quantitative research on NSSI in Aotearoa New Zealand informed the methodology for my first study; I decided that it was important to ground my empirical work for this thesis in stories of self-injury told by people who have experiential knowledge of these behaviours. More specifically, I was interested in clarifying the functions of NSSI through learning about the temporal process of discrete self-injurious episodes. Gathering information about single episodes of NSSI to functionally analyse the behaviour is a technique that is used in DBT (Linehan, 1993a), but I have not found any research studies that have employed this method of analysis with typically developing populations who self-injure. 96 In DBT, therapists complete chain analyses with clients to elucidate the specific events that lead to problem behaviours (including self-injury), along with the consequences that reinforce and maintain those behaviours (Linehan, 1993a; Lynch, Chapman, Rosenthal, Kuo, & Linehan, 2006). A chain analysis is a precise, highly detailed form of behaviour analysis whereby individuals identify the momentary changes that occurred in a particular behaviour chain (Linehan, 1993b; Lynch et al., 2006). Identifying the behavioural trajectory of a single episode of self- injury is necessary for two reasons. First, to formulate hypotheses about the functions of a problem behaviour, one needs to focus on specific episodes as particular behaviours, such as cutting, can serve multiple functions for the same person in different contexts (Cone, 1997). Second, the functions fulfilled by a particular behaviour may change over time (Cipani & Schock, 2007; Dougher & Hayes, 2000). For example, an adolescent may initially self-injure in a group context to access peer approval, but continue to self-injure privately to escape overwhelming, negative emotions. In the current study, I concentrated on single episodes of self-injury because I wanted to elicit rich, detailed narratives about the process of self-injurious behaviours, rather than a description of the antecedents and consequences of people’s amalgamated experiences of NSSI. 1. INTRODUCTION To achieve this, I interviewed people about their most recent episode of NSSI, focusing specifically on what led up to these episodes and the consequences that followed. Identifying the antecedents and consequences of NSSI enabled me to analyse whether people’s descriptions of the temporal process of self-injury were congruent with the trajectory depicted in the EAM and international studies, and to hypothesise about how many of these episodes fulfilled an experientially avoidant function. 2. STUDY OVERVIEW Ethics approval for the current study was granted by the Multi-region Ethics Committee, a New Zealand Health and Disability Ethics Committee administered by the Ministry of Health. Gaining ethics approval involved extensive consultation with 97 individuals and teams within the mental health services at the Capital and Coast District Health Board (CCDHB) and the Hutt Valley District Health Board (HVDHB), as well as with the Student Counselling Service at Victoria University. Despite the inevitable delays that occurred during this consultation, the process was immensely beneficial in that it enabled me to refine and develop my research method in response to the questions and concerns posed by experienced consumer advocates and clinicians. Additionally, it prompted me to consider thoroughly the impact that my research may have on participants who identified as Māori given that consultation with Māori was a critical component of this ethical review process. The current study was conducted in four stages. First, people who expressed interest in participating in the study were screened using the DSHI (Gratz, 2001), a self-report questionnaire designed to assess the form, frequency, and severity of non- suicidal self-injurious behaviours. Second, if people indicated that they had injured themselves on purpose in the previous 12 months, they were invited to participate in an interview. Third, following each interview, I emailed or telephoned the participants to ask whether there was anything else they had thought of that they wanted to share. Finally, I sent each participant a questionnaire asking for feedback on their experiences of taking part in the study. To preserve the structure and flow of the original research process, I have chosen to present the method and results for each stage separately, before discussing whether the findings of this study are consistent with the EAM (Chapman et al., 2006) and international research on the functions of NSSI. 3. STAGE ONE: SCREENING FOR SELF-INJURIOUS BEHAVIOURS 3.1.1 Recruitment strategy In an attempt to interview a diverse group of people for this study, I recruited participants through mental health services and from the community. Recruiting participants from the community was vital to ensure that people who had not actively sought help for NSSI, who had not been referred to mental health services, 98 or who were no longer engaged with such services were given the opportunity to participate. It also became apparent when consulting with consumer advocates and clinicians that restricting my recruitment efforts to mental health services may limit participation to people that clinicians considered ‘well enough’ to take part. One consumer advocate suggested that by granting clinicians power as the gatekeepers of study participation, I was privileging their authority over the autonomy of mental health consumers. Furthermore, recruiting participants from the community ensured that I was not relying solely on clinicians remembering to pass study information on to their clients. or who were no longer engaged with such services were given the opportunity to participate. It also became apparent when consulting with consumer advocates and clinicians that restricting my recruitment efforts to mental health services may limit participation to people that clinicians considered ‘well enough’ to take part. One To obtain ethics approval to recruit participants from the local mental health services, I had to submit locality assessments for each service that participated in the study. This involved first meeting with the heads of the CCDHB mental health service, the HVDHB mental health service, and the Student Counselling Service, and then consulting with consumer representatives, Psychology Advisors, and mental health clinicians from teams within these services. I was also required to consult with Māori representatives or organisations within each service to ensure that my study was culturally appropriate. Following email contact with team leaders and my attendance at team meetings, participant information sheets (see Appendix A) were given to a range of mental health teams. The following CCDHB teams agreed to hand out my information sheets to clients who met the study inclusion criteria and might be interested in taking part: Youth Speciality Service, Community Alcohol and Drug Services, Regional Rangatahi Adolescent Inpatient Services, General Adult Mental Health Services, Team for Assertive Community Treatment, and Pember House Community Mental Health Team. Within the HVDHB, the adult community mental health teams, the Youth Speciality Service, and the Central Region Eating Disorders Service all agreed to distribute information sheets. 3.1.1 Recruitment strategy Additionally, clinicians from the Student Counselling Service at Victoria University of Wellington agreed to distribute information sheets on my behalf. The information sheet invited people to contact me 99 if they wanted more information about the research or if they wanted to participate in the study. To recruit community-based participants, I advertised the study in four local community newspapers and placed posters about the study in relevant community venues (e.g., Evolve, which is a youth health service; the offices of the Wellington Mental Health Consumers Union; the Wellington People’s Centre) and at various locations around Victoria University’s Kelburn campus. The information about my study was also disseminated through a local youth mental health consumer network and to the members of a community public health organisation. 3.1.2 Participants and procedure Adolescents and young adults between the ages of 16 to 34 years who had engaged in at least one type of non-suicidal, self-injurious behaviour within the past 12 months were invited to participate. This age range was selected because research has shown that deliberate self-harm (i.e., non-suicidal self-injury and suicide attempts) is most prevalent among females between the ages of 15 to 24 years and males between the ages of 25 to 34 years of age (Schmidtke et al., 1996). The lower age limit of 16 was selected because within Aotearoa New Zealand, people need to be at least 16 years of age to participate in research without parental consent. Adolescents and young adults between the ages of 16 to 34 years who had engaged in at least one type of non-suicidal, self-injurious behaviour within the past 12 months were invited to participate. This age range was selected because research has shown that deliberate self-harm (i.e., non-suicidal self-injury and suicide attempts) is most prevalent among females between the ages of 15 to 24 years and males between the ages of 25 to 34 years of age (Schmidtke et al., 1996). The lower age limit of 16 was selected because within Aotearoa New Zealand, people need to be at least 16 years of age to participate in research without parental consent. Additionally, only people who could speak English fluently were invited to participate. Other exclusion criteria listed on the information sheet were Intellectual Disability, engaging in NSSI exclusively during episodes of mania or psychosis, or current evidence of mania or psychosis. 3.1.1 Recruitment strategy Although these criteria were not formally assessed, it was clear during the interviews that none of the participants met these criteria. Additionally, only people who could speak English fluently were invited to participate. Other exclusion criteria listed on the information sheet were Intellectual Disability, engaging in NSSI exclusively during episodes of mania or psychosis, or current evidence of mania or psychosis. Although these criteria were not formally assessed, it was clear during the interviews that none of the participants met these criteria. Potential participants were provided with a copy of the information sheet, in which they were assured of their right to withdraw from the study at any time without having to explain their reason(s) for withdrawal. Of the 34 people who contacted me to enquire about the study, one male and one female did not meet the inclusion criteria because they were older than 34 years of age. The 32 people (28 100 female, 4 male) who consented via email or telephone to complete the screening survey were posted a copy of the survey, which included basic demographic questions and the DSHI (Gratz, 2001) (see Appendix B), along with a list of support organisations (see Appendix C) they could contact if they found answering the questions distressing. This list contained the contact details of six different support organisations including LifeLine, Youthline, and Warmline (i.e., a helpline run by people who have experienced mental illness). A total of 31 people completed the survey; one female self-excluded after reading the questions because she engaged primarily in indirect forms of self-injury such as disordered eating, rather than direct forms such as cutting. As a result, she decided that her experiences were not relevant to the current study. Each person received a movie voucher for completing the survey.9 9 One participant received a $10 voucher for the Warehouse instead of a movie voucher because she had hearing difficulties. 3.1.3 The Deliberate Self-harm Inventory As mentioned in the study overview, the DSHI (Gratz, 2001) is designed to measure the form, frequency, and severity of people's engagement in non-suicidal self-injurious behaviours. It was chosen for this study because the content best reflected the definition of self-injury used in this thesis (see p. 26). The DSHI contains 17 index questions about different types of NSSI (e.g., ‚Have you ever intentionally (i.e., on purpose) cut your wrist, arms, or other area(s) of your body (without intending to kill yourself)?‛) and if people endorse a behaviour, they are asked to respond to a further five questions about the age of onset, frequency, recency, duration, and severity of that particular type of NSSI. The final index question gives people the opportunity to list any other self-injurious behaviours that they have engaged in and then answer further questions about those behaviours. Responses on the DSHI can be used to derive a continuous variable by adding the frequencies of NSSI types to give a total score or a dichotomous variable by categorising participants on the basis of whether or not they endorse having self- 9 One participant received a $10 voucher for the Warehouse instead of a movie voucher because she had hearing difficulties. 101 injured (Gratz, 2001). Although the validity and reliability of the DSHI has not been extensively assessed, psychometric analyses to date have found adequate construct validity, good internal consistency (α = .79 - .82), and high test-retest reliability for the rates of NSSI reported across two administrations (r = .91 - .92) (Fliege et al., 2006; Gratz, 2001; Gratz et al., 2011). For the purposes of this study, I modified question 10 of the DSHI (i.e., ‚Have you ever intentionally (i.e., on purpose) used bleach, comet, or oven cleaner to scrub your skin?‛) to exclude the word ‚comet‛ because it is an American product that is not sold in Aotearoa New Zealand. 3.2 Results 10 Although some participants had experienced psychotic episodes in the past, they were still included in the study as the majority of their NSSI, including their most recent episodes, had not occurred in the context of psychosis. 3.2.1 Demographic and diagnostic information Twenty-seven (87.1%) of the 31 people who completed the DSHI were female. Participants ranged in age from 16 to 33 years (M = 21.65, SD = 3.99) and 27 (87.1%) identified as Pākehā/New Zealand European. The remaining four participants identified as New Zealand/Samoan (N = 1), Indian (N = 1), Asian (N = 1), and Chinese (N = 1). Twenty-five (80.6%) participants reported that they had received at least one mental health diagnosis; the mean age of first diagnosis was 17.48 years (N = 23, SD = 4.14). The modal number of reported diagnoses per participant was one (range: 0-5). Twenty-one (67.7%) people reported having been diagnosed with depression, nine (29.0%) with an anxiety disorder, four (12.9%) with an eating disorder, three (9.7%) with a personality disorder, two (6.5%) with Bipolar Disorder, one with Schizophrenia (3.2%), and three (9.7%) with other diagnoses (e.g., Gender Dysphoria, Cyclothymia with psychosis).10 3.2.2 Prevalence of different types of self-injury 3.2.2 Prevalence of different types of self-injury Interpreting the responses on the DSHI (Gratz, 2001) proved challenging, as many people seemed unable to accurately recall the information required to answer the questions. As a result, 30 participants responded to some or all of the frequency 10 Although some participants had experienced psychotic episodes in the past, they were still included in the study as the majority of their NSSI, including their most recent episodes, had not occurred in the context of psychosis. 102 questions (e.g., ‚How many times have you done this?‛) with estimates (e.g., ‚100- 150?‛, ‚unsure—3-5‛, ‚approx. 10‛) or written explanations (e.g., ‚countless‛, ‚too many times to remember‛, ‚don't know‛). Similar responses were obtained for some of the other questions, such as those requiring people to identify when they last engaged in a particular form of self-injury (e.g., ‚sometime this year‛, ‚no idea, months back‛) and the number of years that they had used a particular form of self- injury (e.g., ‚1-2 years (occasionally)‛, ‚on and off but not a regular occurrence‛). Given the difficulties of analysing this data, I emailed Kim Gratz to ask whether she had received similar responses and, if so, how she quantified these. She replied that since modifying the DSHI, she seldom receives qualitative answers (personal communication, August 22, 2008). The revised version of the DSHI instructs people to ‚Please write an actual number (e.g., 1, 5, or 15 NOT some, many, or few)‛ when reporting the number of times they have engaged in a form of NSSI and to ‚Please write the actual number of years you engaged in this behavior‛ when reporting the number of years that they have self-injured in a particular way. If I had used the revised questionnaire11, I may have received fewer written explanations but, in all likelihood, the frequencies reported by participants would still have been estimates. Unless people had kept a detailed record of their self- injurious behaviours, it is unlikely they would have been able to quantify how many times they had injured themselves in a particular way, or when these injuries occurred. The following comments, written at the end of two separate DSHI’s, illustrate these difficulties: ‚Some of the ‚last time you did this‛ are guestimated due to not keeping any record of these events. Sorry.‛ and ‚These are all approximate! 11 I did not use the revised questionnaire because I was unaware that it had been developed. 11 I did not use the revised questionnaire because I was unaware that it had been developed. 3.2.2 Prevalence of different types of self-injury To the best of my recollection.‛ During one of the interviews, one participant who was reflecting on the process of filling in the DSHI commented that being asked to remember how many times you have self-injured was analogous to being asked how If I had used the revised questionnaire11, I may have received fewer written explanations but, in all likelihood, the frequencies reported by participants would still have been estimates. Unless people had kept a detailed record of their self- injurious behaviours, it is unlikely they would have been able to quantify how many times they had injured themselves in a particular way, or when these injuries occurred. The following comments, written at the end of two separate DSHI’s, illustrate these difficulties: ‚Some of the ‚last time you did this‛ are guestimated due to not keeping any record of these events. Sorry.‛ and ‚These are all approximate! To the best of my recollection.‛ During one of the interviews, one participant who was reflecting on the process of filling in the DSHI commented that being asked to remember how many times you have self-injured was analogous to being asked how 103 many times you have worn high-heeled shoes; an impossible task considering you do not make a note of every time you wear a particular type of shoe. An additional concern raised by asking people to recall the exact number of times they had injured themselves was highlighted by another person during an interview who said she had considered counting her scars to determine how many times she had injured herself. However, she had realised that this would still be inaccurate as some of the injuries had not left scars or the scars had faded over time. Knowing that people may feel compelled to count their scars for the purposes of a research study appalled me and, as a result, I modified the wording of the DSHI for my subsequent two studies. However, I still had to somehow make sense of the DSHI data I had obtained for this study. I was primarily interested in extracting the following information from the DSHI: the different types of NSSI people had used, the number of times they had engaged in each type of behaviour, and the recency of those behaviours. 3.2.2 Prevalence of different types of self-injury As is evident in Table 2, I followed the precedent set by other researchers (e.g., Lundh, Karim, & Quilisch, 2007; Whitlock et al., 2006) in choosing to categorise both the frequency and recency data. Cutting was the most commonly endorsed type of NSSI with 96.8% of the participants reporting that they had cut themselves in their lifetimes, followed by severe scratching (83.9%), and preventing wounds from healing (80.6%). Use of at least one other type of NSSI was reported by 12 (38.7%) participants, including grazing one’s skin by rubbing a key back and forth, pouring boiling water over one’s skin, and self-flagellation. On average, people reported having engaged in 8.03 types of NSSI in their lifetimes (SD = 2.69, range: 3-13). Cutting was the most commonly endorsed type of NSSI with 96.8% of the participants reporting that they had cut themselves in their lifetimes, followed by severe scratching (83.9%), and preventing wounds from healing (80.6%). 3.2.2 Prevalence of different types of self-injury Use of at least one other type of NSSI was reported by 12 (38.7%) participants, including grazing one’s skin by rubbing a key back and forth pouring boiling water over one’s As noted earlier, the most difficult information to analyse from the DSHI was the frequencies of NSSI because of people’s tendency to report estimates or provide Table 2 Frequencies and recency of different types of NSSI NSSI type N (%) reporting NSSI type N (%) reporting frequency of engagement in NSSI types N (%) reporting recency of NSSI types Never 1 time 2-10 times 11-50 times >50 times Within past week Within past month Within past 6 months Within past 12 months >12 months ago Cutting wrists, arms, or other areas of body 30 (96.8) 1 (3.2) 0 (0.0) 8 (25.8) 4 (12.9) 7 (22.6) 6 (19.4) 7 (22.6) 8 (25.8) 4 (12.9) 5 (16.1) Severe scratching to extent of bleeding/scarring 26 (83.9) 5 (16.1) 2 (6.5) 15 (48.4) 1 (3.2) 1 (3.2) 2 (6.5) 4 (12.9) 1 (3.2) 6 (19.4) 11 (35.5) Preventing wounds from healing 25 (80.6) 6 (19.4) 2 (6.5) 5 (16.1) 2 (6.5) 2 (6.5) 4 (12.9) 4 (12.9) 4 (12.9) 4 (12.9) 7 (22.6) Sticking sharp objects into skin 22 (71.0) 9 (29.0) 1 (3.2) 4 (12.9) 7 (22.6) 0 (0.0) 0 (0.0) 4 (12.9) 5 (16.1) 6 (19.4) 6 (19.4) Carving words into skin 19 (61.3) 12 (38.7) 2 (6.5) 13 (41.9) 2 (6.5) 0 (0.0) 0 (0.0) 2 (6.5) 1 (3.2) 3 (9.7) 13 (41.9) Carving pictures/designs/marks into skin 18 (58.1) 13 (41.9) 4 (12.9) 11 (35.5) 1 (3.2) 0 (0.0) 1 (3.2) 2 (6.5) 3 (9.7) 1 (3.2) 11 (35.5) Burning with lighter/match 18 (58.1) 13 (41.9) 1 (3.2) 8 (25.8) 5 (16.1) 0 (0.0) 1 (3.2) 0 (0.0) 3 (9.7) 4 (12.9) 9 (29.0) Punching to extent of bruising 17 (54.8) 14 (45.2) 1 (3.2) 10 (32.3) 1 (3.2) 0 (0.0) 1 (3.2) 1 (3.2) 6 (19.4) 1 (3.2) 8 (25.8) Banging head to extent of bruising 15 (48.4) 16 (51.6) 0 (0.0) 5 (16.1) 4 (12.9) 0 (0.0) 2 (6.5) 1 (3.2) 3 (9.7) 2 (6.5) 7 (22.6) Burning with cigarette 13 (41.9) 18 (58.1) 1 (3.2) 5 (16.1) 4 (12.9) 0 (0.0) 0 (0.0) 1 (3.2) 0 (0.0) 3 (9.7) 8 (25.8) Biting to extent of breaking skin 13 (41.9) 18 (58.1) 6 (19.4) 4 (12.9) 1 (3.2) 0 (0.0) 1 (3.2) 0 (0.0) 1 (3.2) 4 (12.9) 6 (19.4) Rubbing glass into skin 7 (22.6) 24 (77.4) 3 (9.7) 1 (3.2) 1 (3.2) 0 (0.0) 0 (0.0) 0 (0.0) 2 (6.5) 1 (3.2) 4 (12.9) Rubbing sandpaper on body 5 (16.1) 26 (83.9) 4 (12.9) 1 (3.2) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.2) 1 (3.2) 3 (9.7) Dripping acid onto skin 2 (6.5) 29 (93.5) 1 (3.2) 1 (3.2) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.2) 1 (3.2) Using bleach/oven cleaner to scrub skin 2 (6.5) 0 (0.0) 0 (0.0) 2 (6.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (6.5) Breaking own bones 1 (3.2) 30 (96.8) 1 (3.2) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.2) Note. Note. N reporting NSSI type may not equal frequencies as some participants endorsed type of NSSI, but did requencies may not add up to 100% because of missing data. Frequencies may not add up to 100% because of missing data. pe may not equal frequencies as some participants endorsed type of NSSI, but did not report frequency. t 100% b f i i d t pe may not equal frequencies as some participants endorsed type of NSSI, but did not report frequency. up to 100% because of missing data. 3.2.2 Prevalence of different types of self-injury N reporting NSSI type may not equal frequencies as some participants endorsed type of NSSI, but did not report frequency. Frequencies may not add up to 100% because of missing data. N (%) reporting recency of NSSI types N (%) reporting frequency of engagement in NSSI types 104 105 written explanations. When I calculated NSSI frequencies, I only included participants who had provided numerical data.12 For example, 30 people reported having cut themselves but only 19 provided numerical responses when asked how many times they had cut themselves. 12 Whenever participants estimated NSSI frequencies, I chose to include the most conservative value. For example, if a person reported that they had cut themselves 150-200 times, I took 150 as the frequency of cutting. If someone wrote thousands of times, I used 1,000 as the frequency. 13 In my initial study, I referred to non-suicidal self-injurious behaviours as self-harm because this is the term that is most commonly used in Aotearoa New Zealand to describe these behaviours. However, in light of the terminological discrepancies between self-harm and self-injury, which I discussed in Chapter 1, I decided to use the term NSSI for my subsequent studies. 4.1 Participants and procedure All 31 people who completed the DSHI (Gratz, 2001) were invited to take part in one or more interviews. Each person was sent a copy of the interview questions (see Appendix D), as advised by a Clinical Psychologist, because of the sensitive nature of the topic. Receiving a copy of the questions enabled potential participants to make a fully informed decision as to whether they would like to take part. The questions were prefaced by the following paragraph: questions were prefaced by the following paragraph: questions were prefaced by the following paragraph: Please read the following questions carefully. The purpose of this study is to understand how you experience self-harm13 from your perspective, while at the same time addressing the research questions listed below. As a result, Robyn cannot guarantee that she will ask you all of these questions or that these will be the only questions she will ask you during the interview(s). The questions do, however, provide you with an idea about what kinds of topics will be discussed during the interview. Please consider carefully if you would be comfortable answering these questions before you decide whether you would like to participate in an interview. Please read the following questions carefully. The purpose of this study is to understand how you experience self-harm13 from your perspective, while at the same time addressing the research questions listed below. As a result, Robyn cannot guarantee that she will ask you all of these questions or that these will be the only questions she will ask you during the interview(s). The questions do, however, provide you with an idea about what kinds of topics will be discussed during the interview. Please consider carefully if you would be comfortable answering these questions before you decide whether you would like to participate in an interview. Additionally, reviewing the questions beforehand gave participants who consented to be interviewed the opportunity to prepare themselves for the interview process. The benefits to participants afforded by this procedure were considered to outweigh the drawbacks that may have occurred from participants over-preparing answers (e.g., to make them more socially desirable). Additionally, reviewing the questions beforehand gave participants who consented to be interviewed the opportunity to prepare themselves for the interview process. The benefits to participants afforded by this procedure were considered to outweigh the drawbacks that may have occurred from participants over-preparing answers (e.g., to make them more socially desirable). Additionally, reviewing the questions beforehand gave participants who consented The interview schedule was developed for this study and was informed by the research literature on the behavioural functions of self-injury. As discussed 106 earlier in this chapter, I was interested in asking people to recall, and reflect on, their most recent episode of NSSI to enable me to hypothesise about the functions of these episodes. Identifying the functions of the NSSI episodes would then allow me to determine whether these descriptions were consistent with the behavioural process of self-injury outlined in the EAM (Chapman et al., 2006). The questions primarily focused on asking participants to describe what situational, emotional, and cognitive factors had led to their most recent episode of NSSI, how they felt and what they thought about while they were injuring themselves, and whether they had experienced any consequences as a result of the self-injurious behaviour. Participants were also asked about whether they considered their most recent episode of NSSI to be typical of their general pattern of self-injurious behaviour. Retrospective bias is a limitation of the majority of studies on NSSI but given that I was asking detailed questions about a specific behaviour, I did not want to interview people who had last self-injured years before. In the absence of a definitive boundary of what constitutes current versus historical self-injurious behaviour, I followed the precedent set by other researchers (Brown, Williams, & Collins, 2007; Whitlock et al., 2008) and invited only those people who had self-injured within the past twelve months to be interviewed. After being sent the interview questions, two people did not respond to follow-up emails, four people emailed to say that they had decided not to take part in an interview, and 25 people agreed to be interviewed. Unfortunately, due to the small sample size, I was unable to conduct any analyses to determine whether there were significant differences in the types, frequencies, or recency of NSSI between the people who consented and those who declined to take part in an interview. If people who agreed to be interviewed were engaged in a therapeutic relationship with a mental health clinician, they were to asked to sign a consent form (see Appendix E) giving me permission to inform their clinician about their participation in the study. This requirement was introduced following consultation 107 with clinicians who indicated that they would want to know if any of their clients chose to take part. Additionally, reviewing the questions beforehand gave participants who consented Fifteen people (14 female) reported that they were currently seeing a mental health clinician or that they received mental health support from another professional (i.e., GP, support worker). Out of the 14 people who consented to me contacting their clinician or other support person; one person nominated her GP as her contact person since she was currently engaged in group, not individual, therapy, while another person requested that I contact her support worker. One woman who was engaged in therapy and agreed to take part in an interview did not return her clinician consent form and was not interviewed as a result. The remaining 10 participants were not engaged in therapy or counselling, and, as such, were not required to complete the clinician consent form. A total of 24 people (20 female, 4 male) took part in the interview stage of the study which was conducted over a period of approximately six months. People were invited to bring a support person (e.g., their clinician, a friend, or a family member) with them to the interview; one young woman chose to bring her mother with her. None of the other participants brought a support person to the interview. A total of 24 people (20 female, 4 male) took part in the interview stage of the study which was conducted over a period of approximately six months. People were invited to bring a support person (e.g., their clinician, a friend, or a family member) with them to the interview; one young woman chose to bring her mother with her. None of the other participants brought a support person to the interview. Additionally, people were given the choice of being interviewed at Victoria University or the mental health service they attended (if applicable). Twenty-two interviews were conducted in a comfortable, private office in the Psychology Department at Victoria University, one interview was conducted at a mental health service attended by the participant, and one interview was conducted at a health service where the participant worked. Additionally, people were given the choice of being interviewed at Victoria University or the mental health service they attended (if applicable). Twenty-two interviews were conducted in a comfortable, private office in the Psychology Department at Victoria University, one interview was conducted at a mental health service attended by the participant, and one interview was conducted at a health service where the participant worked. Additionally, reviewing the questions beforehand gave participants who consented Prior to commencing each interview, I reiterated that although I had specific questions to ask, I was interested in learning about any of the person’s experiences of NSSI that they wanted to share. That is, the goal of the interview was to understand each person’s individual experience of self-injury, while at the same time addressing the research aims. I emphasised that my role as a researcher was to listen to their experiences of self-injurious behaviours; it was not my role to intervene therapeutically unless, as per the standard limits of confidentiality, they were in 108 danger of harming themselves or someone else following the interview. Each participant was informed that while I expected the interview to take approximately an hour, we could schedule a second interview if they felt that there was more they wanted to discuss.14 Participants were also informed that they could take a break or stop the interview at any time. I acknowledged that talking about self-injurious behaviours may be distressing and asked whether there was anything I could do to support them if they did become distressed during the interview, or whether they could think of any helpful strategies (e.g., having a cup of tea, taking a cigarette break) to use if they felt upset.15 Each person was asked to read through the consent form (see Appendix F) before signing it and given the opportunity to ask questions about the research. On the consent form, people were invited to tick whether they wanted to receive a copy of their interview transcript and/or whether they would like a brief summary of the study results. The majority (70.8%) of the participants requested a copy of their transcript and all but one (95.8%) requested the results summary. To begin each interview, I asked the person if there was anything that they wanted to talk about first in relation to their self-injurious behaviours or whether they would rather start with the questions I had sent them. This was done in an attempt to give people the space and time to talk about any experiences or issues they felt were relevant to their history of NSSI, rather than immediately focusing on what I wanted to know. The majority of participants opted to begin the interview with the research questions. 15 This approach was adapted from the University of Washington Risk Assessment Protocol developed by Marsha Linehan and colleagues (Reynolds, Lindenboim, Comtois, Murray, & Linehan, 2006). 14 No second interviews were scheduled. 15 This approach was adapted from the University of Washington Risk Assessment Protocol Additionally, reviewing the questions beforehand gave participants who consented All of the interviews were audiotaped (Mtime = 63 minutes; range: 29-92 minutes) using a digital audio recorder, the audio files were then uploaded on to a computer, and the original file on the recorder was deleted. The files were stored on a password-protected drive. Using Transcription Buddy V4.0, I transcribed the interviews according to a basic, orthographic notation system, 109 which included all verbal utterances (including repeated and cut-off words) and notable non-verbal utterances (e.g., laughter).16 The transcripts, which totalled more than 700 pages, were then checked against the original recordings to ensure accuracy. At the end of the interview, participants were reimbursed with a Farmers, Warehouse, or Motor Trade Association voucher to the value of $30 for taking part and given a list of support organisations (see Appendix C) to contact if they felt distressed. Participants were also invited to contact their clinician (if they had one) or me if they had any concerns. Participants who did not have mental health clinicians, and one participant who was dissatisfied with his clinician, were offered the option of a referral to the Psychology Clinic, which is located in the Department of Psychology at Victoria University. This is a teaching clinic, staffed by two experienced Clinical Psychologists, which is free to students at Victoria and charges up to $40 for clients who are not students. Fees were waived for study participants. Participants who declined to accept referrals were informed that they could contact me in the future if they changed their minds. Two male participants requested referrals following their interviews and one female participant emailed me approximately two months after being interviewed to request a referral. 16 My choice of transcription conventions reflects my interest in what participants said rather than how they said it, which precluded the need to transcribe linguistic details such as the timing of pauses and syllable lengths. Words in single brackets signify my best interpretation of the word(s). The word unclear in single brackets signifies my inability to interpret what was said. 4.2 Analysing the interview transcripts Initially, I attempted to analyse the interview transcripts thematically (Braun & Clark, 2006), but found that identifying group-level themes across the data corpus precluded an individual-level analysis of the temporal process of each person’s most recent episode of NSSI. Rather I needed an analytic method that would allow me to examine how each episode had unfolded over time. Drawing on the behaviour analysis literature, I developed a qualitative method called Interpretative Functional Analysis. Before outlining the rationale, epistemological assumptions, and steps of 16 My choice of transcription conventions reflects my interest in what participants said rather than how they said it, which precluded the need to transcribe linguistic details such as the timing of pauses and syllable lengths. Words in single brackets signify my best interpretation of the word(s). The word unclear in single brackets signifies my inability to interpret what was said. 110 this method in detail, it is first necessary to discuss the theoretical underpinnings and practical limitations of conducting functional analyses with typically developing populations. 4.2.1 What is a functional analysis? As an idiographic approach to the assessment and treatment of problem behaviours, a functional analysis involves examining how a person’s behaviour varies according to their unique learning history and environment (Farmer & Latner, 2007). The aim of a functional analysis is to establish which contingencies are maintaining a person’s problem behaviour so that the associated antecedents and consequences can be targeted to decrease the behaviour (Hanley, Iwata, & McCord, 2003). Accordingly, the three phases of a functional analysis involve: (1) gathering information about the problem behaviour and variables that may impact on that behaviour, (2) hypothesising about what contextual variables cause and maintain the behaviour to determine the function of the behaviour, and (3) manipulating particular variables (i.e., antecedents and consequences) in an attempt to alter the behaviour (Cone, 1997). 4.2.2 Utilising functional analyses with typically developing populations Functional analyses have been the mainstay of therapeutic assessment and intervention with developmentally disabled populations since the 1960s, but this approach has only more recently been applied to typically developing populations with the advent of Clinical Behaviour Analysis (Anderson, 2007; Dougher & Hayes, 2000). Originating in the 1990s, Clinical Behaviour Analysis can be understood, in part, as a reaction against the structuralist system used to assess, diagnose, and treat mental illness (Dougher & Hayes, 2000). From a structuralist perspective, problem behaviour is perceived as an indicator of underlying pathology; as such, causes of behaviour can be found within people (Follette, Naugle, & Linnerooth, 2000; Sturmey et al., 2007a). In contrast, functionalists maintain that behaviour is an adaptive response to the environmental context and, as such, causes of behaviour lie within the environment (Sturmey et al., 111 2007a). Clinicians working within a behavioural paradigm view functional approaches as non-pathologising compared to structuralist approaches, because the target for change is the environment rather than the person (Sturmey et al., 2007a). Structuralism, however, is privileged over functionalism within the domain of mental health research and clinical practice because of the hegemony of the DSM (American Psychiatric Association, 2000). Classifying problem behaviours on the basis of shared topographical features has advantages: it provides a shared, common language for clinicians; indicates which behaviours are likely to covary; and points to possible interventions (Nelson-Gray & Farmer, 1999). However, structural approaches to psychopathology also have a number of disadvantages. As a syndromal classification system, the DSM-IV-TR (American Psychiatric Association, 2000) provides insight into what a person has but not how they interact with the environment and failing to identify the functions of problem behaviours makes it more difficult to alter these behaviours (Cipani & Schock, 2007; Cone, 1997). Given that one behaviour can serve many functions and a single function (e.g., escape) can maintain a range of topographically dissimilar behaviours, functional classification systems (see Cipani & Schock, 2007) arguably have more utility than syndromal classification systems when it comes to understanding problem behaviours (Cone, 1997). Clinical Behaviour Analysis (CBA) is informed by Applied Behaviour Analysis (ABA) in that it involves ‚the application of the assumptions, principles and methods of modern functional contextual behavior analysis‛ to mental disorders (Dougher, & Hayes, 2000, p. 17 Within the field of radical behaviourism, private events such as thoughts, emotions, and somatic sensations qualify as behaviours that can be functionally assessed (Cuper, Merwin, & Lynch, 2007). 18 Although I functionally assessed, rather than analysed, each episode of NSSI, I chose to call my method Interpretative Functional Analysis, not Interpretative Functional Assessment, because I went beyond the realm of individual assessment to then compare the antecedents and consequences of NSSI episodes both within and between functions. 4.2.2 Utilising functional analyses with typically developing populations 11), but it also has three key differences: (1) ABA focuses on external stimuli that can be observed (Kohlenberg, Tsai, & Dougher, 1993); CBA focuses on intrapersonal stimuli (e.g., thoughts, emotions) because these internal processes17 are extremely relevant to the development and maintenance of psychopathology (Hayes et al., 2006). 112 (2) To gather information about problem behaviours, applied behaviour analysts typically depend on direct observations (Hanley et al., 2003). In contrast, clinical behaviour analysts working within mental health settings are seldom able to observe problem behaviours because they are internal processes or they occur outside of therapy (Farmer & Latner, 2007; Miltenberger, 2005). Consequently, self-report is an important source of information utilised by clinical behaviour analysts (Miltenberger, 2005). (3) Applied behaviour analysts manipulate specific variables to test whether or not they reinforce the problem behaviour (Kohlenberg et al., 1993), whereas clinical behaviour analysts are typically unable to manipulate variables that may reinforce problem behaviours as these are often internal (Miltenberger, 2005). (3) Applied behaviour analysts manipulate specific variables to test whether or not they reinforce the problem behaviour (Kohlenberg et al., 1993), whereas clinical behaviour analysts are typically unable to manipulate variables that may reinforce problem behaviours as these are often internal (Miltenberger, 2005). (3) Applied behaviour analysts manipulate specific variables to test whether or not they reinforce the problem behaviour (Kohlenberg et al., 1993), whereas clinical behaviour analysts are typically unable to manipulate variables that may reinforce problem behaviours as these are often internal (Miltenberger, 2005). 4.2.3.1 Rationale Given that the overarching research question in this thesis is whether NSSI functions primarily as an experientially avoidant behaviour within Aotearoa New Zealand, it was necessary for me to assess whether the descriptions of specific self- injury episodes aligned with the operant process described in the EAM (Chapman et al., 2006). My interview questions were designed to elicit the antecedents and consequences of each person’s most recent episode of self-injurious behaviour and, as such, assessing these descriptions in order to hypothesise the function(s) of the behaviours appeared to be the most appropriate form of analysis. To analyse the interviews, I developed a method called Interpretative Functional Analysis which was informed by the principles of CBA. Although I have named this method Interpretative Functional Analysis18, it does not meet the 113 requirements for a full functional analysis as defined within CBA because I was unable to work therapeutically with people to bring about changes in their behaviours. As discussed earlier, a functional analysis consists of three phases: gathering information about a problem behaviour, developing a hypothesis about the function(s) of that behaviour, and manipulating relevant variables in an attempt to decrease or eliminate the behaviour (Cone, 1997). I only conducted one interview with each person in a research rather than therapeutic context; accordingly, I did not complete the treatment phase of a functional analysis. Instead, I focused on the first two phases of information gathering and hypothesis formulation, collectively called a functional assessment (Cipani & Schock, 2007; Cone, 1997), the outcome of which is a ‚hypothesis statement identifying environmental variables that likely evoke and maintain the behavior of concern‛ (Anderson, 2007, p. 459). Assessing the context in which the self-injury occurred (e.g., the antecedents) in conjunction with the consequences that followed the behaviour, allowed me to hypothesise about the functions of participants’ NSSI (Cone, 1997). Once I had formulated a set of hypotheses about the functions of each self- injury episode, I compared the hypothesised functions across the group of participants to identify similarities and differences between antecedents and consequences both within the same function and across different functions. In sum, the aims of conducting an Interpretative Functional Analysis were to: (1) assess each person’s most recent episode of NSSI to derive a hypothesis statement about the function(s) of their behaviour and (2) compare the antecedents and consequences of the self-injurious behaviours both within and between the different functions. 4.2.3.1 Rationale Functionally assessing discrete episodes of self-injury to compare behavioural functions across a group of research participants is unorthodox. Certainly, functional analyses of problem behaviours within typically developing populations have been published as case studies (e.g., Farmer & Latner, 2007) and within treatment manuals (e.g., Linehan, 1993a), but I have been unable to identify any studies where 114 these individual analyses were then compared at a group level. However, I believe that the form of analysis I developed for this study was the most appropriate method to answer my research question. 4.2.3.2 Epistemological assumptions I included the word ‘interpretative’ when labelling my method as an explicit recognition of the impact of my values and perspectives when writing the interview questions, interviewing the participants, transcribing the data, formulating hypotheses about the functions of the behaviours, and writing up the results. Each of these phases involved differing levels of interpretation, all of which were informed by my own personal epistemologies, values, and understanding of the self-injury literature. I concur with Angen (2000) that: I included the word ‘interpretative’ when labelling my method as an explicit recognition of the impact of my values and perspectives when writing the interview questions, interviewing the participants, transcribing the data, formulating hypotheses about the functions of the behaviours, and writing up the results. Each of these phases involved differing levels of interpretation, all of which were informed by my own personal epistemologies, values, and understanding of the self-injury literature. I concur with Angen (2000) that: Truth, from an interpretive perspective, is no longer based on a one-to-one correspondence to objective reality. It is acknowledged that what we can know of reality is socially constructed through our intersubjective experiences within the lived world, which results in a form of truth that is negotiated through dialogue. (p. 386) Truth, from an interpretive perspective, is no longer based on a one-to-one correspondence t objective reality. It is acknowledged that what we can know of reality is socially constructed through our intersubjective experiences within the lived world, which results in a form of truth that is negotiated through dialogue. (p. 386) Additionally, the absence of a treatment phase in the functional analyses I conducted prevented me from testing my hypotheses, which instead remained as functional interpretations. Additionally, the absence of a treatment phase in the functional analyses I conducted prevented me from testing my hypotheses, which instead remained as functional interpretations. 4.2.3.1 Rationale Arguably the most important epistemological assumption underlying Interpretative Functional Analysis, which I touched on when comparing ABA to CBA, is that CBA considers self-report to be a valid form of data. While applied behaviour analysts privilege independent observation and dismiss self-report as an unreliable and invalid approach to assessing behaviour (Cipani & Schock, 2007), clinical behaviour analysts maintain that it is possible to identify reinforcers through self-report while remaining cognisant of the limitations associated with this method (Sturmey, Ward-Horner, Marroquin, & Doran, 2007b). 4.2.3.3 Coding system Arguably the most important epistemological assumption underlying Interpretative Functional Analysis, which I touched on when comparing ABA to CBA, is that CBA considers self-report to be a valid form of data. While applied behaviour analysts privilege independent observation and dismiss self-report as an unreliable and invalid approach to assessing behaviour (Cipani & Schock, 2007), The three term contingency of antecedent-behaviour-consequence formed the basis of my coding system, but I also took into account relevant historical antecedents that provided insight into each person’s current episode of NSSI. 115 Behaviours were then categorised according to whether they served an escape/avoidant or access function (Cipani & Schock, 2007). 4.2.3.3.1 Relevant learning history Historical variables are relevant to completing a functional assessment if they impact on, or point to, significant, contemporary antecedents that are amenable to change (Farmer & Latner, 2007; Follette et al., 2000). Since historical events (e.g., trauma) can have an impact on current behaviour and each person’s learning history determines which behaviours are maintained over time (Sturmey et al., 2007b), I extracted historical information from the interview transcripts that I interpreted as relevant to participants’ most recent episode of NSSI. However, it should be noted that it is often difficult to determine the extent to which these antecedents impact on current behaviour, especially when the information about how the behaviour functions is gathered through retrospective self-report. 4.2.3.3.2 Antecedents Identical behaviours can fulfill different functions depending on the antecedents of those behaviours (Cipani & Schock, 2007); consequently, it is possible to infer the functions of a behaviour by identifying the antecedents (Stickney & Miltenberger, 1999). Temporally remote antecedents can function as setting events which then have an effect on more immediate antecedents (Stickney & Miltenberger, 1999). 4.2.3.1 Rationale For example, receiving a low grade for a report might lead someone to question their academic abilities and subsequent thoughts of inadequacy and failure then lead to an episode of NSSI. Two types of antecedents were identified in the interview transcripts: Establishing Operations (EO) and Discriminative Stimuli (SD). Establishing operations are conditions (e.g., deprivation, aversive stimulation) that establish particular consequences as reinforcers (Dougher & Hackbert, 2000; Follette et al., 2000); people are motivated to engage in specific behaviours because of EOs (Follette et al., 2000; Miltenberger, 2005). To identify an EO, a useful question to ask is: ‚Why does the person ‚want‛ this consequence?‛ (McGill, 1999, p. 399). When a person 116 engages in self-injury to gain the consequence of relief, it is likely that they wanted that relief because they were experiencing aversive internal stimulation (e.g., anxiety, negative thoughts). In this instance, the aversive internal stimulation would be classified as an EO because it establishes the relief as a reinforcer for self-injury. Establishing operations have a value-altering effect in that they determine how effective or potent a particular reinforcer will be in a specific situation (Laraway, Snycerski, Michael, & Poling, 2003; Michael, 1993; Miltenberger, 2004). A reinforcer’s value thus shifts along a continuum depending on the intensity of the EO (Laraway et al., 2003). For example, food deprivation is an EO but people can be more or less food-deprived. The more hungry a person is, the more likely it is that food will be an effective reinforcer. Establishing operations also have a behaviour-altering effect in that they increase the likelihood of the person engaging in behaviours that are typically followed by that reinforcer (Laraway et al., 2003; Michael, 1993; Miltenberger, 2004). The second type of antecedents, SD’s, signal the likelihood that a behaviour will elicit a particular consequence because the stimulus has typically been present when that behaviour was reinforced in the past (Farmer & Latner, 2007; Kearney, 2008). The presence of a SD (e.g., NSSI implements) therefore signals to the person that there is an opportunity for reinforcement or, in other words, the likelihood that a particular reinforcer is available (Miltenberger, 2005). Since a specific behaviour can be triggered by a number of different SD’s (Cuper, Merwin, & Lynch, 2007), the capacity of stimuli to function as reinforcers depends on the EOs. Discriminative stimuli therefore work in conjunction with EOs to bring about particular behaviours. 4.2.3.1 Rationale An SD will only impact on behaviour when an EO is present because a person has to want a particular consequence in order to be to be influenced by the opportunity to experience that consequence (Laraway et al., 2003). For example, a razor blade may have been established as an SD in that it signals the opportunity for the release of intense anger following cutting behaviour. If the EO of intense anger is absent, it is unlikely that the person will use the razor blade to cut themselves because they will not be motivated to seek relief. 117 4.2.3.3.3 Target behaviour 4.2.3.3.3 Target behaviour Since the focus of my analysis was the functions of people’s self-injury, not the topographical features of these behaviours, I did not purposefully seek specific details beyond asking how they had hurt themselves and, occasionally, how many times they had hurt themselves during their most recent episode. Some participants did chose to volunteer detailed information about how they had injured themselves and the implements they had used for this purpose, while others provided little information about the actual self-injurious behaviour. 4.2.3.3.4 Consequences Particular behaviours are selected for through operant conditioning; that is, behaviours impact on the environment to produce consequences and are subsequently more or less likely in particular contexts because of those consequences (Cipani & Schock, 2007; Sturmey et al., 2007b). The likelihood of a behaviour re- occurring depends on whether an individual views the particular consequences of that behaviour as positive or aversive. Positive consequences maintain or increase behaviour, whereas aversive consequences temporarily or permanently suppress behaviour (Goldfried & Sprafkin, 1976). There are four general categories of consequences—positive reinforcement, negative reinforcement, positive punishment, and negative punishment (Sturmey et al., 2007b)—all of which can be intrapersonally or interpersonally mediated. As a result, I coded for four categories of reinforcement: intrapersonal negative reinforcement, intrapersonal positive reinforcement, interpersonal negative reinforcement, and interpersonal positive reinforcement. I coded aversive consequences (i.e., punishers) more generally because when considering clinical behaviour problems (e.g., substance abuse), the aversive consequences are usually delayed and so do not function to suppress the behaviour (Goldfried & Sprafkin, 1976). For example, aversive consequences following NSSI, such as others’ reactions to scarring, are typically delayed compared to positive consequences, such as an adrenalin rush or relief of emotional distress, which are immediate. 118 4.2.3.3.5 Escape or access: Functions of the target behaviour 4.2.3.3.5 Escape or access: Functions of the target behaviour The function of a behaviour refers to the way in which that behaviour impacts on the environment (Hanley et al., 2003). As summarised by Cipani and Schock (2007), behaviour serves two overarching functions: to escape or avoid negative events and to access positive events. Escape or avoidance behaviours are maintained via negative reinforcement, while access behaviours are maintained via positive reinforcement (Cipani & Schock, 2007). Furthermore, behaviours can be intrapersonally or interpersonally mediated (Cipani & Schock, 2007). Each episode of self-injury therefore was coded according to whether it functioned as intrapersonal or interpersonal escape/avoidance or access. 19 All of the participants were given pseudonyms to protect their identities. 4.2.3.3.3 Target behaviour I then completed schematic representations (c.f., Braun & Clarke, 2006) of the behavioural process of each person’s most recent episode of self-injury (see Figure 3 for an example). The structure of these diagrams was adapted from those presented in Follette et al. (2000) and Farmer and Latner (2007). The black circle represents the probability that the discriminative stimuli and establishing operations evoked the target behaviour in the context of the relevant learning history. (5) An iterative and reflexive process was followed whereby I re-read the extracts to check that I had summarised all of the information that was relevant to completing a functional assessment of each episode and that I had included all of this information in the diagrams. I also re-read the original transcripts to ensure that extracts and the diagrams represented each person’s most recent episode as they had described it and that I had not inadvertently left out pertinent information. (5) An iterative and reflexive process was followed whereby I re-read the extracts to check that I had summarised all of the information that was relevant to completing a functional assessment of each episode and that I had included all of this information in the diagrams. I also re-read the original transcripts to ensure that extracts and the diagrams represented each person’s most recent episode as they had described it and that I had not inadvertently left out pertinent information. (5) An iterative and reflexive process was followed whereby I re-read the extracts to check that I had summarised all of the information that was relevant to completing a functional assessment of each episode and that I had included all of this information in the diagrams. I also re-read the original transcripts to ensure that extracts and the diagrams represented each person’s most recent episode as they had described it and that I had not inadvertently left out pertinent information. (6) Based on the information depicted in the schematic representations, I formed a hypothesis as to the primary function of each person’s most recent episode of self-injurious behaviour in the context of their relevant learning history. For example, I hypothesised that Melanie’s19 most recent episode of NSSI (see Figure 3) functioned as an escape from aversive thoughts and emotions. 4.2.3.3.3 Target behaviour 4.2.3.4 Steps in the Interpretative Functional Analysis In order to complete the Interpretative Functional Analysis, I followed the seven steps outlined below: (1) I read through all of the interview transcripts to identify any extracts that could be relevant to each person’s most recent episode, including their learning history, any experiences that motivated them to self-injure, the antecedent events or circumstances that occurred before they self-injured, descriptions of the target behaviour, and the consequences that followed that behaviour. I also identified any information related to whether they considered their most recent episode to be typical of their general pattern of self-injury. (2) The relevant extracts from each interview transcript were then cut and pasted into a word document for each person under the following headings: relevant learning history, establishing operations, discriminative stimuli, target behaviour, consequences, and typicality of episode. Any surrounding, contextual information that was important in order to understand and interpret the extract was retained, including any of my questions or comments that led to particular explanations or elaborations from participants. (2) The relevant extracts from each interview transcript were then cut and pasted into a word document for each person under the following headings: relevant learning history, establishing operations, discriminative stimuli, target behaviour, consequences, and typicality of episode. Any surrounding, contextual information that was important in order to understand and interpret the extract was retained, including any of my questions or comments that led to particular explanations or elaborations from participants. 119 (3) Once tabulated, I read through the selected extracts for each person and summarised any information from the extracts that I deemed relevant to a functional assessment of each person’s most recent episode of NSSI. I then completed schematic representations (c.f., Braun & Clarke, 2006) of the behavioural process of each person’s most recent episode of self-injury (see Figure 3 for an example). The structure of these diagrams was adapted from those presented in Follette et al. (2000) and Farmer and Latner (2007). The black circle represents the probability that the discriminative stimuli and establishing operations evoked the target behaviour in the context of the relevant learning history. (3) Once tabulated, I read through the selected extracts for each person and summarised any information from the extracts that I deemed relevant to a functional assessment of each person’s most recent episode of NSSI. 4.2.3.3.3 Target behaviour (6) Based on the information depicted in the schematic representations, I formed a hypothesis as to the primary function of each person’s most recent episode of self-injurious behaviour in the context of their relevant learning history. For example, I hypothesised that Melanie’s19 most recent episode of NSSI (see Figure 3) functioned as an escape from aversive thoughts and emotions. (7) The hypothesised functions were then compared across participants to identify any similarities or differences in the antecedents and consequences of the NSSI episodes within the same function, and to identify any similarities or differences between functions. (7) The hypothesised functions were then compared across participants to identify any similarities or differences in the antecedents and consequences of the NSSI episodes within the same function, and to identify any similarities or differences between functions. Figure 3. Functional schematic of Melanie’s most recent episode of NSSI. 4.2.3.3.3 Target behaviour Relevant Learning History  Long history of NSSI since age 12; NSSI escalated over time  Self-injures in stages  Gets into an NSSI mindset  NSSI is a coping mechanism  Uses NSSI for tension release and to punish self  Self-injures when stressed and upset  Negative events triggers NSSI ideation  Negative thoughts trigger NSSI  Different tools serve different purposes  Grazing calms her down; cutting makes her feel relieved and in control  NSSI helps her to sleep  Uses NSSI to stop mind racing and to settle herself down  NSSI is like an addiction; if in NSSI mindset, difficult to prevent it  Had Bulimia  Cut deeper than usual which scared her  Felt almost ashamed, had to cover up cuts  Angry with ex-boyfriend for telling mother  Mother was very upset Intrapersonal Negative Reinforcers  Felt calmer, more settled  Thoughts stopped racing Interpersonal Negative Reinforcers  None identified Interpersonal Positive Reinforcers  None identified Intrapersonal Positive Reinforcers  None identified Aversive Consequences Reinforcing Consequences Discriminative Stimuli  Thought that NSSI would help  Implement Establishing Operations  Had a fight with her new boyfriend  Felt really anxious  Mind was racing  Felt upset, jittery, and could not settle down  Had thoughts of not being good enough  Blamed self, everything was her fault  Wanted to settle herself down Target Behaviour Cut self on the arm multiple times, approximately two months prior to the interview 120 Intrapersonal Negative Reinforcers  Felt calmer, more settled  Thoughts stopped racing Intrapersonal Positive Reinforcers  None identified Reinforcing Consequences Interpersonal Negative Reinforcers  None identified Interpersonal Positive Reinforcers  None identified Target Behaviour Cut self on the arm multiple times, approximately two months prior to the interview Aversive Consequences Figure 3. Functional schematic of Melanie’s most recent episode of NSSI. Figure 3. Functional schematic of Melanie’s most recent episode of NSSI. 120 120 121 4.3 Interview results Of the 24 people who were interviewed, one participant provided too little information about their most recent episode for a functional assessment and three participants did not identify experiencing any form of reinforcement following their self-injurious behaviour. As a result, only 20 episodes of self-injury are included in the following analysis. Figure 4 shows which participants’ episodes were Of the 24 people who were interviewed, one participant provided too little information about their most recent episode for a functional assessment and three participants did not identify experiencing any form of reinforcement following their self-injurious behaviour. As a result, only 20 episodes of self-injury are included in the following analysis. Figure 4 shows which participants’ episodes were hypothesised to fulfil each function. The colour purple is used to specify participants whose episode was hypothesised to only fulfil one function, blue specifies participants whose episode fulfilled two functions, and green specifies participants whose episode fulfilled three functions. Escape/Avoidance Intrapersonal Natalie Karen Jenny Nora Melanie Lucy Ella Luke Angela Nicola Josh Emily Tara Paula Sophia Owen Matt Maria Interpersonal Belinda Angela Nicola Josh Emily Tara Owen Matt Maria Debra Paula Sophia Owen Matt Maria Access Figure 4. Functions fulfilled by each participant’s most recent episode of NSSI. Figure 4. Functions fulfilled by each participant’s most recent episode of NSSI. 122 Although the most commonly identified function was intrapersonal escape/avoidance, many episodes appeared to serve multiple functions. Of the episodes that only served one function, eight were categorised as intrapersonal escape/avoidance, one was categorised as intrapersonal access, and one as interpersonal access. Furthermore, as is evident from Figure 4, no episodes were hypothesised to serve an interpersonal escape/avoidant function. In the following sections, I present the detailed findings of my Interpretative Functional Analysis, using relevant interview extracts to illustrate my arguments. The EOs, SD’s, and reinforcing consequences identified within the transcripts for each of the three functions—intrapersonal escape/avoidance, intrapersonal access, and interpersonal access—are discussed. I also briefly examine two historical examples (i.e., not the most recent episode) where self-injury functioned as a form of interpersonal escape/avoidance. Finally, I discuss the aversive consequences described by participants, which may have functioned to punish their most recent episode of self-injurious behaviour. 4.3.1 Intrapersonal escape/avoidance 4.3.1 Intrapersonal escape/avoidance When I considered the antecedents and consequences of the NSSI episodes within the context of each person’s learning history, I hypothesised that the majority of these episodes (N = 18) had functioned as a form of escape from aversive emotions and thoughts. People described an array of both distal and proximal negative events which, along with negative intrapersonal experiences, had established the motivation for them to engage in NSSI. They also identified a variety of stimuli that had signalled to them the potential for negative reinforcement following self- injurious behaviour. All of these participants reported that their episode of self- injury was followed by the reduction or elimination of unwanted emotions and/or thoughts. Although it is difficult to be certain that the distal events reported did have an effect on people’s most recent episode of self-injury, in some cases it is likely that these events partly established the conditions for self-injury to function as a form of 123 escape or avoidance. For example, one participant reported experiencing flashbacks before self-injuring and two participants mentioned traumatic events that they had not fully come to terms with. It is probable that the consequences of these events influenced their most recent episode of NSSI. 4.3.1.1 Establishing Operations 4.3.1.1 Establishing Operations Emily first cut herself on purpose at the age of 11 and had since self-injured in stages, while concurrently struggling with depression. Her self-injury, however, got ‚a lot worse‛ after her marriage ended; she attributed this escalation and her most recent episode of NSSI to the stressful transition she was going through at the time: Emily: I think the type of stress I was experiencing was different<um it was a it wasn’t stress and anxiety directly related to to you know a um a clinical depression it was<it was related to n- to a trauma in my life you know<to to my husband leaving me<my life was ((laughing)) cha- it was changing massively<um you know during that especially that six months<I felt like it was changing for the better<um but it didn’t make the the transition ((laughing)) ((laugh)) easy you know20 mily: I think the type of stress I was experiencing was different<um it was a it wasn’t stress and anxiety directly related to to you know a um a clinical depression it was<it was related to n- to a trauma in my life you know<to to my husband leaving me<my life was ((laughing)) cha- it was changing massively<um you know during that especially that six months<I felt like it was changing for the better<um but it didn’t make the the transition ((laughing)) ((laugh)) easy you know20 Maria had similarly experienced traumatic loss and her grief, following the unexpected and untimely death of a friend, most likely served as a distal EO for her most recent episode of NSSI. Although she recalled self-injuring between the ages of approximately 11 to 14 by banging her head against things and scratching herself, she had not continued to self-injure through her later adolescence. Indeed, she had only begun hurting herself again after her friend’s death. At the time of her most recent episode of NSSI, Maria was also supporting her flatmate who had been the victim of a rape and attempting to focus on a very difficult year of university studies. 4.3.1.1 Establishing Operations Although these stressors, from what she described as ‚the year from hell‛, combined to establish the conditions where she began self-injuring again, it is likely that only the loss of her friend functioned as a distal EO for her most recent episode of cutting because her grief remained unresolved: Maria: I think it started out of my frustration with my friend and not<just with her but with the Maria: I think it started out of my frustration with my friend and not<just with her but with the 20 The ellipses in this and all following interview extracts signify deleted text. Text was deleted when it was considered extraneous to the points being discussed. Standalone minimal encouragers (e.g., mhmm, yeah) were deleted as these disrupt the readability of extracts. 20 The ellipses in this and all following interview extracts signify deleted text. Text was deleted when it was considered extraneous to the points being discussed. Standalone minimal encouragers (e.g., mhmm, yeah) were deleted as these disrupt the readability of extracts. 124 situation she was in<but I think what it really was for me<was the the grief of my friend dying<I think that was that was the huge thing ‘cos it was such a hard thing to grasp situation she was in<but I think what it really was for me<was the the grief of my friend dying<I think that was that was the huge thing ‘cos it was such a hard thing to grasp Although the effect of these distal EOs on both Emily and Maria’s subsequent episodes of NSSI cannot be conclusively determined, these events did appear to exert at least some influence over their engagement in their most recent episode of self-injurious behaviour. Along with distal events, participants talked about how specific proximal events or ongoing stressors, followed by the experience of negative emotions and thoughts, had motivated them to seek relief or release through self-injury. The events and conditions that served as EOs were typically unique to each person and included: fighting, or breaking up, with a boyfriend; being left alone at home without family for two weeks; experiencing an upsetting therapy session; moving back to Wellington; failing at university; ongoing physical and mental health issues; and changing to a different anti-psychotic medication. Each of these EOs was typically accompanied by intense, negative emotions and thoughts which culminated in self-injury. 4.3.1.1 Establishing Operations The accumulating impact of aversive intrapersonal experiences was summed up by Owen who described what led to his most recent episode of self-injurious behaviour, where he burnt himself on the hand multiple times, as follows: Owen: the reason I did it recently was because um was because I thought I was getting ill again<I thought I was getting unwell um I was on a med change and it wasn’t going particularly well<and um I just didn’t wanna um go through that again and and I felt like I was yeah useless and this was going to be the end of my life ra ra rarara ((sniffs)) um and so I um yeah so I just I d- I I just completely broke down really<and um just started to to do that Owen: When I asked Owen what indications he had that he was becoming unwell again, he g g identified that he thought he had heard a voice and had experienced ‚raw anxiety that is just really paralysing.‛ One of the reasons becoming unwell again was so frightening for him was because of his previous psychotic episodes which he described as an ‚open wound‛: if I refer to it people think ah why is he still talking about that why does it what does it matter<it was years ago you move on blah blah blah blah they don’t understand that it Owen: if I refer to it people think ah why is he still talking about that why does it what does it matter<it was years ago you move on blah blah blah blah they don’t understand that it 125 was such a big trauma Potentially hearing a voice in the context of a medication change reminded Owen of the trauma of his previous two psychotic episodes. These memories were accompanied by intense anger at the unfairness of the situation, self-hatred, and self- blame because Owen thought he had partly caused his Schizophrenia by smoking marijuana and partying too hard during adolescence: Potentially hearing a voice in the context of a medication change reminded Owen of the trauma of his previous two psychotic episodes. These memories were accompanied by intense anger at the unfairness of the situation, self-hatred, and self- blame because Owen thought he had partly caused his Schizophrenia by smoking marijuana and partying too hard during adolescence: Owen: it it just didn’t seem fair<and I just hated myself because because for a lot of like it also links into the fact that I think um to a degree that I was a player in my illness<I dunno if I caused it but I definitely made it worse um so there was that feeling that because it’s been a real tough four or five years and so it’s the realisation that the uh you know the feeling that this is going to be like this for the rest of my life<um and that I’ve done it to myself Given his past learning history, it is understandable that Owen chose to burn himself in the context of such intense, negative emotions, self-recrimination, and catastrophising about what the future held for him. Owen: Anger, frustration, self-hatred, and self-blame were some of the most common emotions discussed in the interviews as EOs for the self-injury episodes and, more often than not, were accompanied by a range of self-critical thoughts: Ella: I was just feeling really frustrated and really really overwhelmed and really angry at my body<for um not working the way ((laugh)) it’s meant to I guess 126 Nora: I think the biggest thing was I was um angry with who I was<and I didn’t feel like I fit anywhere<in that I didn’t really have a place in the world<I just didn’t really like anything about myself or the situation that I was in<um and just thinking about um if people really actually did like me or if it was all fake<um and if my parents loved me Lucy: Robyn: Lucy: I was just really upset and decided everyone hated me and stuff like that<the stuff that triggered it was all just to do with kind of rejection and stuff like that<I was angry at the fact that what seemed like little insignificant things were still affecting me mhmm so when you’re feeling like that what kind of thoughts run through your head ((laugh)) not very nice ones ((laughing))<useless worthless just stuff along those lines ((laugh)) Tara: I was kinda frustrated with myself because<I’m not normal and I just wanted to kind of be better and fine and that was all and so I dunno it was like a well it kind of was a way of getting over that anger and frustration at myself by doing that<I think it was quite a hateful thing to do to myself Josh: I guess the main thing that really sort of triggered off me actually um cutting was uh it’s kind of a sort of disgusted at my own thoughts at the time<about how I was um sort of reacting to things at the moment<as far as uni went and um like that I’m not actually really trying at ((laugh))<at anything ((laughing))<I was just sort of uh trying to figure out why I was doing that which led onto thinking back to all sorts of ((clears throat)) past instances where I’ve been doing something and then kind of given up and then<um yeah sort of got quite angry at myself for that and um yeah just kind of got a bit disgusted with the fact that I was ((laughing)) doing it um which sort of turned into thoughts about being sort of useless<and uh not actually sticking anything through Paula: um I think the main thoughts that were going through my head were um the areas that um have triggered my eating disorder for instance<um neglect from parents (unclear)<um disruptive childhood and um certain events that have happened<to me like um abuse and stuff like that and so those sort of things replay in my mind and<um then like the the thoughts of how much I hate myself Sophia: Robyn: Sophia: I think I probably just felt quite exhausted<um and um frustrated at feeling like I wasn’t coping<I think I felt angry...um directed at myself um yeah and when you feel angry like that about yourself<um what kind of thoughts would go through your head in relation to to that anger< <I feel frustrated that well it feels like to me that little things are affecting me<and I feel frustrated that I that even though I don’t want them to that they do<um and I think that probably leads to me thinking that in general I don’t cope very well<um as a person and that you know and and doubting whether how competent I am as a mum<um and how supportive I am as a wife and it it yeah it just I think it just brings into question lots of those things that I um put my self value on The combined pressure of intense, high arousal emotions such as anger and frustration along with thoughts about being useless and worthless led to people I think the biggest thing was I was um angry with who I was<and I didn’t feel like I fit anywhere<in that I didn’t really have a place in the world<I just didn’t really like anything about myself or the situation that I was in<um and just thinking about um if people really actually did like me or if it was all fake<um and if my parents loved me I was just really upset and decided everyone hated me and stuff like that<the stuff that triggered it was all just to do with kind of rejection and stuff like that<I was angry at the fact that what seemed like little insignificant things were still affecting me mhmm so when you’re feeling like that what kind of thoughts run through your head ((laugh)) not very nice ones ((laughing))<useless worthless just stuff along those lines ((laugh)) I was just really upset and decided everyone hated me and stuff like that<the stuff that triggered it was all just to do with kind of rejection and stuff like that<I was angry at the fact that what seemed like little insignificant things were still affecting me g g g mhmm so when you’re feeling like that what kind of thoughts run through your head ((laugh)) not very nice ones ((laughing))<useless worthless just stuff along those lines ((laugh)) I was kinda frustrated with myself because<I’m not normal and I just wanted to kind of be better and fine and that was all and so I dunno it was like a well it kind of was a way of getting over that anger and frustration at myself by doing that<I think it was quite a hateful thing to do to myself I guess the main thing that really sort of triggered off me actually um cutting was uh it’s kind of a sort of disgusted at my own thoughts at the time<about how I was um sort of reacting to things at the moment<as far as uni went and um like that I’m not actually really trying at ((laugh))<at anything ((laughing))<I was just sort of uh trying to figure out why I was doing that which led onto thinking back to all sorts of ((clears throat)) past instances where I’ve been doing something and then kind of given up and then<um yeah sort of got quite angry at myself for that and um yeah just kind of got a bit disgusted with the fact that I was ((laughing)) doing it um which sort of turned into thoughts about being sort of useless<and uh not actually sticking anything through Paula: um I think the main thoughts that were going through my head were um the areas that um have triggered my eating disorder for instance<um neglect from parents (unclear)<um disruptive childhood and um certain events that have happened<to me like um abuse and stuff like that and so those sort of things replay in my mind and<um then like the the thoughts of how much I hate myself um I think the main thoughts that were going through my head were um the areas that um have triggered my eating disorder for instance<um neglect from parents (unclear)<um disruptive childhood and um certain events that have happened<to me like um abuse and stuff like that and so those sort of things replay in my mind and<um then like the the thoughts of how much I hate myself I think I probably just felt quite exhausted<um and um frustrated at feeling like I wasn’t coping<I think I felt angry...um directed at myself um yeah and when you feel angry like that about yourself<um what kind of thoughts would go through your head in relation to to that anger< I think I probably just felt quite exhausted<um and um frustrated at feeling like I wasn’t coping<I think I felt angry...um directed at myself um yeah and h f l l k h b lf h k d f h h ld <I feel frustrated that well it feels like to me that little things are affecting me<and I feel frustrated that I that even though I don’t want them to that they do<um and I think that probably leads to me thinking that in general I don’t cope very well<um as a person and that you know and and doubting whether how competent I am as a mum<um and how supportive I am as a wife and it it yeah it just I think it just brings into question lots of those things that I um put my self value on The combined pressure of intense, high arousal emotions such as anger and frustration, along with thoughts about being useless and worthless, led to people feeling overwhelmed and desperate to alleviate their distress. Owen: After all, a long, established pattern of self-injury since the age of 13 years old had reinforced for him that self- injury was ‚the answer‛ to his self-hatred and depression: Given his past learning history, it is understandable that Owen chose to burn himself in the context of such intense, negative emotions, self-recrimination, and catastrophising about what the future held for him. After all, a long, established pattern of self-injury since the age of 13 years old had reinforced for him that self- injury was ‚the answer‛ to his self-hatred and depression: Owen: when you hate yourself that much or when you’re that messed up and you’re that down<the feeling of pain like just associated with with um the way you’re feeling seems it seems justifiable and it seems the way to do it<it seems the answer<it just feels like sort of what you deserve as well<it’s kind of weird but it feels like it’s what you what you um what you what what would feel the best for you at that time is to feel in pain Although some of the EOs experienced by Owen were unique to him and motivated him to self-injure because of his particular learning history (i.e., the possibility that he had heard a voice was especially anxiety-provoking because of the trauma associated with his previous psychotic episodes), other EOs, such as the negative emotions and self-denigrating thoughts that he experienced, were similarly described by many of the participants. Owen: Ella captures how 127 intensely she felt prior to self-injuring in the following description of her reaction to ongoing health problems: intensely she felt prior to self-injuring in the following description of her reaction to ongoing health problems: Ella: I was quite indignant like how dare you ((laughing))<you know how dare you put me through this shit and<and why aren’t you doing what you’re ‘sposed to and why won’t you work and w- what the fuck is wrong with you kind of<so it was very um yeah it was al- almost as if I had taken kind of every shit experience in my entire life and compounded it into like hatred at this one particular area Ella: I was quite indignant like how dare you ((laughing))<you know how dare you put me through this shit and<and why aren’t you doing what you’re ‘sposed to and why won’t you work and w- what the fuck is wrong with you kind of<so it was very um yeah it was al- almost as if I had taken kind of every shit experience in my entire life and compounded it into like hatred at this one particular area Another participant specifically linked her episode of self-injury to the intensity of her anxiety which manifested itself both psychologically and physically: Robyn: Melanie: Robyn: Melanie: mm ok um so er can you describe how you were feeling before you cut yourself um really anxious my mind was racing like it would take tiny little ideas<and just run off with them absolutely no reason<and yeah yeah yuh so you’re feeling really anxious what else were you feeling well I was quite upset considering<everything that was going on and<I think I was crying quite a bit and just really jittery and couldn’t settle myself down at all Melanie: that I wasn’t good enough I don’t know it was just my mind was racing so much I don’t really know what I was thinking<I was just all over the place all sorts of thoughts about not being good enough and<that everything was my fault sort of<jumping into my head ((laughing)) Melanie explained that in the years that she has been hurting herself, self-injury has developed into a coping mechanism which she uses to regulate her emotions. Thus in describing her motivation for her most recent episode of cutting she simply said: ‚I wanted to settle myself down‛. Owen: In addition to these high arousal emotions, some people also described experiencing negatively valenced, low arousal emotions (e.g., feeling down or hopeless) prior to their episode of NSSI, while others exclusively identified experiencing low arousal, negative emotions: 128 Ella: I was feeling really just self-destructive and just overwhelmed an- and really really hopeless Karen: I just got more and more overwhelmed and depressed<I was feeling really useless<I've just come back down to Wellington to see all my friends and they’re all really successful and have good jobs now and I've come back and I'm just a bum and I'm not doing anything and I'm stressing out ‘cos I can't pay my bond and ((laughs))<just um it seemed like everybody was on top of everything and I was just slowly drowning in it all ((laughing)) Nicola: I think it was just like even just a day that I was just feeling like real<kind of empty<like just a day that I was feeling useless pretty much Paula: I remember how I was feeling and stuff like that<so I think that was just me so in-depth with being overwhelmed by being so depressed and stuff that I<I sort of felt dead<like I couldn’t feel anything Emily: it was still one of those things of those moments of getting stressed out and um feeling really down and and and fragile Natalie: I was feeling really really low<I was just alone for two weeks<I dunno it kinda just got overwhelming<I kind of felt like everyone had abandoned me ((laugh))<I kind of thought that I dunno maybe it was my fault they had gone away and I kind of blow things up in my head ((laughs))<it’s very annoying<so everyone was gone and then I was like ah it must be my fault<I guess it made me sad<that everyone was gone and then when I get sad I kind of withdrawal into myself withdraw<whatever the word is ((laughs)) and I kind of just feel like I’m an empty shell walking around Ella: I was feeling really just self-destructive and just overwhelmed an- and really really hopeless Karen: I just got more and more overwhelmed and depressed<I was feeling really useless<I've just come back down to Wellington to see all my friends and they’re all really successful and have good jobs now and I've come back and I'm just a bum and I'm not doing anything and I'm stressing out ‘cos I can't pay my bond and ((laughs))<just um it seemed like everybody was on top of everything and I was just slowly drowning in it all ((laughing)) Nicola: I think it was just like even just a day that I was just feeling like real<kind of empty<like just a day that I was feeling useless pretty much Paula: I remember how I was feeling and stuff like that<so I think that was just me so in-depth with being overwhelmed by being so depressed and stuff that I<I sort of felt dead<like I couldn’t feel anything Emily: it was still one of those things of those moments of getting stressed out and um feeling really down and and and fragile Natalie: I was feeling really really low<I was just alone for two weeks<I dunno it kinda just got overwhelming<I kind of felt like everyone had abandoned me ((laugh))<I kind of thought that I dunno maybe it was my fault they had gone away and I kind of blow things up in my head ((laughs))<it’s very annoying<so everyone was gone and then I was like ah it must be my fault<I guess it made me sad<that everyone was gone and then when I get sad I kind of withdrawal into myself withdraw<whatever the word is ((laughs)) and I kind of just feel like I’m an empty shell walking around I was feeling really just self-destructive and just overwhelmed an- and really really hopeless I think it was just like even just a day that I was just feeling like real<kind of empty<like just a day that I was feeling useless pretty much I remember how I was feeling and stuff like that<so I think that was just me so in-depth with being overwhelmed by being so depressed and stuff that I<I sort of felt dead<like I couldn’t feel anything it was still one of those things of those moments of getting stressed out and um feeling really down and and and fragile I was feeling really really low<I was just alone for two weeks<I dunno it kinda just got overwhelming<I kind of felt like everyone had abandoned me ((laugh))<I kind of thought that I dunno maybe it was my fault they had gone away and I kind of blow things up in my head ((laughs))<it’s very annoying<so everyone was gone and then I was like ah it must be my fault<I guess it made me sad<that everyone was gone and then when I get sad I kind of withdrawal into myself withdraw<whatever the word is ((laughs)) and I kind of just feel like I’m an empty shell walking around Additionally, two participants, who were depressed at the time of their most recent episode of NSSI, acknowledged thinking about suicide prior to cutting themselves. Owen: Melanie explained that in the years that she has been hurting herself, self-injury has developed into a coping mechanism which she uses to regulate her emotions. Thus in describing her motivation for her most recent episode of cutting she simply said: ‚I wanted to settle myself down‛. All of the emotional EOs (e.g., anger, frustration) discussed so far could be categorised as negatively valenced, high arousal emotions. These emotions were typically self-referential and accompanied by negative, self-castigating thoughts. Owen: Angela who described herself as having been in a ‚really bad episode of depression for a long time‛ and largely house-bound because of her intense social anxiety, explained how her suicidal ideation was linked to feelings of hopelessness and self-stigmatising thoughts: Angela: I didn’t self-harm intending to kill myself but I um had been thinking about suicide and things like that<so um with those yeah just all of that sort of going round in my mind that I wouldn’t be able to um live a a normal um normal life sort of have have a job do all<of the normal things sort of having the um thought that I’m not doing the things that most eighteen year olds should be doing<um and yeah just feeling very hopeless and uh not really seeing an end an end to it ngela: I didn’t self-harm intending to kill myself but I um had been thinking about suicide and things like that<so um with those yeah just all of that sort of going round in my mind that I wouldn’t be able to um live a a normal um normal life sort of have have a job do all<of the normal things sort of having the um thought that I’m not doing the things that most eighteen year olds should be doing<um and yeah just feeling very hopeless and uh not really seeing an end an end to it For the participants who identified thinking about suicide prior to self-injuring, it is possible that suicidal ideation was one of the EOs they were motivated to escape from through hurting themselves. For the participants who identified thinking about suicide prior to self-injuring, it is possible that suicidal ideation was one of the EOs they were motivated to escape from through hurting themselves. 129 From people’s descriptions of what led up to their most recent episode of NSSI, it is clear that they had to contend with multiple EOs. None of the participants identified only a single event, emotion, or thought as the trigger for their self-injury but rather explained how numerous aversive experiences, what Luke described as ‚layers of stress‛, established the conditions for their self-injurious behaviour to function as a form of escape/avoidance. Owen: Sophia summed up the emotional and cognitive maelstrom that preceded her episode of NSSI as follows: my head’s just going so fast and<just knowing that I’m getting stressed or angry at you know but that my emotions are out of control and not<and not f- feeling like I can um rein them in<having that sense of um yeah yeah kind of a desperateness because you just feel like it’s just never gonna stop<like I just wanna go to sleep I’ve got you know I’ve got enough to do tomorrow as it is and you know there’s all this stuff and I should j- not even be thinking about it but uh it’s just gonna it’s just gonna get out of hand Her perception that the pressure would keep building up resonated with a metaphor used by two of the participants in relation to their general experiences of self-injury (i.e., not their most recent episode). They explained how one aversive experience would snowball into other negative thoughts and emotions, growing in size until the magnitude of the situation became overwhelming. Owen: Ella had a different way of explaining the mounting pressure of her frustration, stress, and anger; to her, it was analogous to leaving a lid on a pot of boiling liquid: Ella had a different way of explaining the mounting pressure of her frustration, stress, and anger; to her, it was analogous to leaving a lid on a pot of boiling liquid: ustration, stress, and anger; to her, it was analogous to leaving a lid on a pot of oiling liquid: Ella: I use the boiling pot analogy where if you leave the lid on a pot<and it just boils up and then<it just if you don’t let a bit of steam out it explodes and shit hits all over the kitchen you know Ella: I use the boiling pot analogy where if you leave the lid on a pot<and it just boils up and then<it just if you don’t let a bit of steam out it explodes and shit hits all over the kitchen you know Before her most recent episode of self-injury, Ella described herself as wracking her brain to come up with a solution to prevent the impending explosion: Ella: I was almost kind of desperately thinking you know I just need to do something and if I do something<it I’ll feel better In her self-described quest for ‚something to kind of take the edge off‛, she considered smoking (even though she had given up approximately a year and half before), overdosing on a bottle of sleeping pills, or drinking a bottle of bourbon, but finally settled on self-injury. 130 Although Ella was the only participant who reported actively weighing up alternatives to self-injury, she was not the only one who tried problem-solving strategies. Nicola attempted to talk herself out of self-injury while Lucy went for a run to try to calm down. Unfortunately, neither of these participants was able to prevent themselves from engaging in self-injury. When I asked Lucy if going for a run usually helps, she replied: ‚Sometimes<but ((laugh)) it’s more just kind of preventing the inevitable ((laughing))‛. The perception that self-injury was inevitable when people surpassed a particular threshold of distress was reported by several participants. Owen: The rule becomes a SD because its presence makes it more likely that a certain behaviour and I I was kind of like looking back on like you didn’t need to make such a big deal out of it but I know at the time it was just the headspace I was in and that was all I could do to<kind of get through<I knew that it would make me feel better at least for a little bit it would make all of that stuff go away like it would stand still for a little while hia: I guess it feels quite a desperate situation even though in reality it’s not<um and you know i- if I could be objective would be able to see other options<at the time it feels really really like it just feels like the emotion and the frustration or the stress whatever it is is just gonna just keep getting bigger and bigger and bigger and bigger and just it feels like I’ll self-destruct um and I feel like I need to stop that and um cutting although it doesn’t make me feel better um like its it it does s- stop that sense of it getting bigger and bigger and bigger<um but maybe it feels like I have had a bit of control of it Even Ella who was able to weigh up alternative solutions only considered harmful options, such as taking an overdose or becoming intoxicated, which potentially would have served the same escape/avoidant function as self-injury. 4.3.1.2 Discriminative Stimuli One of the reasons why people might have experienced constricted cognition is because of their verbal rules about NSSI. Verbal rules are SD’s that specify a particular relationship between a behaviour and consequence (Farmer & Latner, 2007; Miltenberger, 2004; Sturmey, Ward-Horner, Marroquin, & Doran, 2007c). The rule becomes a SD because its presence makes it more likely that a certain behaviour will occur; that is, the verbal rule allows a person to discriminate when there is opportunity for reinforcement (Sturmey et al., 2007c). An example of a verbal rule is: If I cut myself, I will feel better. The rule serves as a signal to the person that relief (i.e., a negatively reinforced consequence) is more likely to be available if the person self-injures than if they do not self-injure. Owen: Similarly, Nora attributed her inability to prevent her most recent episode of self-injury to the ‚headspace‛ she was in: 131 Nora: and I I was kind of like looking back on like you didn’t need to make such a big deal out of it but I know at the time it was just the headspace I was in and that was all I could do to<kind of get through<I knew that it would make me feel better at least for a little bit it would make all of that stuff go away like it would stand still for a little while The inability to think of alternative solutions when emotionally overwhelmed was also described by Sophia: Sophia: I guess it feels quite a desperate situation even though in reality it’s not<um and you know i- if I could be objective would be able to see other options<at the time it feels really really like it just feels like the emotion and the frustration or the stress whatever it is is just gonna just keep getting bigger and bigger and bigger and bigger and just it feels like I’ll self-destruct um and I feel like I need to stop that and um cutting although it doesn’t make me feel better um like its it it does s- stop that sense of it getting bigger and bigger and bigger<um but maybe it feels like I have had a bit of control of it Even Ella who was able to weigh up alternative solutions only considered harmful options, such as taking an overdose or becoming intoxicated, which potentially would have served the same escape/avoidant function as self-injury. 4.3.1.2 Discriminative Stimuli One of the reasons why people might have experienced constricted cognition is because of their verbal rules about NSSI. Verbal rules are SD’s that specify a particular relationship between a behaviour and consequence (Farmer & Latner, 2007; Miltenberger, 2004; Sturmey, Ward-Horner, Marroquin, & Doran, 2007c). Owen: Once the negative emotions and thoughts became overwhelming, a type of cognitive constriction (Schneidman, 1996) occurred where self-injury was judged by the participants to be the only solution that would result in relief: Nora: I did wanna stop but I knew that it was all I had at that time I kind of get to this point and I know that I was gonna do that<you know and that doing that would mean that I could go to sleep afterwards...even though I know there’s gonna be a fall-out from it over the next few days over the next week but I felt like I was getting better ((sighs)) you know so I felt like well I’ll be able to deal with it by then but right now I can’t<and I need I need to do this Owen: I would self-harm with whatever um by whatever means<um if I was in that state of mind<you’re in that mindset that it’s the only thing to do<and and that that um you deserve it and that you think of all the bad things that have happened and that you think you’ve been the cause of all these bad things<and so it seems like the the right thing to do Owen’s belief that self-injuring was the ‚only thing to do‛ was echoed by Melanie who described that she would get into the ‚mindset of thinking that every time something went wrong‛, the solution was to cut herself. Owen’s belief that self-injuring was the ‚only thing to do‛ was echoed by Melanie who described that she would get into the ‚mindset of thinking that every time something went wrong‛, the solution was to cut herself. Owen: Four participants, however, provided more explicit examples of verbal rules: Four participants, however, provided more explicit examples of verbal rules: Nicola: I think it was just like even just a day that I was just feeling like real<kind of empty<and<I was just like ah uh uh a- that’ll help uh ((laughing)) Four participants, however, provided more explicit examples of verbal rules: Nicola: I think it was just like even just a day that I was just feeling like real<kind of empty<and<I was just like ah uh uh a- that’ll help uh ((laughing)) Nora: it just progressed and then I got to the stage when I knew that it was the only way I was gonna make myself feel better was to cut myself so I did Tara: the idea just came of<of course that’s what I’ll do to make myself feel better Melanie: um I guess over the years I’ve sort of developed it as a means to cope when I do get like that<(mean) to settle myself down um<so yeah ((laughs)) I wanted to settle myself down ((laughing)) I think it was just like even just a day that I was just feeling like real<kind of empty<and<I was just like ah uh uh a- that’ll help uh ((laughing)) Nora: it just progressed and then I got to the stage when I knew that it was the only way I was gonna make myself feel better was to cut myself so I did it just progressed and then I got to the stage when I knew that it was the only way I was gonna make myself feel better was to cut myself so I did Tara: the idea just came of<of course that’s what I’ll do to make myself feel better Melanie: um I guess over the years I’ve sort of developed it as a means to cope when I do get like that<(mean) to settle myself down um<so yeah ((laughs)) I wanted to settle myself d l h the idea just came of<of course that’s what I’ll do to make myself feel better Melanie: um I guess over the years I’ve sort of developed it as a means to cope when I do get like that<(mean) to settle myself down um<so yeah ((laughs)) I wanted to settle myself down ((laughing)) um I guess over the years I’ve sort of developed it as a means to cope when I do get like that<(mean) to settle myself down um<so yeah ((laughs)) I wanted to settle myself down ((laughing)) These extracts can all be interpreted as versions of the same verbal rule: If I want to feel better, then I need to hurt myself. Owen: Several people provided examples of these rules in their descriptions of their most recent episode of NSSI, although the rules were rarely stated in cause and effect terms (i.e., if I do this, then this will happen). Instead, I typically extrapolated from what people had said to identify rules that, in all likelihood, served as SD’s for their self-injury. People often talked about deciding to self-injure because they knew it would help. Ella commented that she 132 had let the situation build up to a point where she ‚needed to do something quite drastic‛ to calm herself down. To her, cutting seemed to be the most appropriate action at the time and it is likely that the thought that cutting would help her calm down signalled an opportunity, as it had in the past, for relief following the behaviour. Similarly, cutting seemed ‚like the best option‛ to Sophia because, as she stated, when she had used it in the past it had worked to help her release negative emotions. Owen: These SD’s would have worked in conjunction with the EOs in that the participants were motivated to gain relief from their overwhelming emotions and thoughts, and the verbal rules signalled to them that this relief was accessible through NSSI, as it had been in the past. A more detailed example of how particular SD’s and EOs interact to influence behaviour is evident in the effect that Emily’s inability to sleep had on her. In the past, she had found sleep to be ‚a real escape‛ but as she began recovering from depression, she found it more and more difficult to get to sleep: mily: I think when when when you feel that releafs release and relief from from cutting usually what I’d find is that it would be uh so euphoric that it would tire me out<I found that that as I was getting healthier and as I was getting recovering from being quite so depressed<um if I was tired and stressed out and wanted to sleep I wasn’t I wasn’t g- able to do it so easily Emily’s desire for sleep was one of the motivating factors behind her episode of self- injury: 133 Emily: it was still one of those things of those moments of getting stressed out and um feeling really down and and and fragile um where it was kind of like you know I kind of get to this point and I know that I was gonna do that<you know and that doing that would mean that I could go to sleep afterwards In Emily’s case the verbal rule appeared to be: If I cut myself, then I will be able to sleep. As such, the verbal rule (SD) and difficulty getting to sleep (EO) interacted to precipitate Emily’s self-injurious behaviour. Other SD’s mentioned by participants were being alone; having access to implements to injure themselves with, or, as Angela phrased it, a ‚weapon of choice‛; and the time of day. Owen: Tara talked about how once the thought that self-injury would make her feel better had popped into her head the search for something to hurt herself with became all consuming: Tara: the idea just came of<of course that’s what I’ll do to make myself feel better and I think even as soon as that comes in I actually forget that I’m frustrated and it’s just like that’s the goal is of the feeling<is to then go and do that and so I then have to find a way it’s like this little weird mission of my brain’s that I’m on ((laughing)) that I’ve gotta find a way to then do that For her most recent episode of NSSI, Tara found a piece of glass which she then used to cut herself: Tara: I can’t trust myself when I get stressed out to not break the really expensive razor open and do something so at the time I didn’t have anything like that around and I think I found a piece of glass or something like that<and used that Although the SD (i.e., the razor), which had been present in the past when Tara had self-injured, was not available, she had also previously used glass to cut herself when she did not have access to other implements: Robyn: Tara: yuh so do you remember that time where you found the glass like the (unclear) yeah I think I was actively looking for it ‘cos I think I’d actually found I’d done that before when I had<no other options Pieces of glass had thus acquired discriminative status. Owen: Tara acknowledged that despite having practised other coping strategies, she became fixated on cutting as the solution, another example of constricted cognition prior to self-injuring: Tara: I can look at it and go I could’ve stopped myself from even going and looking for that but I just kind of was so set on the idea that once I’d done that I’d be o- like that I could then get over it and be fine I didn’t really want to do it either because I’d been doing well at Robyn: Tara: yuh so do you remember that time where you found the glass like the (unclear) yeah I think I was actively looking for it ‘cos I think I’d actually found I’d done that before when I had<no other options despite having practised other coping strategies, she became fixated on cutting as the solution, another example of constricted cognition prior to self-injuring: I can look at it and go I could’ve stopped myself from even going and looking for that but I just kind of was so set on the idea that once I’d done that I’d be o- like that I could then get over it and be fine<I didn’t really want to do it either because I’d been doing well at 134 trying to<I guess use other ways to cope and I was actively like practising those but I guess this time it was just ((laughs))<was a bit much<um and that was for some reason why I decided ((laughing)) that this time I needed to for that reason trying to<I guess use other ways to cope and I was actively like practising those but I guess this time it was just ((laughs))<was a bit much<um and that was for some reason why I decided ((laughing)) that this time I needed to for that reason The power of specific SD’s to influence behaviour was particularly highlighted by two participants. Owen: Karen described how she was unpacking her belongings after moving back to Wellington when she found a ‚little box of cutty things‛ which, in the context of her feeling depressed and overwhelmed, signalled the opportunity for relief following self-injury: Karen: I found this little box that I've kept a lot of like razor blades and glass and things in and automatically it triggered me wanting to cut so um yeah I sat down on my bed cranked my music right up and I just started to cut When I asked her if she still had the box, she replied: Karen: yeah I do I don't know why I keep it um it’s almost like a memento of times past <and I mean I wouldn't use half the things in there ((laugh))<‘cos half of them are like gross and old ((laughs))<um but yeah that reminds me of it and I don't have the heart to throw it away Karen: yeah I do I don't know why I keep it um it’s almost like a memento of times past <and I mean I wouldn't use half the things in there ((laugh))<‘cos half of them are like gross and old ((laughs))<um but yeah that reminds me of it and I don't have the heart to throw it away Jenny had a very different attitude to one of the objects that she had repeatedly used to injure herself. After her most recent episode where she had stapled herself in the hand, Jenny had made the decision not to use staplers at all anymore: Jenny: I made an informed decision after that that I’m not going to use that particular tool anymore<I call it a tool ‘cos it’s been it’s become such a threat to me<I don’t go near it at all<just just to be sure that I don’t do anything you know Jenny: I made an informed decision after that that I’m not going to use that particular tool anymore<I call it a tool ‘cos it’s been it’s become such a threat to me<I don’t go near it at all<just just to be sure that I don’t do anything you know Sophia also reported trying to restrict her access to implements that she could use to hurt herself. Owen: The night before her most recent episode of NSSI, she had cut herself and had put the razor blades that she had used in a safe that only her husband could open. The next night when she wanted to injure herself again, she did not have access to the blades. However, she did have a small glass with her which she broke and then used to cut herself. It was not clear whether she had done this before. She had gone to bed early and being alone at the time may also have signalled to her that reinforcement would be available following NSSI. Sophia also reported trying to restrict her access to implements that she could use to hurt herself. The night before her most recent episode of NSSI, she had cut herself and had put the razor blades that she had used in a safe that only her husband could open. The next night when she wanted to injure herself again, she did not have access to the blades. However, she did have a small glass with her which she broke and then used to cut herself. It was not clear whether she had done this before. She had gone to bed early and being alone at the time may also have signalled to her that reinforcement would be available following NSSI. 135 Certainly, being alone functioned as an SD for Paula’s most recent episode as she was able to buy razor blades and go to the beach where she cut herself. The voice that Paula is referring to is her negative mindset which she called her ‚Anorexic voice Owen: Usually, she was carefully monitored by her caregiver: she was carefully monitored by her caregiver: the opportunity I got was when um my caregiver was at work ‘cos she’s quite the Nazi woman ((laughing)) [[Robyn laugh]] so she ((laughing)) she keeps an eye on me and she she’s usually really good on picking up on um when I’m out of control as such and I don’t have to say anything she can just tell<but yeah she was at work and so I saw I just took that opportunity and just I went on a walk and all of sudden the voice21 was just so much stronger and I was just so aching to feel that I went and brought blades and<I just sat at the beach and I tried everything to cry because I felt like I just needed to like all the pain I was holding was like I n- I needed like I I thought I should’ve been just sitting there exploding in tears and<I just couldn’t there was just nothing nothing was happening and so I just sat there and I just kept dragging it across my like my wrist and my arm and then I just yeah just let rip and did it really quite badly really deeply and stuff It is clear that particular SD‘s (e.g., being alone, blades) allowed participants to discriminate when reinforcement was likely following self-injury, but that this reinforcement was only desired because of the EOs (e.g., feeling numb). Owen: Nicola encapsulated this interaction between the SD of a craft knife and the EO of feeling down in a matter-of-fact way, when describing the lead up to her most recent episode: Nicola: I just broke up with my ex like<back then and it’s like always emotional you know like ((laughing)) but um yeah and so I was down a l- ((laughing)) and I had a craft knife ((laughing))<and its I cut my leg Nicola: I just broke up with my ex like<back then and it’s like always emotional you know like ((laughing)) but um yeah and so I was down a l- ((laughing)) and I had a craft knife ((laughing))<and its I cut my leg For Nicola, having access to a craft knife while feeling down led her to cut her leg multiple times, a behaviour that functioned as an escape from her negative intrapersonal experiences because it was followed by a sense of relief. 4.3.1.3 Consequences For Nicola, having access to a craft knife while feeling down led her to cut her leg multiple times, a behaviour that functioned as an escape from her negative intrapersonal experiences because it was followed by a sense of relief. 4.3.1.3 Consequences All of the 18 participants whose episodes of NSSI functioned as escape or avoidance from negative intrapersonal experiences reported a reduction in, or the elimination of, negative emotions and/or thoughts during or following their self- injurious behaviour. After cutting herself, Tara described feeling almost as though she had ‚a whole new start to the day as if the rest of the day hadn’t gone so bad 21 The voice that Paula is referring to is her negative mindset which she called her ‚Anorexic voice‛. 21 The voice that Paula is referring to is her negative mindset which she called her ‚Anorexic voice‛. 136 and<nothing else had really happened‛. The most common words used by participants to depict these negatively reinforced consequences of NSSI were ‚relief‛ and ‚release‛: and<nothing else had really happened‛. Owen: The most common words used by Natalie: I guess it’s sort of like a relief a it kind of lifts my spirits a little bit Paula: just a massive amount of release Nora: um I think I was feeling relief Ella: I think um you know because of adrenalins and endorphins and stuff that you do get some sense of release from it Emily: I think it was the usual kind of just relieved and tired Maria: there is kind of a sense of relief I guess For Josh, the sense of relief that followed cutting was analogous to taking off a really tight hat: Josh: then when I do cut it’s kind of uh a release it’s like it’s as strange it might be ((laughing)) to describe it it’s like wearing a really really tight hat and then<taking it off and just going like ah<sweet you know sort of ((laughing)) yeah sub- it it kind of feels like that<um sort of yeah as a emotionally and it can feel like that physically as well ((laughing)) In their descriptions of the consequences of self-injury, participants articulated several, possible underlying mechanisms of self-injurious behaviours. Some people reported that self-injury distracted them from aversive internal stimulation, thus providing respite from their intense emotions and thoughts. For instance, Nicola talked about how focusing on the act of cutting, to ensure she did not hurt herself too severely, was distracting: I guess it’s just like takes my mind off everything ‘cos its like…I don’t wanna like hurt myself like really badly or anything<it’s like kind of like I guess your mind is on something q- quite intently you know<whereas and if if you’re finding it if like if I’m finding it really hard to focus on everything you know<and then I finally find something that I can like focus on ola: I guess it’s just like takes my mind off everything ‘cos its like…I don’t wanna like hurt myself like really badly or anything<it’s like kind of like I guess your mind is on something q- quite intently you know<whereas and if if you’re finding it if like if I’m finding it really hard to focus on everything you know<and then I finally find something that I can like focus on ilarly described being distracted from her thoughts and feelings while mm so in terms of um keeping on those sort of same lines about talking about the feelings and thoughts when you had finished cutting<can you describe whether those thoughts and feelings say that led up to it change 137 not really they always end up coming back but just like for a while it gives me something else to focus on not really they always end up coming back but just like for a while it gives me something else to focus on Lucy: The degree of respite experienced by Lucy depended on the severity of her self- injury and how much pain she experienced: The degree of respite experienced by Lucy depended on the severity of her self- injury and how much pain she experienced: Robyn: Lucy: so how quickly do they usually come back depends how bad I’ve cut really ((laughing)) like<if it’s not bad they come back pretty quickly but if it’s bad sometimes it kind of take a couple of hours<‘cos I’m sort of focussing on stopping myself ((laughing)) bleeding and focussing on the pain Robyn: Lucy: so how quickly do they usually come back depends how bad I’ve cut really ((laughing)) like<if it’s not bad they come back pretty quickly but if it’s bad sometimes it kind of take a couple of hours<‘cos I’m sort of focussing on stopping myself ((laughing)) bleeding and focussing on the pain While Luke also commented on the interrelationship between the severity of While Luke also commented on the interrelationship between the severity of his cutting and the relief he experienced, he additionally described how for him the letting of blood was a cleansing ritual: his cutting and the relief he experienced, he additionally described how for him the letting of blood was a cleansing ritual: Luke: I cut good and proper like<because I knew that I would feel relief and I did like it’s almost from the moment you hit a point it may be one cut or four or five and usually for me it’s about five<when there’s enough blood coming and and I can feel the sting of the razor and that it’s just like a it washes everything away<when nothing matters right at that time nothing<matters at all it even sort of afterwards and as I’m healing Like Luke, Josh also cut himself multiple times to achieve a release. ilarly described being distracted from her thoughts and feelings while He described how one of his techniques was to cut in the same place over and over as he found that the continuous pain provided him with a more powerful distraction. For his most recent episode, he had cut himself in three different locations, but had also cut seven to eight times in each of those sites. For these participants, distraction from emotional pain was achieved through the physical experiences of pain, witnessing blood, and wound-care. Like Luke, Josh also cut himself multiple times to achieve a release. He described how one of his techniques was to cut in the same place over and over as he found that the continuous pain provided him with a more powerful distraction. For his most recent episode, he had cut himself in three different locations, but had also cut seven to eight times in each of those sites. For these participants, distraction from emotional pain was achieved through the physical experiences of pain, witnessing blood, and wound-care. ilarly described being distracted from her thoughts and feelings while Other participants specified that they felt emotionally numb during cutting, which provided them with relief from the chaos of their overwhelming thoughts and feelings: Karen: Robyn: Karen: Robyn: Karen: I was just feeling just numb and it was so much better than feeling ((laughing)) overwhelmed<and it was just yeah it was like a release just to feel numb just to get the headache out of my head kind of thing ((laughing)) mm yeah so when you were f- you say you were feeling numb was that while you were actually cutting you felt numb yeah it was<and it was like I was able to think more rationally and everyth- all the problems seemed to go away ah ok so is that during the cutting or afterwards yuh that was during<it made me think what am I so stressed about you know ((laughing))<all these things can be fixed Other participants specified that they felt emotionally numb during cutting, which provided them with relief from the chaos of their overwhelming thoughts and feelings: Karen: Robyn: Karen: Robyn: I was just feeling just numb and it was so much better than feeling ((laughing)) overwhelmed<and it was just yeah it was like a release just to feel numb just to get the headache out of my head kind of thing ((laughing)) mm yeah so when you were f- you say you were feeling numb was that while you were actually cutting you felt numb yeah it was<and it was like I was able to think more rationally and everyth- all the problems seemed to go away ah ok so is that during the cutting or afterwards Other participants specified that they felt emotionally numb during cutting, which provided them with relief from the chaos of their overwhelming thoughts and feelings: 138 Robyn: Nora: mhmm so you kinda you said that you go numb<in what ways um I think ((sighs)) ((laugh)) it’s weird ‘cos I kind of find with cutting myself it numbs me but it kind of wakes me up as well<like it works in two different ways and yeah I think just the feelings that I got when I cut myself would enable me to just it just feels like nothing else matters and my head that was all cloudy and stuff<is just silenced and all the thoughts aren’t there for like a few moments<um yeah and it just numbs all the feelings that I had inside to one level that I don’t have to deal with at that time<um it just puts them into one thing<so I can not think about them For both Karen and Nora, the relief of having a break from their thoughts seemed to indirectly enable problem-solving. ilarly described being distracted from her thoughts and feelings while Cutting appeared to help Karen put her problems in perspective and as she said, ‚all the problems seemed to go away‛. In comparison, Nora still experienced similar thoughts following her episode of NSSI, but felt she was more able to deal with them because she was no longer overwhelmed by her emotions: Robyn: Nora: Robyn: Nora: Robyn: Nora: did you find that um your thoughts changed [[Nora coughs]] like once you had cut yourself um not really ((laughing)) not really no they were kind of yeah they were kind of still there<um definitely still there but I guess I felt like I could deal with them in a little bit more depth<um after I had cut myself<um it kind of I felt like it enabled me to get more of a clear headspace in terms of dealing with that and um not feeling so overwhelmed by everything that I was feeling at the time<yuh and just get clarity and try and pull out what emotion I was feeling and then be able to deal with that instead of it all being one big thing<and feeling like I couldn’t deal with it ‘cos it was so big and so hard um so yeah the thoughts were still there but I didn’t feel as if they were so big ok<so it sounds like the thoughts stay stayed pretty much the same but the just the emotions become more manageable mm yuh yuh um yeah and that enables me to kind of think about the thoughts more clearly and um what I can do what I could do to try and make them easier or better For both Karen and Nora, the relief of having a break from their thoughts seemed to indirectly enable problem-solving. Cutting appeared to help Karen put her problems in perspective and as she said, ‚all the problems seemed to go away‛. In comparison, Nora still experienced similar thoughts following her episode of NSSI, but felt she was more able to deal with them because she was no longer overwhelmed by her emotions: For both Karen and Nora, the relief of having a break from their thoughts seemed to indirectly enable problem-solving. Cutting appeared to help Karen put her problems in perspective and as she said, ‚all the problems seemed to go away‛. ilarly described being distracted from her thoughts and feelings while Similarly, making the decision to cut impacted on Lucy’s mood: Robyn: Lucy: Robyn: Lucy: so what happens to those thoughts and feelings once you decide that you’re going to cut it differs sometimes they go away sometimes they don’t sometimes they get stronger and then kind of affects how bad my cutting is if they go away and I just kind of stop feeling it’s not as bad so this episode last night<did they go away or did they get worse no they went away In the past when the thoughts had remained present, she acknowledged that she needed to force herself to stop cutting. While the majority of participants reported feeling distracted during or after injuring themselves, two people described being distracted from their thoughts and emotions following their decision to cut and before they had actually injured themselves. As detailed earlier in this chapter, Tara acknowledged that once she had thought of self-injury as the solution, she became so focused on finding something to injure herself with that she forgot about how intensely frustrated she had been feeling. Similarly, making the decision to cut impacted on Lucy’s mood: While the majority of participants reported feeling distracted during or after injuring themselves, two people described being distracted from their thoughts and Robyn: Lucy: Robyn: Lucy: so what happens to those thoughts and feelings once you decide that you’re going to cut it differs sometimes they go away sometimes they don’t sometimes they get stronger and then kind of affects how bad my cutting is if they go away and I just kind of stop feeling it’s not as bad so this episode last night<did they go away or did they get worse no they went away In the past when the thoughts had remained present, she acknowledged that she needed to force herself to stop cutting. In the past when the thoughts had remained present, she acknowledged that she needed to force herself to stop cutting. In the past when the thoughts had remained present, she acknowledged that she needed to force herself to stop cutting. ilarly described being distracted from her thoughts and feelings while In comparison, Nora still experienced similar thoughts following her episode of NSSI, but felt she was more able to deal with them because she was no longer overwhelmed by her emotions: Melanie described a similar experience of being able to problem-solve once she was feeling less emotionally aroused and her thoughts had stopped racing. While she was cutting, she began to feel a bit calmer, and more settled and in control of what was going on. Following her episode of self-injury, she reported being able to think more clearly, and, although the negative thoughts of self-blame and inadequacy were still present, she did not believe them as much: Melanie: um and but you know I was able to think more clearly<about everything so you know I rang my boyfriend up and got him to come back so we could talk and sort things out like I was able to think<so it felt better<after ((laughing)) Melanie described a similar experience of being able to problem-solve once she was feeling less emotionally aroused and her thoughts had stopped racing. While she was cutting, she began to feel a bit calmer, and more settled and in control of what was going on. Following her episode of self-injury, she reported being able to think more clearly, and, although the negative thoughts of self-blame and inadequacy were still present, she did not believe them as much: um and but you know I was able to think more clearly<about everything so you know I rang my boyfriend up and got him to come back so we could talk and sort things out like I was able to think<so it felt better<after ((laughing)) 139 While the majority of participants reported feeling distracted during or after injuring themselves, two people described being distracted from their thoughts and emotions following their decision to cut and before they had actually injured themselves. As detailed earlier in this chapter, Tara acknowledged that once she had thought of self-injury as the solution, she became so focused on finding something to injure herself with that she forgot about how intensely frustrated she had been feeling. 4.3.1.4 Summary She described being in hospital as ‚a good break‛: Sophia: Robyn: Sophia: Robyn: Sophia: often just want to be um in hospital for a couple of days or you know something like that where the expectations like my own and what I think other people have on me I’ve got a it seems to then give a yeah give a legitimate reason why they they can’t be met< so have you been to hospital a few times<(from) medication and taking too much medication yuh yuh<yuh over the years it’s been quite a few times and um and although you know it’s never a very pleasant experience um it does mean that things stop <you know it does it does interject in your life and um uh you know often you’re constrained by what the medical staff you know what whether you know like like where they i- they’ll discharge you or not and so<there you know there’s those things that you kinda have to just go along with um<which are quite good ((laughing)) um yuh yeah kind of removes that pressure of decision-making yuh yuh<yuh it takes the responsibility away Overdosing on medication as a legitimate form of escape had resulted in Sophi Although both Karen and Sophia had purposefully hurt themselves in the past to escape or avoid interpersonal demands, they had self-injured using types of self-harm that would result in hospitalisation and which are beyond the scope of this thesis. However, it is worthwhile noting that they felt they needed to resort to hospitalisation to gain sufficient respite and, in Sophia’s case, to a type of self-harm that would render her physically incapable of carrying out her responsibilities as a wife and mother. 4.3.1.4 Summary The majority of the self-injury episodes analysed appeared to serve an escape/avoidant function. Intense, negative emotions and thoughts, associated with both distal and recent events, established the motivation for self-injury, while specific stimuli (e.g., being alone, implements) signalled an opportunity for relief or release following self-injury. Participants’ ability to problem-solve and think of alternative solutions to their distress often became constricted, and verbal rules about needing to self-injure in order to gain relief predominated. 4 3 2 I l E /A id None of the participants’ most recent episodes of NSSI appeared to function as a way to avoid, or escape from, interpersonal obligations or responsibilities. However, two participants did acknowledge that they had been motivated to self- injure in the past for this purpose: that's another way of self-harm for me is that I can actually make my potassium that low if I want to but um and I've done that in the past year I've just been so<miserable and I've wanted to hurt myself so I've done that I haven't done it deliberately to want to kill myself<I've done it make myself sick so I go to hospital<just to get myself out of situations ((laugh)) 140 I have taken medications at other times and um taken too much medication<um and that’s often seems like quite a easy option because um one it it’s accessible and m- most of the medications that I have are s- sleep-inducing<um so it really is an escape in that sense<and it means that I’m also kinda out of play so if I’m feeling like I can’t cope with things that’s kind of a legitimate question-mark legitimate but le- uh uh in my head it can feel like that’s a legiti- you know I l- like I am like being actually physically sick<‘cos if I you know if I take a whole lot of um drugs that I’m not meant to take in that amount then it has a real physical effect on me<and I can’t actually you know like if if I’m awake I can’t actually function that well so I can’t be responsible for the kids so you know it kind of has that a follow-on effect of has that a follow-on effect Overdosing on medication as a legitimate form of escape had resulted in Sophia being hospitalised on numerous occasions. 4.3.3 Intrapersonal access Intrapersonal, positively reinforcing consequences (e.g., an adrenalin rush) were described by eight people following their episodes of NSSI, leading me to hypothesise that these episodes functioned as a form of intrapersonal access. It is important to emphasise that only one of these eight episodes functioned exclusively 141 as intrapersonal access; most of the episodes in this functional category also featured in the intrapersonal escape category. as intrapersonal access; most of the episodes in this functional category also featured in the intrapersonal escape category. 4.3.3.1 Establishing operations and discriminative stimuli 4.3.3.1 Establishing operations and discriminative stimuli The types of EOs reported by Belinda, the one participant whose self-injury episode functioned exclusively as intrapersonal access, were comparable to those reported by participants whose episodes of NSSI functioned as forms of intrapersonal escape. Although Belinda had an extensive history of NSSI—she reported that at one stage she was self-injuring up to thirty times a day by cutting or hitting her head against hard surfaces—she conceptualised her most recent episode of NSSI as a relapse. 4.3.3 Intrapersonal access In the months preceding her episode of NSSI, she had experienced several miscarriages, which had served as distal EOs for her self-injury: Belinda: what led up to it was I was trying to have a baby and I had had two or no three miscarriages in a period of um like an- nine months<and they couldn’t find any reason why and I was really stressed<and I felt really it was just something for me to blame myself for I thought ah it must be me I’m I must<just be really stuffed up and wrong and um I think yeah I was internalising a lot of a lot of blame<f- for whatever was happening B li d t t d ib i l EO th t ti t d h t i j h lf what led up to it was I was trying to have a baby and I had had two or no three miscarriages in a period of um like an- nine months<and they couldn’t find any reason why and I was really stressed<and I felt really it was just something for me to blame myself for I thought ah it must be me I’m I must<just be really stuffed up and wrong and um I think yeah I was internalising a lot of a lot of blame<f- for whatever was happening Belinda went on to describe more proximal EOs that motivated her to injure herself. 4.3.3.2 Consequences In the context of Belinda’s learning history, I hypothesised that the pain of smashing her head on the kitchen floor was a positively reinforced consequence. When discussing her self-injurious behaviour, Belinda repeatedly described using self-injury as a way to punish herself: when I have self-harmed it’s like yeah good you deserved that you<deserved this punishment and I really think that it’s been quite closely linked for me to um well right back to to being a kid but probably ((clears throat)) early adolescence<where um I had some pretty full-on experiences around abusive relationships an- and being told I was really useful and hopeless and crap<I kind of started you know internalising a lot of messages that I heard<um and repeating them and kind of becoming my own worst enemy and self-harm was a way of actually feeling that I had been sufficiently punished for being innately bad<or useless or stupid or<fat or whatever the hell it was that I I was deciding to pick on myself for at that time Belinda explained how the severity of her self-injury depended on how intense her Belinda explained how the severity of her self-injury depended on how intense her negative thoughts were at the time: Belinda: the words are screaming in my head<um different phrases like um you’re st- you know you’re fat and ugly you’re stupid you’re an idiot so it’s a whole lot of words that actually screaming in my head while I’m doing it<so I’m not even really concentrating on what I’m doing<actually there’s just words screaming in my head telling me this and the louder they scream the more I would they’re not external they’re in my own head<but um the more I would the deeper I would cut or the harder I would hit my head Eventually, the cutting or hitting would alleviate her distress: Belinda: there’s a point where endorphin pain endorphin kicks in<and um you actually are at yeah you feel ok sufficiently punished<so you’re like ok well yeah it’s like this this this feeling of release<so if its yeah it’s this big huge tension release a bit of a endorphin high<you feel relaxed Belinda thus appeared to experience release through pain, which she stated ‚feels kind of good as well as painful‛. s preparing a salad for Christmas lunch when she burnt the sunflower seeds: nda: suddenly it just kind of all compac- compounded on me and I was going ah you’re so stupid you know you’re such an idiot and you know now you’ve burnt the sunflower seeds which is like a small thing but it was just like the last straw<and I just found myself on the floor of the kitchen basically smashing my head against the um kitchen floor<and it was just like really it was quite sudden ((laugh))<then I was was like ah it really hurt Belinda: suddenly it just kind of all compac- compounded on me and I was going ah you’re so stupid you know you’re such an idiot and you know now you’ve burnt the sunflower seeds which is like a small thing but it was just like the last straw<and I just found myself on the floor of the kitchen basically smashing my head against the um kitchen floor<and it was just like really it was quite sudden ((laugh))<then I was was like ah it really hurt Apart from Belinda, all of the participants whose most recent episode was hypothesised to function as intrapersonal access endorsed experiencing relief or release from aversive intrapersonal experiences following their self-injury. The same EOs that I discussed in the intrapersonal escape section thus established the conditions for NSSI to function as an adrenalin rush. That is, participants were motivated to engage in self-injury while experiencing negative emotions and thoughts because past self-injury had been followed by desired intrapersonal consequences. Similarly, the objects or occasions that functioned as SD‘s (e.g., being alone, implements) for these particular participants, described in section 4.3.1.2, 142 would have signalled to them the opportunity for positive reinforcement following self-injury. would have signalled to them the opportunity for positive reinforcement following self-injury. 4.3.3.2 Consequences 4.3.3.2 Consequences 4.3.3.2 Consequences da: the words are screaming in my head<um different phrases like um you’re st- you know you’re fat and ugly you’re stupid you’re an idiot so it’s a whole lot of words that actually screaming in my head while I’m doing it<so I’m not even really concentrating on what I’m doing<actually there’s just words screaming in my head telling me this and the louder they scream the more I would they’re not external they’re in my own head<but um the more I would the deeper I would cut or the harder I would hit my head Eventually, the cutting or hitting would alleviate her distress: there’s a point where endorphin pain endorphin kicks in<and um you actually are at yeah you feel ok sufficiently punished<so you’re like ok well yeah it’s like this this this feeling of release<so if its yeah it’s this big huge tension release a bit of a endorphin high<you feel relaxed Belinda thus appeared to experience release through pain, which she stated ‚feels kind of good as well as painful‛. kind of good as well as painful‛. Given that Belinda began hitting her head on the kitchen floor in the context of experiencing thoughts about being stupid and an idiot, the pain she then experienced, in all likelihood, would have positively reinforced her self-injury as it had in the past when she punished herself. Although she did not state that she 143 experienced release following her most recent episode, this may have been because her self-injury was interrupted by her husband who came running into the kitchen. Historically, she had only experienced release when she felt sufficiently punished for her perceived failings. If she had stated that she had experienced tension release, her episode of self-injury would have then functioned as both a form of intrapersonal escape and access. Two other participants also discussed pain as a positive consequence following their most recent episode of self-injury. As I discussed in the previous section on intrapersonal escape, Owen burned himself most recently in the context of self-blame, self-directed anger, and the fear of becoming psychotic again. 4.3.3.2 Consequences 4 3 3 3 S uh it’s really bad I I quite I enjoy it like it<it is like it’s weird but it’s like kind of almost fun ((laugh))<I guess I probably feel like better A for adrenalin and what not I know how to get myself in into sort of the right state of mind where I can do it quite calmly<and just get the adrenalin rush<and then I burn myself and then I’m only focussing on that nothing else I’m just focussing on that pain and so I feel kind of calm and kind of high it felt quite exhilarating like<I mean I I think you know all the adrenalin and everything<as well you physically feel quite hyped up but then afterwards I’d feel really exhausted it was like but during it it w- uh um yeah it felt very exhilarating and yeah I just felt very I ‘spose alive Based on these descriptions, it is likely that these episodes of self-injurious behaviour would have been reinforced by the positive, internal stimulation experienced by participants. Based on these descriptions, it is likely that these episodes of self-injurious behaviour would have been reinforced by the positive, internal stimulation experienced by participants. 4.3.3.2 Consequences Self-injury provided Owen with a sense of release from his negative emotions and thoughts, a release he seemed to attribute to the pain he experienced while burning himself: Owen: yeah I was actually feeling quite quite good ((laugh))<I was feeling a bit of release<a bit um ((sigh)) yeah it just it felt right when it’s when it was when I was doing it<and so I kept on doing it and it felt it felt like um it felt like it was justified<as well like you know this was this is what I want this is what I wanna feel<it’s just what you wanna feel<you just wanna feel that pain In the context of his intense negative emotions, the pain felt good because Owen was motivated to punish himself. He noted how he would have reacted differently to the pain, had he not been experiencing self-hatred and self-blame: Owen: it’s weird because I couldn’t do it now<like if I tried to do it now I couldn’t hold it there that long<but when I’m feeling like that hatred or that intensity of emotion it’s feels good<it feels I can hold it there The EOs he experienced therefore had a direct impact on the value of the pain and because the consequence of pain was positively reinforcing, he was able to burn himself for longer. The EOs he experienced therefore had a direct impact on the value of the pain and because the consequence of pain was positively reinforcing, he was able to burn himself for longer. The EOs he experienced therefore had a direct impact on the value of the pain and because the consequence of pain was positively reinforcing, he was able to burn himself for longer. Self-punishment was also a recurring theme in my interview with Tara. She described sometimes using self-injury to teach herself a lesson, and that her self- injury would increase in severity over time as punishment for not learning her lesson from past self-injury. 4.3.3.2 Consequences Apart from pain, other intrapersonal positive reinforcers identified by participants as having occurred during, or following, their episode of self-injury, were experiencing an adrenalin rush, feeling alive, or sublime: Angela: you just feel a bit more alive when you’re doing something like that Nicola: uh it’s really bad I I quite I enjoy it like it<it is like it’s weird but it’s like kind of almost fun ((laugh))<I guess I probably feel like better A for adrenalin and what not Josh: (unclear) it’s sort of sublime Matt: I know how to get myself in into sort of the right state of mind where I can do it quite calmly<and just get the adrenalin rush<and then I burn myself and then I’m only focussing on that nothing else I’m just focussing on that pain and so I feel kind of calm and kind of high Maria: it felt quite exhilarating like<I mean I I think you know all the adrenalin and everything<as well you physically feel quite hyped up but then afterwards I’d feel really exhausted it was like but during it it w- uh um yeah it felt very exhilarating and yeah I just felt very I ‘spose alive Based on these descriptions, it is likely that these episodes of self-injurious behaviour would have been reinforced by the positive, internal stimulation experienced by participants. 4.3.3.2 Consequences When I asked what kind of lesson she was trying to teach herself, she replied: 144 Tara: hmm to be more like in control and more perfect and like ((laughing)) perfect’s a really bad word to use but I do realise that I quite often think that I should be perfect<um be yeah more in control of and better at things than perhaps I am<which is kind of an issue that I know I have ((laughs)) is trying to be really great at everything Tara: hmm to be more like in control and more perfect and like ((laughing)) perfect’s a really bad word to use but I do realise that I quite often think that I should be perfect<um be yeah more in control of and better at things than perhaps I am<which is kind of an issue that I know I have ((laughs)) is trying to be really great at everything In regards to her most recent episode of self-injury, she acknowledged that pain was an important part of learning her lesson: Tara: straight afterwards I kind of was just sort of checking to see a couple of minutes afterwards it’s like that’s when I it does start to hurt and so I was like well is that gonna hurt yeah ok and I kind<of almost feel like I need that afterwards just to remind myself I guess still part of that teaching myself a lesson thing For Tara, it is likely that pain served as a positively reinforced consequence because it was evidence for her that she had punished herself sufficiently. For Tara, it is likely that pain served as a positively reinforced consequence because it was evidence for her that she had punished herself sufficiently. 4.3.3.3 Summary For a number of participants, positive reinforcers, such as experiencing an adrenalin rush following self-injury, appeared to go hand-in-hand with negative reinforcers, such as experiencing a sense of relief. In these cases, it seems that 145 negative emotions dissipated to be replaced with positive emotions. Pain was also identified as positively reinforcing for those participants who were motivated to punish themselves through self-injury. 4.3.4 Interpersonal access Several participants identified that they had accessed attention, care, and support from other people following their most recent episode of NSSI; however, only two participants mentioned that they had been motivated to seek support prior to hurting themselves. Debra, who was the only person whose episode of self-injury appeared to function exclusively as a form of interpersonal access, had been visiting her ex-boyfriend when she became really upset. She left his apartment because she did not want to ‚make a scene‛ in front of his flatmate and sat around the corner of his building where she then hit her head repeatedly on the concrete in front of her. 4.3.4 Interpersonal access 4.3.4.1 Establishing Operations The EOs that Debra described were comparable to those identified by other participants whose NSSI episodes had fulfilled intrapersonal escape and access functions: Debra: I just I got really kind of upset I was with him in his room and I was just you know I was sort of thinking about what would be to come in the break-up like<and I knew how hard my previous break-ups had been so I was just thinking about how sort of difficult I was expecting it to be and I just got really upset Debra: I just I got really kind of upset I was with him in his room and I was just you know I was sort of thinking about what would be to come in the break-up like<and I knew how hard my previous break-ups had been so I was just thinking about how sort of difficult I was expecting it to be and I just got really upset Debra: when I’m upset sort of my ability to to rationalise things<sort of just goes and and um I dunno like things that are that are sort of seem like little problems just suddenly seem really big and so I I dunno I would guess I’d say I wasn’t really thinking very straight<and I just I wasn’t thinking about what I was doing and I just yeah I was just thinking about how bad things were Debra: when I’m upset sort of my ability to to rationalise things<sort of just goes and and um I dunno like things that are that are sort of seem like little problems just suddenly seem really big and so I I dunno I would guess I’d say I wasn’t really thinking very straight<and I just I wasn’t thinking about what I was doing and I just yeah I was just thinking about how bad things were Although she struggled to remember exactly what she had been thinking, she Although she struggled to remember exactly what she had been thinking, she guessed that it would have been: just everything is hopeless and I’ve just fucked everything up and like yeah I sort of felt like er uh he’d broken up with me because he wanted to be single<and so he said you know this is nothing to do with you<but I just sort of felt like I’d screwed up everything and I had no idea what to do about it and and that I was just that I was just totally useless 146 Self-blame and thoughts of being useless, which were antecedents commonly identified by other participants, thus also preceded Debra’s episode of NSSI. However, one of the differences about the episode Debra described was that she was motivated to self-injure in order to access support from her ex-boyfriend: Debra: I was just you know I was really upset that he didn’t wanna be together anymore and<and I dunno I mean I guess I sort of wanted to to show him how upset I was maybe but I also sort of just felt like like I didn’t really have any options I mean I mean he’d broken up with me I couldn’t you know it was sort of out of my control Other participants described accessing support as a result of their self-injury, which may have reinforced their behaviour, but they did not report self-injuring in order to access support. Another participant, whose self-injury was more likely to have been reinforced by the attention he received rather than by the concurrent negatively reinforced consequence he reported of feeling calm, was Matt. He was the only participant who had injured himself most recently in front of another person. guessed that it would have been: His self-injury occurred in the context of feeling ‚deflated‛ because he had applied for two jobs and had been turned down for both: Matt: I was really I was sort of annoyed I was like so the journalistic world doesn’t want me and the government doesn’t want me and I guess my reaction was well fuck it I may as well just keep doing what I do it’s like this was gonna be excuse to straighten myself out to<act properly and I was like obviously I just don’t fit into that world I may as well just keep being weird and I was just like fuck it I’ll just burn myself again Matt: my flatmate was like ah I don’t wanna watch and her friend was like was like wow cool can I watch and I was like yeah sure um and so I put my knives on and I did like like three lines across my arm Matt: my flatmate was like ah I don’t wanna watch and her friend was like was like wow cool can I watch and I was like yeah sure um and so I put my knives on and I did like like three lines across my arm The phrase ‚I put my knives on‛ refers to what Matt called his ‚favourite way‛ of self-injuring; he would put two bread knives on a stove element to heat them up and then burn himself with the thin edge of the knives. The phrase ‚I put my knives on‛ refers to what Matt called his ‚favourite way‛ of self-injuring; he would put two bread knives on a stove element to heat them up and then burn himself with the thin edge of the knives. Matt was atypical in that he had regularly self-injured in front of other people; Debra was the only other participant who identified that she had 147 occasionally hurt herself when others were present. guessed that it would have been: Whether Matt self-injured alone or front of others depended on the types of EOs (e.g., frustration versus a desire to be the centre of attention) he was experiencing: Matt: I was more likely to be on my own when I was cutting<that I would be sitting in my room I’d be kind of frustrated or in a bad mood and I would just pick up my craft knife and I’d try and cut myself whereas the burning is more likely when I’d been drinking and there were like other people around<and it was kind of a show-offy kinda thing Matt: I discovered YouTube and I thought it would be cool to like video myself doing it and putting it on the Internet and because I didn’t like I had two dots on my arm and I didn’t like them so I decided<to cover them over with a line erm and that was more just a performance thing I think Subsequent to his video posting, he had engaged in discussions with people who had left comments about his video and he had also told people about his posting. 4.3.4.2 Discriminative stimuli Subsequent to his video posting, he had engaged in discussions with people who had left comments about his video and he had also told people about his posting. 4.3.4.2 Discriminative stimuli Subsequent to his video posting, he had engaged in discussions with people who had left comments about his video and he had also told people about his posting. 4.3.4.2 Discriminative stimuli It was also evident from the discussion I had with Debra about her history of NSSI that she had developed the verbal rule that if she hurts herself, then people will understand how distressed she is. guessed that it would have been: Instead, she commented that the headache, which follows her head hitti i d h th t it i ‚ d b thi t d ‛ Owen: I mean maybe maybe if the probably the least likely but a little bit of attention<um because I was voicing that I wasn’t feeling very good and wasn’t very well for five days before I did it<and that I thought no-one was listening and the here I am getting unwell again and all the rest of it um so it was a little bit of that<in this time when previously it was it was none of that it was more just hate Owen: I mean maybe maybe if the probably the least likely but a little bit of attention<um because I was voicing that I wasn’t feeling very good and wasn’t very well for five days before I did it<and that I thought no-one was listening and the here I am getting unwell again and all the rest of it um so it was a little bit of that<in this time when previously it was it was none of that it was more just hate Owen: I mean maybe maybe if the probably the least likely but a little bit of attention<um because I was voicing that I wasn’t feeling very good and wasn’t very well for five days before I did it<and that I thought no-one was listening and the here I am getting unwell again and all the rest of it um so it was a little bit of that<in this time when previously it was it was none of that it was more just hate because I was voicing that I wasn’t feeling very good and wasn’t very well for five days before I did it<and that I thought no-one was listening and the here I am getting unwell again and all the rest of it um so it was a little bit of that<in this time when previously it was it was none of that it was more just hate her head was hurting, she told him what had happened: Debra: I was like ah it was really stupid and yeah he was just like yeah I thought that you might have been doing that mm just sort of gave me a hug I was like ah it was really stupid and yeah he was just like yeah I thought that you might have been doing that mm just sort of gave me a hug Compared with other participants, Debra did not identify pain as a positive consequence. guessed that it would have been: 4.3.4.3 Consequences After hitting her head, Debra texted her ex-boyfriend to ask him to meet her: Debra: he came down and was hugging me making sure I was ok<he was there and that made me<feel better It is likely that his comforting response was positively reinforcing. Later on, when her head was hurting, she told him what had happened: Debra: I was like ah it was really stupid and yeah he was just like yeah I thought that you might have been doing that mm just sort of gave me a hug Compared with other participants, Debra did not identify pain as a positive consequence. guessed that it would have been: She believed that she was a fairly unstable person and because she got upset often, she asserted that people were unable to distinguish between her varying levels of distress: Debra: I guess for other people it’s kind of hard to tell the times I’m really really upset from the times I’m just quite upset or whatever<and so I dunno it’s sort of a like a way of showing people how upset I am This verbal rule, apparent prior to Debra’s most recent episode of NSSI in her acknowledgment that she wanted to show her ex-boyfriend how upset and sad she was feeling, would have served as a cue to Debra that there was the opportunity for her to access support following self-injury. This verbal rule, apparent prior to Debra’s most recent episode of NSSI in her acknowledgment that she wanted to show her ex-boyfriend how upset and sad she was feeling, would have served as a cue to Debra that there was the opportunity for her to access support following self-injury. This verbal rule, apparent prior to Debra’s most recent episode of NSSI in her acknowledgment that she wanted to show her ex-boyfriend how upset and sad she was feeling, would have served as a cue to Debra that there was the opportunity for her to access support following self-injury. Owen was the only other participant who explicitly reported that he had self- injured most recently as a way of communicating his distress to others: Owen was the only other participant who explicitly reported that he had self- injured most recently as a way of communicating his distress to others: 148 Owen: I mean maybe maybe if the probably the least likely but a little bit of attention<um because I was voicing that I wasn’t feeling very good and wasn’t very well for five days before I did it<and that I thought no-one was listening and the here I am getting unwell again and all the rest of it um so it was a little bit of that<in this time when previously it was it was none of that it was more just hate However, as acknowledged by Owen, support-seeking was not his primary motivation for self-injuring and therefore may not have reinforced his behaviour. guessed that it would have been: Instead, she commented that the headache, which follows her head hitting, reminds her that it is ‚a dumb thing to do‛. Compared with other participants, Debra did not identify pain as a positive consequence. Instead, she commented that the headache, which follows her head hitting, reminds her that it is ‚a dumb thing to do‛. Owen similarly received support following his episode of self-injury when he told his case manager what had happened. This consequence, however, was temporally distant from his self-injurious behaviour when compared with the other consequences (e.g., release of negative emotions) he experienced. Temporally distant consequences are less likely to act as reinforcers (Anderson, 2007) and, as such, it is more likely that the negatively reinforced, intrapersonal consequence of emotional release would have reinforced his behaviour, rather than positively reinforced, interpersonal consequence of accessing support. In contrast, Paula identified experiencing a ‚massive amount of release‛ after cutting herself, but still felt dissociated, and as though her self-injury was not enough, until her caregiver came home and comforted her: she realised what I’d done and stuff and she cleaned it up and all that and she did make it feel a lot better<and just um was comforting me for the rest of the afternoon and evening 149 It is therefore more likely that her cutting functioned as a form of intrapersonal access rather than escape, even though the care and support she received from her caregiver was more temporally distant from the self-injury than the release she experienced. Attention, rather than support, in all likelihood reinforced Matt’s self- injurious behaviour. It was evident that he had received substantial attention over the years for self-injuring, which had partially served to maintain his behaviour. guessed that it would have been: Given that he had been feeling rejected and deflated prior to burning himself most recently, the attention from his flatmate’s friend, who he asserted was really impressed by his ability to burn himself without flinching, most likely served as further positive, interpersonal reinforcement: she was really impressed ‘cos she was like wow you didn’t even flinch that was amazing but then I was like but that was kinda lame because I was kinda gonna do it anyway but the fact that she thought this was cool and wanted to watch kind of< encouraged me it was like yeah I’ve got an audience now I’m like performing<and then like her reaction like yeah that was really cool you didn’t even flinch I was just like yeah I’m tough Other participants similarly received attention from family, friends, or clinicians following their self-injury but in most cases it was not clear whether this attention was a reinforcing consequence, especially since these participants had not explicitly identified feeling unsupported or neglected prior to their NSSI episode. For some of the episodes, attention from others seemed to occur by chance, such as when Belinda received comfort from her husband when he interrupted her banging her head on the kitchen floor. In these instances, the person inadvertently communicated their distress to others but the goal of the behaviour was not necessarily to access support. However, the support that was received may still have served as a positive reinforcer for future self-injurious behaviour. A few participants actively sought out support by telling people what had happened (e.g., Owen who told his case manager about the episode) and the attention they received may have reinforced their behaviour. However, in some 150 cases, the attention was deemed to be an aversive consequence when the people they confided in were judgemental and critical of their behaviour. 4.3.4.4 Summary Although some participants were motivated to seek attention through self- injuring, for the most part they appeared to want to communicate their need for support. Matt was the only participant who self-injured specifically for attention rather than support. In some cases, the temporal delay between the self-injurious behaviour and the support received by participants made it difficult to assess the likelihood that these behaviours functioned as forms of interpersonal access. 22 Four participants did not report any aversive consequences. guessed that it would have been: These included disappointment, anger, and 151 guilt at having self-injured again: Karen: I always feel really horrible afterwards like I feel like I've let everybody down ((laughs)) I feel like I've let myself down<and I think I should've I should’ve been better I should've waited I should've held on longer I should've done this I should've done that ((laughing)) <why have I done this you know I'll start reflecting and I always feel really guilty<I was so pissed off at myself when I did it a month ago I was like dammit it's almost been a year you know ((laugh)) I've just gone<and done it again dammit Natalie: it’s more disappointment at myself<because I gave in<I guess<I get really angry at myself because<it’s not something I enjoy doing ((laugh)) Owen: the consequences really are<the realisation that you’ve done it yourself<that’s the stuff that’s really hard to deal with because you’re you’re you’re attacking the person that you should care the most about<and that’s that’s quite detrimental< also it made me think that I was going backwards in my recovery<which was quite damaging Nora: I think the most of would’ve just been with myself and<um feeling bad that I’d done it again and um yeah ‘cos I did wanna stop but I knew that it was all I had at that time<um yeah I think that was probably the biggest consequence was my own self All of these participants judged themselves negatively for their perceived failure to resist self-injuring. Moreover, Karen stated that her episode of self-injury ‚kinda opened a can of worms‛ and, as a result, she believed that she would self-injure again before the year’s end. guessed that it would have been: 4.3.5 Aversive Consequences As I mentioned earlier, the aversive consequences of clinical behaviour problems are usually delayed (Goldfried & Sprafkin, 1976), which makes it difficult to ascertain whether they function as punishers. Despite this limitation, it is still useful to present the consequences that may have served as punishers for the participants in the current study because they provide insight into why people may stop self-injuring. Additionally, I will briefly review the aversive consequences experienced by the three participants who did not report any reinforcing consequences as it is likely that their self-injury was punished rather than reinforced. The majority of the aversive consequences22 that followed participants’ most recent episode of NSSI represented four themes: negative intrapersonal experiences, the wound and scarring, restricted clothing choices, and unhelpful reactions from others. Since my intention in this chapter was to focus on the consequences that potentially reinforced and maintained participants’ self-injurious behaviours, I will only briefly discuss the consequences that may have punished the behaviour. 4.3.5.1 Negative emotions and thoughts Many people identified experiencing negative emotions and/or thoughts following their episode of self-injury. guessed that it would have been: I always feel really horrible afterwards like I feel like I've let everybody down ((laughs)) I feel like I've let myself down<and I think I should've I should’ve been better I should've waited I should've held on longer I should've done this I should've done that ((laughing)) <why have I done this you know I'll start reflecting and I always feel really guilty<I was so pissed off at myself when I did it a month ago I was like dammit it's almost been a year you know ((laugh)) I've just gone<and done it again dammit it’s more disappointment at myself<because I gave in<I guess<I get really angry at myself because<it’s not something I enjoy doing ((laugh)) the consequences really are<the realisation that you’ve done it yourself<that’s the stuff that’s really hard to deal with because you’re you’re you’re attacking the person that you should care the most about<and that’s that’s quite detrimental< also it made me think that I was going backwards in my recovery<which was quite damaging I think the most of would’ve just been with myself and<um feeling bad that I’d done it again and um yeah ‘cos I did wanna stop but I knew that it was all I had at that time<um yeah I think that was probably the biggest consequence was my own self All of these participants judged themselves negatively for their perceived failure to resist self-injuring. Moreover, Karen stated that her episode of self-injury ‚kinda opened a can of worms‛ and, as a result, she believed that she would self-injure again before the year’s end. All of these participants judged themselves negatively for their perceived failure to resist self-injuring. Moreover, Karen stated that her episode of self-injury ‚kinda opened a can of worms‛ and, as a result, she believed that she would self-injure again before the year’s end. Aversive emotional consequences not only appeared to stem from participants being unable to meet their own expectations, but also from the inability to meet societal expectations. Tara, in particular, reported feeling ashamed that she had transgressed the social boundaries of what is considered normal, a transgression which then impacted on how she perceived herself: which then impacted on how she perceived herself: I mean at the time it’s kind of dealing with my feelings but then I have to afterwards deal with the shame that I’ve done something that’s not really<considered normal to myself and admit that to other people and<admit that even though at that time I did have other options available to myself because I had been trying other ways of coping I had for some reason I didn’t use that<and so it kind of in some ways it was kind of hard ‘cos it reinforces my whole thoughts of I’m not normal I don’t do things normally I should do things normally kind of<a little bit of a merry-go-round that I go on In contrast, Ella was not ashamed of, or disappointed in, self-injuring, bu rather was frustrated that it did not have the same effect that it used to: I guess you kind of you you go back to your old coping strategies<or your old comforts um habitually and so I thought I think I thought that it would do more than what it did <I think rationally um I kind of yeah I kind of thought well that was a waste of time um 152 <‘cos it it didn’t yeah I mean I ((sighs)) it helped a little but uh I don’t know maybe I’d talked myself into thinking it was gonna like magically<make me feel wonderful <which of course it’s not gonna do A couple of the other participants similarly commented that their most recent episode of self-injury had failed to live up to their expectations, which in Josh’s case led to him feeling as though he needed to self-injure again to get the desired effect: Josh: the sort of main I guess problem with last week was um having done drugs<as well as um self-harm so it’s kind of um I didn’t get as much of the sort of sublime<feeling um as I normally would’ve<I just more s- sort of had like a uh sort of awkward feeling really ((laughing))<normally I wouldn’t actually continually think about you know um it afterwards I’d just kind of ((clears throat)) go with the fl- go with it basically<whereas um yeah with with the drugs I was still kind of aware of<the reason why I was doing that which kind of made me feel like I needed to do it again Josh’s expectations of how he would feel following cutting were unfulfilled because of the dampening effect of drugs, whereas Nora’s expectations were unfulfilled because self-injuring did not solve her problems: Josh’s expectations of how he would feel following cutting were unfulfilled because of the dampening effect of drugs, whereas Nora’s expectations were unfulfilled because self-injuring did not solve her problems: Nora: I felt quite bad because I knew that it hadn’t solved anything like<this problems that were there before it were still there<um yeah kind of disillusioned that they hadn’t gone <um and that it hadn’t just disappeared when I cut myself<that all the feelings hadn’t just gone<out of me Nora: I felt quite bad because I knew that it hadn’t solved anything like<this problems that were there before it were still there<um yeah kind of disillusioned that they hadn’t gone <um and that it hadn’t just disappeared when I cut myself<that all the feelings hadn’t just gone<out of me 4.3.5.2 The wound and scarring Physical wounds and scarring were identified by participants as both short- and long-term negative, consequences of their most recent episode of self-injury. For some participants, the short-term consequences focused on the discomfort associated with the wound: Ella: it’s quite uncomfortable while y- you know you’re healing and stuff Debra: I guess yeah the times I hit my head my head is sore and I’m kind of just like well that was stupid this now I have a headache well that was a dumb thing to do However for Luke, the location of the wounds was distressing because they were difficult to conceal: However for Luke, the location of the wounds was distressing because they were difficult to conceal: and yeah so this time<it was kind of disappointment<like ah too too many too visual I I got really upset with myself because they were too close to my sleeve line 153 The difficulties with having to conceal wounds were also associated with the aversive, long-term consequence of scarring. Angela considered scarring to be sufficiently adverse for it to deter her from engaging in NSSI more frequently: Angela: afterwards I um I don’t feel sad about doing it or anything like that because I think with depression well with mine anyway um there’s that feeling that you’re just you’re worth nothing basically so it’s not really that I feel sad about having cut myself but I k- I just worry about scarring and things like that<if that wasn’t a worry in my mind I think I would do it more often Karen : I think what have I done to myself these marks are gonna be here forever ‘cos I scar quite dark Of course, the potential for scarring was not a protective factor against future self- injury for all participants; Nicola mentioned that the scar from her most recent episode had ‚pretty much gone now‛ so she did not rate it as a negative consequence. 4.3.5.3 Restricted clothing choices 4.3.5.3 Restricted clothing choices Another set of possibly punishing consequences revolved around how the location of the wounds and the extent of scarring restricted participants’ clothing choices: and then of course it’s like ah no I'm gonna have to hide it now and gonna have to bandage it up and gonna have to wear long sleeves for however long< but of course if I've just cut ((laugh)) I'm not gonna walk around with a blatant bandage on my arm ((laughing)) ‘cos that just makes it obvious and like<I don't want everyone to know so and then of course it’s like ah no I'm gonna have to hide it now and gonna have to bandage it up and gonna have to wear long sleeves for however long< but of course if I've just cut ((laugh)) I'm not gonna walk around with a blatant bandage on my arm ((laughing)) ‘cos that just makes it obvious and like<I don't want everyone to know so and I guess also um another consequence of it is that I think it was well it was just coming up to winter so it wasn’t so bad<but it means that I’d had to wear long sleeve tops for a long time I can’t wear a t-shirt for another three months<thank god it’s winter you know like<like I mean they seem really sort of surface but that that becomes a big issue<when you’re thinking about what you’ve done is ah god here we go<like no t-shirt again for three months like<that can be quite sad 154 desire to keep their behaviour a secret in order to retain control over it or to avoid being judged by others. desire to keep their behaviour a secret in order to retain control over it or to avoid being judged by others. 4.3.5.4 Reactions from others Indeed, the anticipation of being negatively evaluated by others was one of the reasons Debra gave for not having cut herself more often: Debra: the times I’ve cut myself I sort of its it left a scar that stayed for f- three or four months but now<its sort of gone<which I am pretty glad about ‘cos I dunno I mean like I mean I’ll tell my close friends about it but I don’t want just random people<thinking I’m that kind of person<a couple of times when I’ve thought of cutting myself the fact that it would scar has been a factor in me not doing it because<I I mean people will really judge you about that kind of thing When Debra anticipates being judged as ‚that kind of person‛, it is unclear what type of person she is referring to. Other participants, however, did report incidents where they were judged for their most recent episode of NSSI; judgements that were often at odds with how they perceived themselves. When Debra anticipates being judged as ‚that kind of person‛, it is unclear what type of person she is referring to. Other participants, however, did report incidents where they were judged for their most recent episode of NSSI; judgements that were often at odds with how they perceived themselves. 4.3.5.4 Reactions from others Both Karen and Ella were frustrated with how they were evaluated as unstable and mentally unwell by medical and mental health professionals: I was um in hospital um just after I’d done them like two weeks after I’d done them for something ah what did I go in for ah it was some kind of health check thing anyway um they the scars only just healed<so they were still a little bit new and um the guy was like I was like ah they’re two weeks old man you know and he‘s no they’re not and I’m like yes they are ((laugh)) no we need the CAT team now and it was like nah oo they ah for god’s sake you know ((laughing)) so frustrating like no they’re old I you know they’ve healed<so that kind of thing really frustrates me it’s like ask me first and take my opinion seriously ((laughs)) when I said I actually feel like I’m ok and<you know I I did this yuh I I did this once and for me I feel like it was a slip-up and I feel like I’ve got past it and my psychiatrist went yeah but you’ve said that before and I was just like ah what the hel- like and it was almost like I was written off completely<and I was back to being that you know really small little crazy mental patient in the corner um just like that over one decision I’d made<which I was ok with ((laughs))<and then they couldn’t understand why I got angry ((laughing)) It was clear that being made to feel like ‚a small little crazy mental patient‛ or someone who cannot be trusted was an extremely disempowering experience for Ella and Karen, both of whom felt unfairly judged by clinicians. 4.3.5.4 Reactions from others 155 Owen was similarly judged solely on the basis of his self-injury when, after his most recent episode, he was questioned as to whether he was a ‚burner‛: Owen: my most recent episode I um burnt myself and then walking around and and everyone going what happened what happened<are you a burner are you wh- what’s going on just all that stuff made it me think nah this is really you know this stuff really isn’t what I wanna be about Having his identity reduced to one particular behaviour choice may have had some punishing value for Owen as he cites this as a reason for stopping self-injury. Having his identity reduced to one particular behaviour choice may have had some punishing value for Owen as he cites this as a reason for stopping self-injury. Conversely, when Melanie was judged negatively for cutting herself, her reaction was to justify her use of self-injury as a coping mechanism. She had texted her ex-boyfriend to tell him what had happened and he subsequently informed her mother that she had self-injured: Melanie: ((laugh)) had a bit to deal with there when she came home ((laughing))<mm she doesn’t really understand it at all so<she was very upset said she thought I had got past all that and<that it wasn’t a good way to deal with things and ((laughing))<I sort of had to tell her yes I know that but ((laughing)) it’s a way to deal with things ((laughing)) The manner in which Melanie dealt with this altercation makes it seem unlikely that her mother’s reaction would have punished her self-injurious behaviour. What seems more likely is that her mother’s reaction would have acted as a punisher for her contacting her ex-boyfriend following any future episodes of self-injury. For Jenny, other people’s reactions to her self-injury did not appear to concern her as much as God’s reaction: the only consequence really after that last one was I was afraid of how God might see me<because I kept asking him to heal me and<but I kept doing what I did and I’d done it the last time I was thinking I wonder what he’s thinking looking down at me thinking there you are I tried to make you better but y- what are you doing you’re still going back to it<and I felt an immense sense of responsibility for that that he might perceive me as being a very um I dunno arrogant in some way uncooperative maybe<just taking advantage of him<that was almost enough as a consequence for me that he might you know send something down on me or<punish me or in some way or be very unhappy with me What is particularly striking about Jenny’s admission is her palpable sense of self- blame and belief that it was her fault for not being healed. What is particularly striking about Jenny’s admission is her palpable sense of self- blame and belief that it was her fault for not being healed. 156 Matt was the only person to experience an admiring reaction to his self-injury as a possibly punishing consequence. 4.3.6 Summary of Interpretative Functional Analysis results Overall, the most frequently identified function was intrapersonal escape/avoidance, but many of the participants’ self-injury episodes were hypothesised to serve more than one function. Interpersonal functions were far less common than intrapersonal functions, with only a few episodes hypothesised to function as a form of interpersonal access and none as interpersonal escape/avoidance. Many of the EOs and SD’s did not appear to differ according to the function of the behaviour, but rather were the same across functions. For example, negative emotions and thoughts commonly precipitated NSSI episodes that served intrapersonal or interpersonal functions, highlighting the importance of individual learning histories in maintaining self-injurious behaviours. Overall, the most frequently identified function was intrapersonal escape/avoidance, but many of the participants’ self-injury episodes were hypothesised to serve more than one function. Interpersonal functions were far less common than intrapersonal functions, with only a few episodes hypothesised to function as a form of interpersonal access and none as interpersonal Having his identity reduced to one particular behaviour choice may have had some punishing value for Owen as he cites this as a reason for stopping self-injury. Self-injuring in front of his flatmate’s friend resulted in him feeling lame and immature: I felt immature<like that’s probably the same thing as b- feeling lame I just felt like I was doing I was doing it because it’s what I used to do and I had no other better reason and that eh and I felt kind of bad about the fact that I was like showing off to this person and not setting a good example<I guess the person who was watching me is a little bit unstable herself um she has her own way of dealing with things usually by getting drunk and you know I think the fact that she was like thought it was really interesting when I was doing it was like an extra reason it was like cool I have an audience<but I realised pretty much immediately afterwards that that wasn’t necessarily good for her and so I didn’t feel particularly guilty about it but again<I was just like ah that’s a bit silly that’s a bit stupid of me Matt’s realisation that he self-injured more out of habit than need and that he has a responsibility to set an example to those younger than him, may deter him from harming himself in similar situations in the future. 4.3.5.5 Exclusive experience of punishing consequences As I mentioned earlier, three participants identified experiencing exclusively punishing consequences following their most recent episode of NSSI, which included feelings of shame, disappointment for giving in to the urge to self-injure, and concerns about the wound getting infected. These consequences were not substantively different from the punishing consequences reported by participants who had also experienced reinforcing consequences following their episode of NSSI. 4.3.5.6 Summary The aversive consequences identified by the participants reflected the following four themes: negative emotions and thoughts, physical effects of the wound and concerns about scarring, needing to restrict their clothing choices to hide the evidence of their self-injury from others, and experiencing unhelpful and stigmatising reactions from others. As such, the majority of these consequences stemmed from the impact of their self-injury on the way in which they viewed themselves, how they anticipated being perceived by others, and, in some cases, the way in which they actually were judged by others. 157 4.3.6 Summary of Interpretative Functional Analysis results 5. STAGE THREE: EMAIL OR TELEPHONE FOLLOW-UP Approximately two days after each interview, I contacted the participants by email or phone to once again thank them for taking part in the study and to ask them whether they had thought of anything else that they wanted to share with me. This was recommended by one of the consumer advocates I consulted as a way of checking in with, rather than checking up on, participants. I also hoped that by initiating contact with participants after the interviews, they would be more likely to contact me if they had any concerns or questions about the study. Some participants responded to my email enquiry by sending follow-up thoughts on diverse issues such as their reasons for self-injuring, other people’s misconceptions and assumptions about NSSI, and the link between direct and non- direct forms of NSSI. One woman emailed me the diary entry she wrote the night of the interview noting that she did not realise how much more she wanted to say. In her diary entry, she reflected on why she cut (e.g., ‚Cutting allowed me to escape‛) and the effect that it had on her: 6.1 Participants and procedure Approximately two weeks after being interviewed, participants were sent a questionnaire (see Appendix G) to provide anonymous feedback about their experiences of taking part in the study, which they were asked to complete and return within two weeks. Twenty-three people (95.8%) completed their questionnaires; the one person who did not return their questionnaire later wrote to apologise that he had been away at the time. Unfortunately, I was unable to calculate the average number of days between the interview participation and questionnaire completion because the questionnaires were anonymous. Each person was posted a movie voucher for completing the questionnaire.23 23 One participant received a $10 voucher for the Warehouse instead of a movie voucher because she had hearing difficulties. and the effect that it had on her: I let cutting become such a big part of me, and lost my emotions, and sense of who I was, and any control I had over that. My body felt numb and empty, but my head was full, and cloudy, and heavy. Kind of like the air when it's about to rain. Cutting enabled me to let some of the cloudyness go, and left me feel fresh, kind of like the air after it's rained. 158 Other participants emailed to comment on their experience of taking part in the study: Other participants emailed to comment on their experience of taking part in the study: Just glad to be a part of something that will help others. I just wanted to say, thanks for the opportunity to tell my story. As nervous as I was, after th experience I felt very liberated and a bit less alien than before I started. I just wanted to say, thanks for the opportunity to tell my story. As nervous as I was, after the experience I felt very liberated and a bit less alien than before I started. I didn't think of anything else yet, that's probably the most I've ever talked about self harm in one go anyway! I felt good afterwards though, I think it was a good opportunity to process some stuff and reflect on it for myself. 6.2 Measure As I was unable to locate a previously published questionnaire that related specifically to how people had responded to taking part in a study about self-injury, I adapted the Reactions to Research Participation Questionnaire for Parents (RRPQ-P; Kassam-Adams & Newman, 2002, 2005) for the purposes of the current study. The RRPQ-R consists of 12 items rated on a 5-point scale from strongly disagree to strongly agree and has demonstrated acceptable internal consistency scores of .78 to .80 (Kassam-Adams & Newman, 2002, 2005). 159 Nine of the 12 items in the RRPQ-P (Kassam-Adams & Newman, 2005) were included in the research evaluation measure used in this study. Of these nine items, two were taken verbatim from the RRPQ-P, one was split into two items, and seven were modified (e.g., ‚I am sorry I was in this study‛ was rewritten to read, ‚I regret participating in this study‛). A further seven questions were added to the measure (e.g., ‚The study was explained thoroughly to me before I took part‛). This resulted in a total of 17 items in the revised measure; participants were asked to respond to the first 15 items on a 5-point likert scale from 1 (strongly disagree) to 5 (strongly agree). These 15 items focused on issues of informed consent, rapport, emotional reactions to the interview, and beliefs about study participation. The final two items were open-ended and asked whether participants had any suggestions about how to improve future research studies on NSSI and whether they had any other comments to add about their experiences of being in the study. 6.3 Results The results of the research evaluation questionnaire are presented in Table 3. All of the participants agreed or strongly agreed that they had given informed consent to participate in the study and that they knew they could skip questions or take a break from the interview whenever they wanted to. Only one participant was not sure that they could stop being in the study at any time. The participants reported feeling comfortable and respected during the interview. People were more likely to endorse feeling sad or upset, rather than distressed, as a result of participating in the study. Almost half of the participants agreed that being in the study had made them feel good about themselves and all but one of the participants believed that their involvement in the study was helpful to other people who self- injure. Despite some people reporting that they felt sad or upset because of their participation in the study, 22 people did not regret taking part and all 23 participants agreed or strongly agreed that with the benefit of hindsight and knowing what it was like to take part in the study, they would still choose to participate. Table 3 Number of participants (N = 23) who endorsed each statement Item Strongly disagree N (%) Disagree N (%) Not sure N (%) Agree N (%) Strongly agree N (%) The study was explained thoroughly to me before I took part. 0 (0.0) 0 (0.0) 0 (0.0) 2 (8.7) 21 (91.3) It was my choice to take part in the study (i.e., I could have said no even if other people wanted me to say yes). 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.3) 22 (95.7) I knew I could skip questions or parts of the study if I wanted to. 0 (0.0) 0 (0.0) 0 (0.0) 4 (17.4) 19 (82.6) I knew I could stop being in the study at any time. 0 (0.0) 0 (0.0) 1 (4.3) 1 (4.3) 21 (91.3) During the interview, I knew that I could ask to take a break whenever I wanted. 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.3) 22 (95.7) The interviewer made me feel comfortable during the interview. 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.3) 22 (95.7) The interviewer showed respect for my feelings and experiences during the interview. 6.3 Results 0 (0.0) 0 (0.0) 0 (0.0) 2 (8.7) 21 (91.3) I am glad that I took part in this study. 0 (0.0) 0 (0.0) 0 (0.0) 10 (43.5) 13 (56.5) Being in this study made me feel distressed. 4 (17.4) 13 (56.5) 6 (26.1) 0 (0.0) 0 (0.0) Being in this study made me feel upset. 3 (13.0) 14 (60.9) 3 (13.0) 2 (8.7) 1 (4.3) Being in this study made me feel good about myself. 0 (0.0) 3 (13.0) 9 (39.1) 10 (43.5) 1 (4.3) I regret participating in this study. 17 ( 73.9) 5 (21.7) 1 (4.3) 0 (0.0) 0 (0.0) Being in this study made me feel sad. 2 (8.7) 10 (43.5) 3 (13.0) 7 (30.4) 1 (4.3) I believe that by being in the study I am helping other people who self-harm. 0 (0.0) 0 (0.0) 1 (4.3) 12 (52.2) 10 (43.5) Knowing what I know now about participating in the study, I would still choose to take part in this research. 0 (0.0) 0 (0.0) 0 (0.0) 3 (13.0) 20 (87.0) Item 160 161 Reasons as to why participants valued participating in the study can be gleaned from their responses to the open-ended questions: It was fantastic to partake in such an important study. Hopefully it will help ascertain and clarify what is going on in the minds of many young people who self harm, and what steps can be taken to actually support people who do. Self harm is a societal taboo in many ways, hopefully studies as these help to dispel the myths that even the medical profession sometimes adheres to still. We need to move away from the belief that asking people about their self harm is ‚dangerous‛ and will cause people to self harm more. There is no evidence to support these assumptions. Many people who self harm would probably rather people asked than made misguided assumptions. I’m really glad I participated. I haven’t had the opportunity to talk about these issues or process it for myself before. I felt ready to reflect on it and very safe and respected in this process. I hope the research is well received and helpful to others. I found that I felt quite relieved after the study as I’ve never really spoken about my self harming. It was good to get it off my chest and I felt like I was helping other self harmers out there. I actually really liked being in this study. It felt really good to be able to talk freely and openly about self harm, in an environment that felt safe and non-judgemental<Although at times, I felt a little bit sad and distressed, it was only slightly, mostly because of taking myself back to my experiences with self-harm, but I liked the fact that I felt that somehow my experiences may eventually be able to help others< I did not find taking part in this research to be at all distressing or triggering, and actually found it quite positive—helped me process. It was easier to talk about self harm in a research participation situation because it was more about being able to possibly help someone else, which bypasses the natural instincts to avoid talking because of feelings of no or low self worth or self importance. Item Taking part in the study thus appeared to be a positive experience for participants because they were able to discuss and reflect on their experiences of self-injury without feeling judged and, at the same time, they felt as though their experiences may help others in the future who are struggling with similar issues. Taking part in the study thus appeared to be a positive experience for participants because they were able to discuss and reflect on their experiences of self-injury without feeling judged and, at the same time, they felt as though their experiences may help others in the future who are struggling with similar issues. 7.1 Forms and frequencies of NSSI behaviours The majority of the people who participated in the current study had an extensive history of NSSI and, on average, had used eight different types of self- injury in their lifetimes. The extent of NSSI in this sample was unsurprising given that several participants were recruited through mental health services and a mental health consumer network, and the prevalence of self-injury is substantially higher in 162 psychiatric compared to community populations (Bebbington et al., 2010; Briere & Gil, 1998; Claes, Klonsky, et al., 2010). A number of university students similarly reported high rates of NSSI in the current study, but most of these students were currently receiving mental health support, or at least, had received such support in the past. Several participants reported types of self-injury that did not meet the definition of NSSI used in this thesis. Two examples of other forms of self-injury, in particular, warrant further discussion. To gain admission into hospital, Sophia acknowledged overdosing on medication several times; while Karen, who had been diagnosed with Bulimia, had ensured that her potassium levels were dangerously low (see section 4.3.2). Both of these participants were motivated to be hospitalised to escape from, or avoid, interpersonal responsibilities and, as such, it is likely that these episodes functioned as forms of interpersonal escape/avoidance. These examples suggest that restricting the types of NSSI to exclude behaviours such as overdosing may have decreased the likelihood of observing certain functions. Indeed, none of the participants reported self-injuring most recently as a form of interpersonal escape/avoidance. Further research is necessary to determine whether particular types of NSSI are aligned with specific functions. 7.2 Is self-injury primarily an experientially avoidant behaviour within Aotearoa New Zealand? New Zealand? Functionally analysing participants’ most recent episode of NSSI enabled me to identify considerable homogeneity among complex, seemingly heterogeneous incidents of self-injury. The majority of the self-injury episodes were hypothesised to fulfil an intrapersonal escape/avoidance function, which is synonymous with experiential avoidance. In contrast, no episodes were hypothesised to serve an interpersonal escape/avoidance function. Access functions commonly occurred in conjunction with escape/avoidance functions. Only one episode functioned exclusively as a form of interpersonal access, while one other episode functioned exclusively as a form of intrapersonal access. For 163 the latter episode, the participant was interrupted by her husband while she was hitting her head on the kitchen floor. The behaviour may have fulfilled an escape/avoidant function, as it had numerous times in the past, if she had not been interrupted. While the findings from my Interpretative Functional Analysis are broadly consistent with the EAM (Chapman et al., 2006) in that the majority of episodes functioned as a form of escape from aversive thoughts and emotions, they also provide insight into the ways in which self-injurious behaviours function that are undervalued within, or excluded from, the contingencies depicted in the EAM (Chapman et al., 2006). These insights include the importance of cognitions and clarifying the role of positive reinforcement in maintaining NSSI. Cognitions are not completely dismissed within the EAM, but Chapman and colleagues (2006) do maintain that self-injury is predominantly an emotionally avoidant behaviour. Additionally, they suggest that emotional avoidance may underlie attempts to escape from negative cognitions; in other words, it is the emotions connected to negative cognitions that motivate people to engage in avoidant behaviours. An explicit focus on the emotional precipitants of NSSI makes for a more parsimonious model, but has far-reaching research and clinical implications. To date, researchers have largely neglected the role of cognitions in NSSI, preferring instead to concentrate on explaining how self-injury is used to regulate emotions (e.g., Adrian et al., 2011; Kamphuis et al., 2007). Models, such as the EAM (Chapman et al., 2006), may perpetuate this limited focus through deemphasising the influence of unwanted cognitions on self-injurious behaviours. In the current study, cognitions featured heavily in participants’ accounts of their most recent episode of self-injury. More research is needed to decipher whether specific types of cognitions and/or particular cognitive processes commonly precipitate NSSI. New Zealand? 164 During the interviews, one of the most frequently mentioned cognitive processes to occur prior to self-injury was that of constriction. Cognitive constriction is typically associated with suicidal behaviours (Leenaars, De Wilde, Wenckstern, & Kral, 2001; O’Connor, Sheehy, & O’Connor, 1999) and is defined by Schneidman (1996) as: ‚a tunneling or a focusing or narrowing of the range of options usually available to that individual’s consciousness when the mind is not panicked into dichotomous (either-or) thinking‛ (p. 134). An example of constricted thinking in the context of a suicidal crisis is: ‚If this disease is incurable, I will end it all‛ (Walsh, 2006, p. 13). This conditional, if-then formula is identical to that of the verbal rules discussed earlier in this chapter (see section 4.3.1.2), which suggests that cognitive constriction may be a key component of NSSI. Although Pattison and Kahan (1983) listed cognitive constriction as one of the symptoms of their deliberate self-harm syndrome, it has been largely overlooked in empirical investigations of NSSI. On the contrary, Walsh (2006) recommends differentiating between suicidal and non- suicidal self-injurious behaviours on the basis of cognitive constriction. According to Walsh (2006), people who engage in NSSI typically demonstrate disorganised, not dichotomous, thought patterns, and consider NSSI as one of a number of coping strategies they could use. Participants in the current study did indeed perceive that they had various options available to them but these perceptions usually occurred in hindsight, after they had engaged in NSSI. When discussing their thought processes prior to self-injuring, many participants reflected on how it seemed that NSSI was the only option they had at the time to effectively cope with distress. More research is needed on how people reach the point where their cognition becomes constricted. For participants in the current study, this process seemed to occur when they surpassed a certain threshold of emotional arousal, which then interfered with their ability to problem-solve alternative solutions. Decreasing their emotional arousal through self-injury then facilitated future problem-solving and provided them with a sense of agency. 165 Understanding more about the role of verbal rules (as a form of cognitive constriction) in precipitating NSSI episodes thus has important clinical implications. As SD’s, verbal rules signal to people when reinforcement is likely to be available following self-injury and therefore help to maintain self-injurious behaviours (Sturmey et al., 2007c). New Zealand? Clinicians therefore may need to explicitly identify and target verbal rules in treatment. 7.3 Strengths of Interpretative Functional Analys The detailed functional assessments undertaken for this study provided valuable insights into specific antecedents (e.g., verbal rules) and consequences (e.g., attention) of self-injurious behaviours, which have been largely ignored, or at times misrepresented, in the literature. To my knowledge, no other researchers have distinguished between EOs and SD’s when examining the role of antecedents in precipitating self-injurious behaviours among typically developing populations, or conducted a group-level comparison of functional assessments of NSSI. Furthermore, interviewing 24 people enabled me to access a wide range of self- injury experiences. 7.2.2 What about positive reinforcement? Several self-injury episodes appeared to facilitate access to positive intrapersonal experiences or support, highlighting the role of positive reinforcement in the maintenance of self-injury. As discussed earlier in this thesis, a number of researchers have investigated whether NSSI is a positively reinforced behaviour (e.g., Lloyd-Richardson et al., 2007; Nock & Prinstein, 2004) and results are consistent with the findings of this study, which suggest that positive reinforcement is important for some people. However, it is notable that positive reinforcement schedules were seldom evident in the absence of negative reinforcement. This suggests that co-existing reinforcement schedules operate for some people who self-injure, making it potentially more difficult to extinguish the behaviour. These results are consistent with Brown et al.’s (2002) study where participants reported multiple reasons for single self-injury episodes. Certainly, Sturmey et al. (2007b) contend that focusing on single antecedents and consequences of operant behaviour is simplistic as concurrent reinforcement schedules are the norm. Another important finding is that very few people engaged in self-injury to access attention, which is contrary to anecdotal perceptions of NSSI as an attention- seeking behaviour. More typically, when people did report receiving attention as a direct result of self-injuring, it was perceived to be an aversive consequence. Only two people acknowledged being motivated to engage in self-injury to gain attention from others, although it is possible that some participants may not have reported such motivations if they perceived them to be socially undesirable (Nock, 2008). 166 Intrapersonal positive reinforcement, however, was more common. For example, experiencing pain in the context of a desire to punish oneself for perceived wrongdoings potentially functions to positively reinforce self-injurious behaviour. 7.3 Strengths of Interpretative Functional Analysis 7.4 Limitations of Interpretative Functional Analysis Conducting idiographic assessments allowed me to hypothesise about which consequences may reinforce participants’ self-injurious behaviours, but to qualify as a reinforcer these consequences would have had to increase the frequency of participants’ self-injurious behaviour (Dougher & Hayes, 2000). Since each participant was only interviewed once, I cannot confirm whether the hypothesised reinforcers actually did reinforce participants’ self-injurious behaviour because I do not know whether they self-injured again. Furthermore, several of the participants had self-injured months before the interview and the extent to which their accounts of their most recent episode were influenced by retrospective biases is unclear. The length of time between the episodes and interviews also precluded a more detailed behaviour chain analysis, similar to those conducted as part of DBT (Lynch et al., 2006). However, chain analyses are likely inappropriate for one-off research participation given that people frequently experience these as arduous; it has even been suggested that they may function to punish NSSI (Lynch et al., 2006). If I had 167 been able to elicit more detailed information from participants, it would have still been difficult to achieve absolute certainty about the functions of the behaviours due to the complexity of concurrent reinforcement schedules (Sturmey et al., 2007b) and the inability to manipulate specific variables. been able to elicit more detailed information from participants, it would have still been difficult to achieve absolute certainty about the functions of the behaviours due to the complexity of concurrent reinforcement schedules (Sturmey et al., 2007b) and the inability to manipulate specific variables. Finally, many more women than men participated in the current study, which is common in much self-injury research (Russell et al., 2010). In light of recent studies that suggest there are no significant gender difference in the prevalence of NSSI (e.g., Andover et al., 2010; Claes, Houben, et al., 2010), it is unclear why so few men participated. One possibility is that males may have felt more uncomfortable disclosing stories about their self-injurious behaviour in a socio-cultural context where NSSI is still viewed as a largely female phenomenon. On the surface, the process and functions of the self-injury episodes described by the male participants in this study did not appear different to those described by the female participants. Although the only participant to self-injure regularly in front of others was male, it is unknown whether self-injury as performance for others is a gendered phenomenon. 7.4 Limitations of Interpretative Functional Analysis One of the few studies to investigate whether self-injury functions differ according to gender found that men were significantly more likely to report sensation-seeking (Klonsky & Glenn, 2009); self-injury as performance could be a manifestation of this function. Further research is necessary to ascertain whether this is typical of males who self-injure. 7.5 Is participating in a study about self-injury harmful? One of the difficulties of researching NSSI is the iatrogenic potential of questions about self-injury. Talking about self-injury is no doubt distressing for some people, and given that self-injury is used to cope with distress, it is a concern that people may self-injure following research participation. Although there is little or no evidence to suggest this is the case, assumptions about the harmfulness of self- injury research are commonplace and, at times, are even perpetuated by researchers themselves: 168 Given the sensitive nature of the topic and the way the field has been dominated by quantitative approaches, it seemed clear that face-to-face interviews would be difficult to arrange and harrowing [emphasis added] for all participants. (Harris, 2000, p. 165) Given the sensitive nature of the topic and the way the field has been dominated by quantitative approaches, it seemed clear that face-to-face interviews would be difficult to arrange and harrowing [emphasis added] for all participants. (Harris, 2000, p. 165) This perspective is antithetical to how the participants in the current study evaluated their research participation (see section 6.3); certainly, none of their responses indicated that the process was the least bit harrowing. While some of the participants did report feeling sad and upset as a result of taking part in this study, none regretted their participation. Instead, they stated that the research provided them with the opportunity to process their history of self- injury in greater depth, as well as potentially help others who are experiencing similar difficulties. These reactions are consistent with a comprehensive review of 46 studies conducted by Jorm, Kelly, and Morgan (2007) who found that participants in mental health-related research were more likely to view their experience as positive, than negative. Furthermore, positive reactions to research participation were largely independent of distress, which provides evidence that people may rate research participation as positive even if they do experience some distress (Jorm et al., 2007). However, participating in research specifically about self-injury may be distressing and, if so, the question remains whether that distress leads people to hurt themselves. Results from a recent study that examined the impact of screening questions about deliberate self-harm and suicidal thoughts on male secondary school students, showed that such questions did not significantly increase distress (Robinson et al., 2011). 7.5 Is participating in a study about self-injury harmful? Only 9% of students reported that these questions were moderately or very distressing, with at-risk students reporting higher distress levels (Robinson et al., 2011). Aside from my research, only one other study to my knowledge has specifically examined whether questions about NSSI upset people who have self- injured (Hanly, Pietrusza, Gluck, & Whitlock, 2011). University students with a history of NSSI were twice as likely as students who had never self-injured to report that completing a survey, which included questions on NSSI, was difficult but that it 169 encouraged them to reflect on their experiences (Hanly et al., 2011). Students with a history of NSSI were also almost three times as likely as those without such a history to report that completing the survey was a negative experience. In both of these studies, some participants did experience distress as a result of answering questions about self-harm or self-injury, but neither study tracked participants over time. As such, it is impossible to determine whether students coped with their distress by self-injuring. Further research is needed to ascertain whether research about NSSI has an iatrogenic effect and, if so, whether this effect differs according to the research methodology (e.g., anonymous self-report survey versus in-depth interview) or the extent of NSSI questions. Certainly, the few negative comments received about the current study were primarily in regards to the DSHI (Gratz, 2001), which participants found overly detailed. 7.6 Conclusion 7.6 Conclusion Based on the results of this study, the EAM (Chapman et al., 2006) is a useful theoretical framework for research and treatment purposes. In the majority of cases, it appeared that NSSI episodes did indeed serve an experientially avoidant function. However, more consideration of the role of positive reinforcement in maintaining self-injury is warranted. Furthermore, researchers and clinicians need to assess the interrelationships between cognitions (in particular, verbal rules) and emotions, to clarify the role of specific thought patterns in the reinforcement and maintenance of NSSI. In the following chapter, I report the results of a national survey conducted to examine whether people with a recent history of NSSI endorse affect regulation as the primary function of self-injury and whether intrapersonal functions of NSSI are more commonly endorsed than interpersonal functions. Additionally, I investigate whether participants report a change in their affect or cognitions following engagement in NSSI. 170 1. INTRODUCTION The Interpretative Functional Analysis presented in the previous chapter highlights the difficulty in functionally assessing individual episodes of NSSI. Despite this complexity, it is evident that self-injury did primarily fulfil an experientially avoidant function for the participants in my first study, thus supporting the overarching premise of the EAM (Chapman et al., 2006) and providing preliminary evidence for the utility of this model within Aotearoa New Zealand. However, both aversive emotions and cognitions served to establish NSSI as a form of escape or avoidance. Furthermore, several participants described experiencing positively reinforcing, intrapersonal consequences of NSSI (e.g., an adrenalin rush) in addition to negatively reinforcing, intrapersonal consequences (e.g., relief), which is consistent with previous research (Klonsky & Glenn, 2009; Nock & Prinstein, 2004). Both negatively and positively reinforced intrapersonal consequences dominated participants’ narratives about their most recent episode of self-injury; interpersonal consequences seldom featured. When interpersonal consequences were described, participants typically reported that these consequences occurred subsequent to intrapersonal ones. The time lapse between the self-injurious behaviour and the experience of interpersonal consequences makes it less likely that these consequences served as reinforcers for self-injurious behaviour (Anderson, 2007). To build on the findings of the Interpretative Functional Analysis, I investigated whether similar functional processes could be identified in the self- injurious episodes of a larger sample of people living in Aotearoa New Zealand. For the remaining two studies of my thesis, I therefore chose to focus on quantitatively 171 testing the key assumptions of the EAM (Chapman et al., 2006), which are that NSSI functions as a negatively reinforced, experientially avoidant behaviour and that it belongs in a functional response class with other experientially avoidant behaviours (e.g., substance use). For the current study, I first hypothesised that participants would endorse affect regulation as the primary function of NSSI (Klonsky, 2009; Klonsky & Glenn, 2009). Second, I proposed that participants would endorse intrapersonal functions of NSSI more strongly than interpersonal functions (Klonsky & Glenn, 2009; Nock & Prinstein, 2004). My third hypothesis was that participants would report decreased negative affect and increased positive affect following NSSI (Klonsky, 2009). Finally, given the important role of self-referent cognitions in precipitating the self-injurious episodes I analysed in Chapter 4, I wanted to identify whether participants in the current study would report that the content of their cognitions had changed following their engagement in NSSI. 1. INTRODUCTION Given that this has never been explicitly tested using quantitative measures, I had no a priori hypotheses as to whether shifts in cognitions would occur. 24 Participants were informed that they could request a paper version of the survey if they did not want to complete it online. 2.1.1 Recruitment strategy Potential participants were invited to take part in a survey24 designed to investigate the thoughts, feelings, and events that lead people to intentionally injure themselves. I approached a number of community and mental health organisations and networks to ask them to post information about the study on their websites, weblogs, on notice boards in their offices or meeting rooms, and/or to include study information in their newsletters. The following organisations agreed to advertise or disseminate information about my study online, in print, and/or by placing posters on their notice boards: the Mental Health Foundation of New Zealand; Supporting 24 Participants were informed that they could request a paper version of the survey if they did not want to complete it online. 172 Families in Mental Illness (Auckland); Supporting Families Wairarapa; Supporting Families Otago; Supporting Families Taranaki; Male Survivors of Sexual Abuse Trust; Balance NZ; North Shore Women’s Centre; Canterbury Men's Centre; Psychiatric Consumer Trust (Christchurch); Awareness (consumer group); Warmline (peer support organisation); Bipolar Support Canterbury; Regional Consumer Network (Auckland); Connect Supporting Recovery; Werry Centre; Eating Awareness Team (Christchurch); Auckland Sexual Abuse HELP; Kapiti Women's Centre; the Problem Gambling Foundation of New Zealand; West Coast Well Women’s Centre; Rainbow Youth; and the Youth One Stop Shop (Palmerston North). Participants from my interview study who consented to be contacted about future research were also invited to complete the survey if they met the inclusion criteria. Families in Mental Illness (Auckland); Supporting Families Wairarapa; Supporting Families Otago; Supporting Families Taranaki; Male Survivors of Sexual Abuse Trust; Balance NZ; North Shore Women’s Centre; Canterbury Men's Centre; Psychiatric Consumer Trust (Christchurch); Awareness (consumer group); Warmline (peer support organisation); Bipolar Support Canterbury; Regional Consumer Network (Auckland); Connect Supporting Recovery; Werry Centre; Eating Awareness Team (Christchurch); Auckland Sexual Abuse HELP; Kapiti Women's Centre; the Problem Gambling Foundation of New Zealand; West Coast Well Women’s Centre; Rainbow Youth; and the Youth One Stop Shop (Palmerston North). Participants from my interview study who consented to be contacted about future research were also invited to complete the survey if they met the inclusion criteria. I also recruited Psychology students from universities across Aotearoa New Zealand and gifted them each a $25 prezzy card25 to put up posters about my study around their campuses. Posters were placed at the Albany and Palmerston North campuses of Massey University, Auckland University, Auckland University of Technology, Waikato University, Canterbury University, and Otago University. 25 Prezzy cards are gift cards which can be used at most stores that accept VISA. 2.1.1 Recruitment strategy I also put up posters advertising the study at various locations around the Kelburn campus of Victoria University. Depending on the format of the advert (e.g., print/email newsletter or online), participants were either invited to type the survey address into their browser or click on the link within the advertisement to be directed to the survey. Online surveys were completed through Surveymonkey. Participants were not individually reimbursed for taking part but could opt into a draw to win one of two iPod Shuffles (2GB). 173 participants so that they could make an informed choice as to whether to take p For example, participants were informed that the survey contained lots of questions about NSSI and were given the following example: ‚You will be asked to think back to the time when you most recently injured yourself on purpose, without intending to kill yourself, and to answer questions about how you felt before and after that episode‛ (see Appendix H). 2.1.2 Inclusion/exclusion criteria People were invited to participate in the study if they were 16 years of age or older, lived in Aotearoa New Zealand, and if they had injured themselves on purpose without suicidal intent, at least once in the past 12 months. They were excluded if they reported that their most recent episode of NSSI had occurred while they were experiencing psychosis (i.e., delusions or hallucinations) or mania. 2.1.3 General ethical considerations 2.1.3 General ethical considera Ethics approval for the current study was granted by the School of Psychology Human Ethics Committee at Victoria University of Wellington. Arguably, the most pressing ethical issue for the current study was the risk of participants becoming distressed as a result of survey completion and the potential for this distress to lead to self-injury. This risk was acknowledged in the information page that preceded the survey: Non-suicidal self-injury can be a very difficult topic to answer questions about and there is a risk that some of the questions asked may bring up past memories or feelings that are unpleasant or distressing. If you do become distressed while completing the survey, you can stop filling it in at any time. Also, there will be a list of options for further support at the end of the survey. Non-suicidal self-injury can be a very difficult topic to answer questions about and there is a risk that some of the questions asked may bring up past memories or feelings that are unpleasant or distressing. If you do become distressed while completing the survey, you can stop filling it in at any time. Also, there will be a list of options for further support at the end of the survey. 2.1.4 Ethical issues specific to Internet Mediated Research Given that participation in the study involved completing an online survey, concerns specific to Internet Mediated Research (IMR; British Psychological Society, 2007) had to be addressed. The following three IMR issues were identified as pertinent to this research: ensuring that participants met the inclusion criteria, 174 debriefing participants who exited the survey before finishing it, and potential breaches of confidentiality (British Psychological Society, 2007; Kraut et al., 2004). 2.1.4.1 Meeting the inclusion criteria One of the risks associated with IMR is the inability to verify that participants meet the designated inclusion criteria (Kraut et al., 2004). To minimise the risk of people, who did not meet the inclusion criteria, participating in this study, question skip logic was used to direct respondents to specific pages based on their answers. When potential participants clicked on the survey link, they initially were directed to an information page about the study, which listed the inclusion/exclusion criteria. One of the risks associated with IMR is the inability to verify that participants meet the designated inclusion criteria (Kraut et al., 2004). To minimise the risk of people, who did not meet the inclusion criteria, participating in this study, question skip logic was used to direct respondents to specific pages based on their answers. When potential participants clicked on the survey link, they initially were directed to an information page about the study, which listed the inclusion/exclusion criteria. After clicking ‘next’, they were then directed to a second page which relisted the four inclusion/exclusion criteria and asked: ‚Are ALL FOUR of these statements true for you?‛ If respondents clicked yes, they were directed to the next page of the survey, but if they clicked no they were directed to a page that contained the following information: ‚Thank you for your time. Unfortunately, you do not meet the criteria to participate in this survey. Please close your browser (e.g., Internet Explorer, Firefox) to exit this survey.‛ 2.1.4.2 Exiting the survey before being debriefed 2.1.4.3 Protecting anonymity Participants were assured on the information page that their survey responses would remain anonymous and would not be linked to their email addresses. To ensure anonymity, once participants entered their email addresses and clicked ‘next’, they were directed to a separate survey where they were not required to enter any identifying information. Additionally, to protect participants’ anonymity and privacy, their Internet Protocol addresses were not recorded. 2 2 Participants 2.1.4.2 Exiting the survey before being debriefed Another risk of IMR is that respondents may choose not to finish the survey, preventing them from accessing any debriefing information because it is typically presented at the end of research participation (Kraut et al., 2004). To address this concern, when people confirmed that they met the inclusion criteria for the study they were required to enter their email address before being allowed to proceed to the survey questions. Participants were informed that their email addresses were compulsory in order to send them a copy of the debriefing information (see Appendix J). This was not a sophisticated solution but survey functionality precluded the use of a pop-up debriefing window, which is a recommended safety strategy to ensure participants who close the survey early are adequately debriefed (British Psychological Society, 2007). Unfortunately, this method was not full-proof as one 175 person entered an incorrect email address and another person simply typed in a series of letters. 2.2.1 Flow of participants through the study Figure 5 depicts the flow of participants through the online version of the study. Some people (N = 45) indicated that they met the inclusion criteria but chose not to complete the survey. There are a number of reasons why people may have decided against proceeding to the survey questions. After reading the information sheet, they may have decided that filling in the survey would be upsetting or perhaps that they simply were not interested in taking part. Alternatively, the time required for survey completion, which was listed as being no more than 45 minutes, may have deterred people from participating. However, it is also possible that people may have revisited the site later when they did have the time available to complete the survey. Requiring people to type in their email address before being directed to the survey questions may have been another deterrent. Although people were informed that their email addresses would not be linked to their survey answers, this may not have been sufficient to reassure some people that their responses would be anonymous. Given that self-injury is usually a secretive behaviour, other people may not have wanted their email addresses to be connected to a study about NSSI. As is evident in Figure 5, the number of people (N = 197) who consented to participate in the survey by providing their email address was one less than the 176 Figure 5. Flow of participants through the online survey. 2.2.1 Flow of participants through the study Self-excluded: did not meet criteria (N = 12) Self-assessed: met inclusion criteria (N = 246) Excluded (N = 49)  Did not enter email address (N = 45)  Repeat entries (N = 4) Implied consent: entered email address for debriefing (N = 197) Excluded (N = 36)  Spoiled survey (N = 1)  ≥ 75% of survey incomplete (N = 9)  Did not meet study definition of NSSI (N = 3)  Had not self-injured within past 12 months (N = 22)  Pervasive Developmental Disorder diagnosis (N = 1) Included in general NSSI dataset (N = 162) Excluded (N = 10)  Did not meet study definition of NSSI for most recent episode (N = 7)  100% of most recent episode sections incomplete (N = 3) Included in most recent NSSI dataset (N = 152) Consent process General NSSI Most recent episode Total online participants (N = 198) Excluded: repeat entries (N = 10) Read information sheet (N = 258) Total started survey (N = 208) Self-excluded: did not meet criteria (N = 12) Self-assessed: met inclusion criteria (N = 246) Excluded (N = 49)  Did not enter email address (N = 45)  Repeat entries (N = 4) Implied consent: entered email address for debriefing (N = 197) Total started survey (N = 208) Excluded: repeat entries (N = 10) Total online participants (N = 198) General NSSI Included in general NSSI dataset (N = 162) Excluded (N = 10)  Did not meet study definition of NSSI for most recent episode (N = 7)  100% of most recent episode sections incomplete (N = 3) Figure 5. Flow of participants through the online survey. 177 number of people (N = 198) who actually started the survey. Although this could be a repeat entry which I have not detected, this is unlikely because I thoroughly compared entries using demographic data (e.g., gender, age, and region) to ensure I removed multiple entries completed by the same people. Rather, it is possible that the question skip logic malfunctioned and directed one person through to the survey even though they had not entered their email address. People were also informed that they could have a paper version of the survey if they preferred to fill in a hard copy. 2.2.1 Flow of participants through the study Only three people requested a paper copy of the survey and only one of these surveys was returned to me. This response was added to the dataset, which resulted in a total of 163 participants. All of the quantitative data analyses reported in this chapter were conducted using SPSS PASW Statistics (v. 18). 2.2.2 Demographics and descriptive characteristics of the sample The participants ranged in age from 16 to 62 years (M = 24.64, SD = 8.48) and were predominantly female (81.6%), with 27 (16.6%) male and 3 (1.8%) transgender participants. Participants belonged to the following ethnic group(s): 134 (82.2%) New Zealand European, 13 (8.0%) Māori, 1 (0.6%) Samoan, 3 (1.8%) Chinese, 4 (2.5%) Indian, and 28 (17.2%) other ethnicities. People from every region in Aotearoa New Zealand participated in the study: 54 (33.1%) from Auckland, 42 (25.8%) from Wellington, 23 (14.1%) from Canterbury, 18 (11.0%) from Waikato, 10 (6.1%) from Manawatu-Wanganui, 2 (1.2%) from the Bay of Plenty, 9 (5.5%) from Otago, and 1 (0.6%) person each from Northland, the East Cape, Hawke’s Bay, Marlborough, and Southland. Over half of the sample (60.7%) reported their current occupation as tertiary student, but there were a range of other occupations as well: 5 (3.1%) were secondary school students, 13 (8.0%) were in part-time employment, 21 (12.9%) were in full- time employment, 20 (12.3%) were unemployed, 4 (2.5%) were stay-at-home parents and 1 (0.6%) person identified their occupation as other. The high number of tertiary students in the sample was reflected in the levels of education attained by 178 participants: 9 (5.5%) had no qualifications, 90 (55.2%) had a high school qualification, 4 (2.5%) had a trade/technical qualification, 38 (23.3%) had a degree or diploma, 13 (8.0%) had a postgraduate qualification, and 9 (5.5%) had other qualifications. Participants’ sexual orientation, which was measured as a single-item variable based on Savin-Williams and Ream’s (2007) conceptualisation, was as follows: 90 (55.2%) heterosexual, 38 (23.3%) mostly heterosexual, 15 (9.2%) bisexual, 10 (6.1%) mostly homosexual, 5 (3.1%) homosexual, and 5 (3.1%) asexual. Two thirds of the sample (66.3%) reported receiving at least one mental health diagnosis, with the following diagnoses listed in order of highest frequency: 95 (58.3%) Depression, 41 (25.2%) Anxiety, 32 (19.6%) PTSD, 24 (14.7%) BPD, 16 (9.8%) Bipolar Disorder, 15 (9.2%) Anorexia, 14 (8.6%) Bulimia, 9 (5.5%) OCD, 5 (3.1%) Substance Use Disorders, 1 (0.6%) Schizophrenia, and 8 (4.9%) other diagnoses. Among those who endorsed being diagnosed with one or more mental health disorders, the number of diagnoses reported ranged from one to six, with an average of 2.41 (SD = 1.28) diagnoses per person. This average may be slightly inflated due to the inclusion of PTSD and OCD as separate categories to anxiety disorders. 2.2.2 Demographics and descriptive characteristics of the sample The rationale for this demarcation was to ensure that people did not underreport these diagnoses if they were unaware that they qualify as anxiety disorders. Finally, I used the first item of the Suicidal Behaviors Questionnaire-Revised (Osman et al., 2001) to assess lifetime suicidality (i.e., ideation and attempts). Finally, I used the first item of the Suicidal Behaviors Questionnaire-Revised (Osman et al., 2001) to assess lifetime suicidality (i.e., ideation and attempts). Participants were asked to choose one statement out of six that best applied to their experiences of suicidality. The majority of the participants had a history of suicidal ideation or behaviours; only 9 (5.5%) had never thought about or attempted to kill themselves. Twenty-three (14.1%) participants had experienced fleeting suicidal thoughts, 26 (16.0%) had made a plan at least once to complete suicide but had not tried to kill themselves, 36 (22.1%) had made a plan at least once and had really wanted to die, 25 (15.3%) had attempted to kill themselves but did not want to die, Finally, I used the first item of the Suicidal Behaviors Questionnaire-Revised (Osman et al., 2001) to assess lifetime suicidality (i.e., ideation and attempts). 179 while 44 (27.0%) of the participants had attempted suicide and had really hoped to die. while 44 (27.0%) of the participants had attempted suicide and had really hoped to die. 2.3.1 The Deliberate Self-harm Inventory A modified 15-item version of the DSHI (Gratz, 2001) was used to assess participants’ lifetime prevalence of different types of NSSI. Modifications were made to shorten the measure and to address the issues that I discussed in the previous chapter (see pp. 101-105). Instead of asking about the age of onset, frequency, recency, and medical severity of each type of behaviour, I simply asked participants to endorse whether they had engaged in a particular behaviour, and, if yes, to then report the number of times they had engaged in that behaviour. Furthermore, after asking people how many times they had engaged in each type of self-injury I added: ‚If you can’t remember, please estimate the number of times (e.g., 5, 10, 100) you have done this‛ (see Appendix H). Other modifications included collapsing some of the questions into single items (Lundh et al., 2007). Specifically, questions two (which asks about burning yourself with a cigarette) and three (which asks about burning yourself with a lighter or match) were collapsed into a single question, which read as follows: ‚Have you ever intentionally (i.e., on purpose) burned yourself with a cigarette, lighter, or a match?‛ Similarly, questions four (which asks about carving words into your skin) and five (which asks about carving pictures, designs, or other marks into your skin) were collapsed into the following single question: ‚Have you ever intentionally (i.e., on purpose) carved words, pictures, designs, or other marks into your skin?‛ The word ‚comet‛ (item 10) was once again excluded. 2.3.2 Inventory of Statements About Self-injury The ISAS (Klonsky & Glenn, 2009) was chosen to assess the functions of participants’ NSSI because it is the most comprehensive functional assessment measure of self-injury available. It consists of two sections, each of which can be used independently. Section one assesses the frequency of different types of NSSI 180 and contextual factors surrounding these behaviours, while section two assesses the functions of non-suicidal self-injurious behaviours. Only section two was used in this study. Section two of the ISAS is comprised of an index question (i.e., ‚When I self- harm, I am<‛) followed by 39 reasons for NSSI. Participants are invited to respond to each statement on a 3-point likert scale according to how relevant it is to their experience of NSSI (i.e., not relevant, somewhat relevant, or very relevant). The statements are grouped into the following 13 functions subscales (3 items per subscale): affect regulation (e.g., ‚<calming myself down‛), interpersonal boundaries (e.g., ‚<creating a boundary between myself and others‛), self-punishment (e.g., ‚<punishing myself‛), self-care (e.g., ‚<giving myself a way to care for myself by attending to the wound‛), anti-dissociation/feeling generation (e.g., ‚<causing pain so I will stop feeling numb‛), anti-suicide (e.g., ‚<avoiding the impulse to attempt suicide‛), sensation-seeking (e.g., ‚<doing something to generate excitement or exhilaration‛), peer-bonding (e.g., ‚<bonding with peers‛), interpersonal influence (e.g., ‚<letting others know the extent of my emotional pain‛), toughness (e.g., ‚<seeing if I can stand the pain‛), marking distress (e.g., ‚<creating a physical sign that I feel awful‛), revenge (e.g., ‚<getting back at someone‛), and autonomy (e.g., ‚<ensuring that I am self-sufficient‛). For the purposes of this study, I changed the words ‚self-harm‛ to ‚self- injury‛ in both the instructions for section two and the index question. The instructions thus read as follows: ‚This inventory was written to help us better understand the experience of non-suicidal self-injury. Below is a list of statements that may or may not be relevant to your experience of self-injury‛. The index question read as, ‚‚When I self-injure, I am<‛. These modifications were made to ensure that the terminology (i.e., NSSI) that I used remained consistent throughout my survey. Given that the word self-harm typically refers to all self-injurious behaviours, regardless of suicide intent, using self-harm interchangeably with self- injury may have confused participants and confounded these two phenomena. 2.3.2 Inventory of Statements About Self-injury 181 Although the ISAS was developed to measure the general functions of NSSI, I also wanted to examine the frequency and type of functions endorsed by participants for a single episode of NSSI. To achieve this, I invited participants to complete the ISAS twice at different points in the survey; once in relation to their general experiences of NSSI and once in relation to their most recent episode of NSSI. The instructions for the latter version were amended to reflect the requirement that participants answer only in relation to their most recent episode (see Appendix H). 2.3.3 Positive and Negative Affect Schedule The expanded form of the Positive and Negative Affect Schedule (PANAS-X; Watson & Clark, 1994) was used to investigate whether participants reported changes in emotions following self-injury. The PANAS-X (Watson & Clark, 1994) is a 60-item measure, with 13 scales that assess positive, negative, and other affect states. Items are rated on a 5-point scale from 1 (very slightly or not at all) to 5 (extremely). There are two general dimension scales with 10 items in each: negative affect (afraid, scared, nervous, jittery, irritable, hostile, guilty, ashamed, upset, and distressed) and positive affect (active, alert, attentive, determined, enthusiastic, excited, inspired, interested, proud, and strong). The four basic negative emotion scales include: fear (afraid, scared, frightened, nervous, jittery, and shaky), hostility (angry, hostile, irritable, scornful, disgusted, and loathing), guilt (guilty, ashamed, blameworthy, angry at self, disgusted with self, and dissatisfied with self) and sadness (sad, blue, downhearted, alone, and lonely). The three basic positive emotion scales are: joviality (happy, joyful, delighted, cheerful, excited, enthusiastic, lively, and energetic), self-assurance (proud, strong, confident, bold, daring, and fearless), and attentiveness (alert, attentive, The three basic positive emotion scales are: joviality (happy, joyful, delighted, cheerful, excited, enthusiastic, lively, and energetic), self-assurance (proud, strong, confident, bold, daring, and fearless), and attentiveness (alert, attentive, concentrating, and determined). Finally, the other affective states included are: shyness (shy, bashful, sheepish, and timid), fatigue (sleepy, tired, sluggish, and drowsy), serenity (calm, relaxed, and at ease), and surprise (amazed, surprised, and astonished). As one of the most widely used measures of affect, the PANAS-X has confident, bold, daring, and fearless), and attentiveness (alert, attentive, concentrating, and determined). Finally, the other affective states included are: shyness (shy, bashful, sheepish, and timid), fatigue (sleepy, tired, sluggish, and drowsy), serenity (calm, relaxed, and at ease), and surprise (amazed, surprised, and astonished). As one of the most widely used measures of affect, the PANAS-X has 182 been extensively tested and consistently demonstrates excellent psychometric properties (Watson & Clark, 1994). Although the PANAS-X (Watson & Clark, 1994) can be applied to multiple time-frames (e.g., past week, in general), it is not typically used to evaluate the affective experience associated with a particular behaviour. However, as I discussed in Chapter 2, one of the limitations of self-injury research is the tendency for researchers to use unvalidated measures. 2.3.3 Positive and Negative Affect Schedule It was therefore deemed preferable to use the PANAS-X and amend the instructions (see Appendix H), rather than create a new affect measure specifically for this study, especially in light of the observation by Watson and Clark (1994) that varying the time instructions does not appear to influence the scales’ reliabilities. For the purposes of this study, I also modified the anchor point 1 to read not at all rather than very slightly or not at all as I wanted the lowest rating to reflect an absence of the emotion being measured. 2.3.4 Revised Automatic Thoughts Questionnaire The revised Automatic Thoughts Questionnaire (ATQ-R; Kendall, Howard, & Hays, 1989) is a 40-item measure that contains 30 negative self-statements (e.g., ‚I wish I were a better person‛) and 10 positive/neutral self-statements (e.g., ‚I’m proud of myself‛). I included the ATQ-R in the survey because the role of negative thoughts in precipitating self-injurious behaviour is seldom investigated and such thoughts were identified as self-injury antecedents in the first study of this thesis. The ATQ-R is designed to assess the frequency of automatic thoughts within the past week and, as such, using this measure to retrospectively identify thoughts before and after episodes of self-injury is unorthodox. However, in the absence of measures designed specifically for this purpose, I chose to use the ATQ-R for a series of exploratory analyses. Participants were asked to identify whether or not specific thoughts had occurred before and/or after their most recent episode of NSSI by simply responding yes or no. 183 3. QUANTITATIVE RESULTS 3.1 Characteristics of participants’ self-injurious behaviour 2.4 Open-ended questions Participants were also asked a series of open-ended questions to enable them to describe the antecedents and consequences of their most recent episode of NSSI in their own words. This qualitative component was incorporated to add depth and scope to my understanding of the events that may establish the conditions for self- injury and the consequences that may subsequently reinforce NSSI. To determine whether a specific event had led to their self-injury, they were asked: ‚Did something specific happen that led to this most recent episode of self-injury?‛ If they answered yes, then they were asked to describe what had happened, when it happened, and how it made them feel. At the end of the survey, participants were asked to describe up to five consequences of their most recent episode of NSSI and to evaluate whether they had experienced these consequences as positive, negative, or neutral. Consequences were defined within the question as anything that had happened after the person injured themselves that was caused by the self-injury. That is, the consequences would not have occurred in the absence of the self-injurious episode. This definition is consistent with operant principles, which define behavioural consequences according to their impact on the environment (Sturmey et al., 2007a). 3.1 Characteristics of participants’ self-injurious behaviour Despite simplifying the DSHI (Gratz, 2001) and explicitly stating that participants could provide estimates, some of the responses about the frequency and other types of NSSI were still challenging to interpret. Once again, I chose to err on the side of caution by including the lowest frequencies provided. For example, non- numeric frequencies (e.g., countless) that were too difficult to quantify were treated as missing data; 10?, 10’ish, or 10+ were included in the data set as 10; 100-200 as 100; less than 10 as 9; a few times as 2; and hundreds of times as 100. I also adhered as closely as possible to Gratz’s (2001) definition of NSSI, which specifies that the behaviour results in tissue damage, when deciphering what 184 qualified as a form of other NSSI. This was difficult because what ‘counts’ as NSSI varies considerably between studies, particularly in relation to self-poisoning (e.g., drinking toxic substances or overdosing) and hair-pulling. I chose to exclude both of these forms of self-injury; self-poisoning because of its ambiguous relationship to suicide (Brown et al., 2004; Freedenthal, 2007) and hair-pulling because it is currently classified within the DSM-IV-TR (American Psychiatric Association, 2000) as trichotillomania. Furthermore, people did not always state the severity of their self- poisoning or hair-pulling behaviours and, as a result, it was unclear whether tissue damage resulted from these behaviours. When reporting types of NSSI within the other category, some participants disclosed that tissue damage had occurred (e.g., ‚rubbed a key over my skin – forming large graze‛, ‚slammed hand in a door repeatedly, intending to break it but only soft tissue injury‛), while others listed behaviours where tissue damage was implied but not directly stated (e.g., ‚dislocated my fingers/thumbs‛, ‚poured cup of boiling water onto arms‛), or highly probable because of the type of injury (e.g., ‚threw myself down the stairs‛, ‚swallowed glass‛) . All behaviours where tissue damage was directly stated, implied, or highly probable were included. Behaviours where the occurrence of tissue damage was ambiguous (e.g., slapping oneself) were excluded. Finally, some participants reported other self-injury where the form, but not the implement used to carry out the injury, was listed in the DSHI. For example, the DSHI specifies burning oneself with a cigarette, lighter, or match. Some participants thus reported burning themselves with petrol, an iron, or hair straighteners in the other self-injury question. 3.1 Characteristics of participants’ self-injurious behaviour These responses were included in the dataset, which may have resulted in a slight inflation in the types of NSSI reported by participants. Note. N reporting NSSI type may not equal frequencies of engagement as some participants endorsed type of NSSI but did not report frequency. Frequencies may not add up to 100% because of missing data. 3.1.1 Global NSSI episodes The mean age of onset for NSSI was 14.45 years (SD = 4.42) and ranged from 4 to 41 years of age. Participants reported having engaged in an average of 5.75 (SD = 2.66) different types of NSSI, although this ranged from one to 14 types. As can be Table 4 Frequencies of different types of NSSI Frequencies of different types of NSSI NSSI type N (%) reporting NSSI type Frequency of participants’ engagement in NSSI types Never 1 time 2-10 times 11-50 times >50 times Cutting wrists, arms, or other areas of body 144 (88.3%) 19 (11.7%) 0 (0.0%) 39 (23.9%) 42 (25.8%) 62 (38.0%) Severe scratching to extent of bleeding/scarring 116 (71.2%) 46 (28.2%) 3 (1.8%) 59 (36.2%) 31 (19.0%) 20 (12.3%) Sticking sharp objects into skin 102 (62.6%) 60 (36.8%) 1 (0.6%) 64 (39.3%) 27 (16.6%) 7 (4.3%) Carving words/pictures/designs/marks into skin 97 (59.5%) 64 (39.3%) 13 (8.0%) 65 (39.9%) 16 (9.8%) 3 (1.8%) Preventing wounds from healing 98 (60.1%) 65 (39.9%) 1 (0.6%) 27 (16.6%) 36 (22.1%) 25 (15.3%) Punching to extent of bruising 86 (52.8%) 78 (47.9%) 9 (5.5%) 42 (25.8%) 21 (12.9%) 8 (4.9%) Banging head to extent of bruising 72 (44.2%) 90 (55.2%) 8 (4.9%) 42 (25.8%) 17 (10.4%) 2 (1.2%) Burning with cigarette/lighter/match 71 (43.6%) 92 (56.4%) 4 (2.5%) 46 (28.2%) 16 (9.8%) 3 (1.8%) Biting to extent of breaking skin 57 (35.0%) 105 (64.4%) 13 (8.0%) 30 (18.4%) 13 (8.0%) 1 (0.6%) Rubbing glass into skin 29 (17.8%) 133 (81.6%) 6 (3.7%) 15 (9.2%) 6 (3.7%) 2 (1.2%) Rubbing sandpaper on body 12 (7.4%) 149 (91.4%) 3 (1.8%) 7 (4.3%) 1 (0.6%) 1 (0.6%) Dripping acid onto skin 6 (3.7%) 155 (95.1%) 4 (2.5%) 1 (0.6%) 0 (0.0%) 0 (0.0%) Breaking own bones 5 (3.1%) 156 (95.7%) 4 (2.5%) 1 (0.6%) 0 (0.0%) 0 (0.0%) Using bleach/ oven cleaner to scrub skin 6 (3.7%) 157 (96.3%) 0 (0.0%) 4 (2.5%) 0 (0.0%) 1 (0.6%) Note. N reporting NSSI type may not equal frequencies of engagement as some participants endorsed type of NSSI but did not report frequency. Frequencies may not add up to 100% because of missing data. Frequency of participants’ engagement in NSSI types 185 186 seen in Table 4, the most common form of self-injury was cutting, which was endorsed by 88.3% of the participants, followed by severe scratching to the extent of bleeding or scarring (71.2%); sticking sharp objects into one’s skin (62.6%); and carving words, pictures, designs, or other marks into one’s skin (59.5%). The least common forms of self-injury were dripping acid onto one’s skin, breaking one’s own bones, and using bleach or oven cleaner to scrub one’s skin; only 3% of the participants endorsed engaging in these types of NSSI one or more times. Frequencies of different types of NSSI Additionally, 37 (22.7%) of the participants reported having engaged in at least one other form of self-injury, such as swallowing glass, punching walls, and dislocating fingers. Similarly to the DSHI (Gratz, 2001) data presented in Chapter 4, the frequencies of NSSI behaviours in the current study were grouped into five categories (see Table 4). 3.1.2 Most recent NSSI episode To assess how participants had self-injured most recently, they were presented with a list of self-injurious behaviours identical to those in the modified version of the DSHI (Gratz, 2001), which was described in section 2.4.1, and asked to endorse any behaviours that applied to their episode. They were asked to identify whether the injury had been severe enough to require medical treatment, if they told anyone that they had self-injured, and whether they had used drugs or alcohol prior to self-injuring. The frequencies of NSSI types for participants’ most recent episode of self-injury are presented in Table 5. Participants reported engaging in a range of NSSI types for their most recent episode of self-injury, with the most common type being cutting (65.6%), followed by severe scratching (21.5%) and carving words, pictures, designs, or other marks into the skin (16.0%). The least commonly endorsed types of NSSI were biting (4.3%), rubbing sandpaper on the body (1.2%), and scrubbing the skin with bleach or oven cleaner (0.6%). A further six (3.7%) people reported engaging in other forms of NSSI (e.g., punching walls) for their most recent episode. No-one endorsed injuring 187 themselves most recently by rubbing glass into, or dripping acid onto, their skin or by breaking their own bones. Frequencies of different types of NSSI for participants’ most recent episode Frequencies of different types of NSSI for participants’ most recent episode NSSI type N (%) Cutting wrists, arms, or other areas of body 107 (65.6%) Severe scratching to extent of bleeding/scarring 35 (21.5%) Carving words/pictures/designs/marks into skin 26 (16.0%) Punching to extent of bruising 20 (12.3%) Sticking sharp objects into skin 16 (9.8%) Preventing wounds from healing 16 (9.8%) Banging head to extent of bruising 13 (8.0%) Burning with cigarette/lighter/match 10 (6.1%) Biting to extent of breaking skin 7 (4.3%) Rubbing sandpaper on body 2 (1.2%) Using bleach/oven cleaner to scrub skin 1 (0.6%) Rubbing glass into skin 0 (0.0%) Dripping acid onto skin 0 (0.0%) Breaking own bones 0 (0.0%) Note. Frequencies total more than 100% because some participants reported using multiple types of NSSI during their most recent episode. Note. Frequencies total more than 100% because some participants reported using multiple types of NSSI during their most recent episode. Note. Frequencies total more than 100% because some participants reported using multiple types of NSSI during their most recent episode. 3.1.2 Most recent NSSI episode On average, people reported using 1.69 (SD = 1.04) types of self-injury during their most recent episode; this ranged from one to six types. The mean number of days that had elapsed between participants completing the survey and their most recent episode of NSSI was 81.06 (SD = 85.20) with a range of zero to 359 days. Approximately 15% of participants reported that their injury was severe enough to require medical treatment but it is unknown whether they actually received treatment. On average, people reported using 1.69 (SD = 1.04) types of self-injury during their most recent episode; this ranged from one to six types. The mean number of days that had elapsed between participants completing the survey and their most recent episode of NSSI was 81.06 (SD = 85.20) with a range of zero to 359 days. Approximately 15% of participants reported that their injury was severe enough to require medical treatment but it is unknown whether they actually received treatment. Almost a third of the participants (30.1%) told someone that they had hurt themselves on purpose; 20 (12.3%) participants told their mental health clinician (e.g., psychologist, social worker), 19 (11.7%) told a friend, and seven (4.3%) told their partner or spouse. A few participants told their health professional (3.7%), boyfriend/girlfriend (2.5%), others such as an online friend (2.5%), a co-worker (1.8%), parent (1.8%), sibling (1.2%), or another relative, teacher/lecturer, or 188 acquaintance (all 0.6%). The majority of participants did not consume more than two standard drinks of alcohol (74.8%), take in excess of a recommended dosage of medication (85.9%), or use illegal drugs (91.4%) before injuring themselves. 3.2 Is affect regulation the primary function of NSSI? Descriptive statistics and reliability coefficients for the ISAS (Klonsky & Glenn, 2009) in reference to participants’ global and most recent NSSI episodes are presented in Table 6. I have tabulated these results side-by-side to allow for ease of comparison, but will discuss the results for the global and most recent episodes of NSSI separately. 3.2.1 Self-reported functions of participants’ global NSSI episodes As is evident from the skewness and kurtosis values presented in Table 6, visual inspection of the histograms and Q-Q plots of each of the 13 ISAS subscales indicated that none of the data was normally distributed. Rather, the majority of the subscales were skewed to the right. Kolmogorov-Smirnov tests with Lilliefor’s correction (Field, 2009) confirmed that all subscales were significantly non-normal, D’s(162-163) ≥ .13 (p’s < .001). Given that the assumption of normality was violated for all subscales, non-parametric statistics were used. Prior to beginning the non-parametric analyses, I calculated the means and standard deviations of each of the subscales to allow for comparisons with other studies. Affect regulation (M = 4.65, SD = 1.61), self-punishment (M = 4.17, SD = 1.90), and marking distress (M = 2.80, SD = 1.97) were the most highly endorsed functions, while sensation-seeking (M = 0.75, SD = 1.17), revenge (M = 0.66, SD = 1.22), and peer-bonding (M = 0.15, SD = 0.57) were the least endorsed functions. Comparable results were obtained through other measures of central tendency—the median and mean rank—that do not rely on the assumption of normality. For example, affect regulation and self-punishment both had the highest median of 5 with mean ranks of 11.43 and 10.94 respectively. The third most endorsed item, marking distress, had a median of 3 and a mean rank of 8.98. ( ) p ( ) p a-l Mean ranks with the same superscripts indicate subscales that do not differ significantly from one ano g p p p p All D’s(162-163) >.13, p’s < .001; * All D’s(140) >.15, p’s < .001. he data in the global episodes section of the table was based on 163 participants apart from the Autonomy D’s(162-163) >.13, p’s < .001; *** All D’s(140) >.15, p’s < .001. Note. * All the data in the global episodes section of the table was based on 163 participants apart a in the global episodes section of the table was based on 163 participants apart from the Autonomy subscale which was based on 162 participants. 2 163) > 13 ’ < 001 *** All D’ (140) > 15 ’ < 001 15, p s < .001. subscales that do not differ significantly from one another. All other subscales were significantly different. * All the data in the global episodes section of the table was based on 163 participants apart from the Auto All D’s(162-163) >.13, p’s < .001; * All D’s(140) >.15, p’s < .001. A ( ) , p 00 ; A ( 0) , p 00 a-l Mean ranks with the same superscripts indicate subscales that do not differ significantly from one another. All other subscales were significantly different. . * All the data in the global episodes section of the table was based on 163 participants apart from the Autonomy subscale which was based on 162 participants. All D’s(162-163) >.13, p’s < .001; * All D’s(140) >.15, p’s < .001. ) , p ; ( ) , p with the same superscripts indicate subscales that do not differ significantly from one another. All other sub ble was based on 163 participants apart from the Autonomy subscale which was based on 162 participants. 15 p’s < 001 3.2.1 Self-reported functions of participants’ global NSSI episodes Table 6 Descriptive statistics and reliability coefficients for the ISAS in reference to participants’ global and most recent episodes of NSSI Descriptive statistics and reliability coefficients for the ISAS in reference to participants’ global and most recent episodes of NSSI Global episodes (N = 163)* Most recent episode (N = 140) ISAS subscale (score range 0-6) Mean (SD) Median Cronbach’s α Skewness Kurtosis Mean Rank Mean (SD) Median Cronbach’s α Skewness Kurtosis Mean Rank Affect regulation 4.65 (1.61) 5.00 .73 -1.23 0.72 11.43a 4.61 (1.72) 5.00 .74 -1.22* 0.60 11.48g Self-punishment 4.17 (1.90) 5.00 .82 -.08 -0.56 10.94a 3.94 (2.13) 5.00 .88 -0.72* -0.87 10.51g Marking distress 2.80 (1.97) 3.00 .78 0.14 -1.12 8.98b 2.68 (1.96) 2.00 .73 0.28* -1.05 9.31h Anti-dissociation/ feeling generation 2.70 (2.17) 3.00 .86 0.20 -1.34 8.56b 2.10 (2.27) 1.00 .91 0.62* -1.12 7.70h Anti-suicide 2.23 (1.97) 2.00 .84 0.47 -0.83 7.71b 2.03 (2.27) 1.00 .93 0.67* -1.01 7.50h,i Self-care 1.50 (1.48) 1.00 .60 0.81 -0.26 6.69c 1.36 (1.53) 1.00 .65 1.07* 0.54 6.92i,j Toughness 1.28 (1.54) 1.00 .73 1.13 0.42 6.21c 1.06 (1.55) 0.00 .85 1.51* 1.68 6.30j Interpersonal influence 1.17 (1.46) 0.00 .68 1.06 0.24 6.00c,d 0.90 (1.32) 0.00 .70 1.59* 2.20 6.02j,k,l Interpersonal boundaries 1.16 (1.51) 1.00 .77 1.40 1.51 6.02c,d 0.88 (1.47) 0.00 .80 1.81* 2.66 5.79j,k,l Autonomy 0.77 (1.17) 0.00 .68 1.50 1.42 5.06d,e 0.62 (1.20) 0.00 .79 2.41* 6.21 5.40k,l Sensation-seeking 0.75 (1.17) 0.00 .59 1.78 3.37 5.12d,e 0.53 (1.20) 0.00 .79 2.75* 7.88 4.97lk,l Revenge 0.66 (1.22) 0.00 .82 2.02 3.94 4.77e 0.60 (1.36) 0.00 .91 2.50* 5.76 5.04k,l Peer-bonding 0.15 (0.57) 0.00 .63 4.90 27.00 3.52 0.16 (0.84) 0.00 .92 5.97* 36.90 4.07 Note. * All the data in the global episodes section of the table was based on 163 participants apart from the Autonomy subscale which was based on 162 participants. All D’s(162-163) >.13, p’s < .001; * All D’s(140) >.15, p’s < .001. Global episodes (N = 163)* Most recent episode (N = 140) 189 190 Sensation-seeking, revenge, and peer-bonding all had medians of 0 and low mean ranks. The subscales demonstrated questionable to good internal consistency (α ranged from .60 for self-care to .86 for anti-dissociation/feeling generation), with the exception of sensation-seeking which demonstrated poor internal consistency (α = .59). 3.2.1 Self-reported functions of participants’ global NSSI episodes To determine whether there were any significant differences between the ISAS subscales, I conducted analyses using the Friedman Test (N = 162) and post-hoc Wilcoxon signed-rank tests (i.e., non-parametric alternatives to ANOVA and t-tests). Results indicated that at least one of the subscales was significantly different from at least one other subscale, χ2(12) = 855.36, p < .001. Given the number of post-hoc comparisons, a Bonferroni corrected alpha of p < .001 was used to control for family- wise error. The results of these comparisons are reported in Table 6. Affect regulation and self-punishment, although not different to each other, differed significantly from all of the other subscales. Marking distress, anti-suicide, and anti-dissociation/feeling generation did not differ, but were all significantly different to the other 10 subscales. Self-care was not significantly different to toughness, interpersonal influence, and interpersonal boundaries, while autonomy was not significantly different to interpersonal influence, interpersonal boundaries, sensation-seeking, and revenge. Peer-bonding was the only subscale that was significantly different to all of the other subscales. Effect sizes were calculated by dividing Z by the square root of the number of observations (Field, 2009). For significantly different subscales, the effect sizes ranged from r = .21 (anti-suicide compared with self-care) to r = .61 (affect regulation compared with peer-bonding). 3.2.2 Self-reported functions of participants’ most recent NSSI episode Visual inspection of the data (i.e., histograms and Q-Q plots), along with the means and standard deviations of the ISAS subscales for participants’ most recent episode of NSSI, showed that it was predominantly skewed to the right. The skewness and kurtosis values for each of the subscales are presented in Table 6; the 191 distributions of all of these subscales were significantly non-normal, D’s(140) ≥ .15 (p’s < .001). While the means and mean ranks for the functions of participants’ most recent episode were similar to those of the global functions they endorsed, the medians tended to be slightly lower. Lower medians are to be expected given that the participants were rating the functions of one specific episode of NSSI as opposed to their general experience of self-injury. The most highly endorsed functions for global episodes of NSSI—affect regulation, self-punishment, and marking distress—were also the most highly endorsed functions for participants’ most recent episode. Mean ranks for affect regulation and self-punishment were 11.48 and 10.51 respectively, and they both had medians of five. 3.2.1 Self-reported functions of participants’ global NSSI episodes Marking distress, the third most endorsed function, had a mean rank of 9.31 and a substantially lower median of 2. Peer bonding was once again the least endorsed function with a mean rank of 4.07 and a median of 0. Sensation- seeking (mean rank = 4.97, median = 0) and revenge (mean rank = 5.04, median = 0) followed peer-bonding as the second and third least endorsed functions. This pattern of results differed only slightly from that obtained in relation to participants’ global episodes of NSSI, where sensation-seeking was the third, not the second, least endorsed item. Apart from this discrepancy, the order in which the functions were endorsed was identical for participants’ global and most recent episodes of NSSI. The internal consistencies of the ISAS subscales for participants’ most recent episode of self-injury tended to be higher than for global episodes, and ranged from questionable (α = .65 for self-care) to excellent (α = .93 for anti-suicide). The Friedman Test was used to determine whether there were any significant differences between the functions endorsed for participants’ most recent NSSI episode. Results showed a significant difference in participants’ (N = 140) endorsement of the subscales, χ2(12) = 697.74, p < .001. Post-hoc Wilcoxon signed- rank tests with a Bonferroni corrected alpha of p < .001 revealed a similar pattern of results to that obtained for global episodes of NSSI (see Table 6). 192 Peer-bonding was once again the only subscale that differed significantly from all of the other subscales. Affect regulation and self-punishment did not differ from each other, but differed significantly from the other eleven subscales. Marking distress, anti-suicide, and anti-dissociation/feeling generation were not significantly different to one another but did differ from the other subscales. Although self-care once again was not significantly different to toughness, interpersonal influence, or interpersonal boundaries, it also did not differ from anti-suicide, which contrasted with the difference observed between self-care and anti-suicide when the participants completed the ISAS in reference to their global episodes of NSSI. The lack of differences between autonomy, interpersonal influence, interpersonal boundaries, sensation-seeking, and revenge was also observed for participants’ most recent episode of NSSI. Effect sizes for the significant differences between functions ranged from r = .21 (autonomy compared with toughness) to r = .62 (revenge compared with self-punishment). Peer-bonding was once again the only subscale that differed significantly 3.3 Are intrapersonal functions more highly endorsed than interpersonal? Self-reported functions of NSSI have been found to load onto two superordinate factors that reflect intrapersonal and interpersonal reasons for self- injury (Klonsky & Glenn, 2009; Nock & Prinstein, 2004). Grouping the single- function models on the basis of intrapersonal and interpersonal functions is theoretically consistent with multi-function models such as the FFM (Nock & Prinstein, 2004, 2005) and the EAM (Chapman et al., 2006), and allows researchers to test these models. Unfortunately, conducting a confirmatory factor analysis with data that is significantly non-normal, such as the functions data collected in this study, is not recommended (Tabachnik & Fidell, 2007). Instead, I conducted cluster analyses, which do not require data to be normally distributed (Norusis, 2010), to determine whether the single function subscales clustered into intrapersonal and interpersonal categories. The purpose of conducting a cluster analysis is: 193 to group entities on the basis of their similarity with respect to selected variables, so that members of the resulting groups are as similar as possible to others within their group (high within-group homogeneity) and as different as possible to those in other groups (low between-group homogeneity). (Clatworthy, Buick, Hankins, Weinman, & Horne, 2005, p. 330) For the current study, I conducted two hierarchical agglomerative cluster analyses using Ward’s method. Squared Euclidean distance was used as the distance measure in both analyses (Norusis, 2010). 3.3.1.1 Cluster analysis of global NSSI functions 3.3.1.1 Cluster analysis of global NSSI functions Inspection of the agglomeration schedule (presented in Table 7) and dendrogram (see Figure 6) for the global functions of NSSI provided evidence of a two cluster solution. The first cluster (agglomeration coefficient = 2095.13, stage 8 of Table 7) contained the following eight subscales: peer-bonding, revenge, sensation- seeking, interpersonal influence, autonomy, interpersonal boundaries, toughness, and self-care. Table 7 Agglomeration schedule, Ward’s method and squared Euclidean distance for global NSSI functions Agglomeration schedule, Ward’s method and squared Euclidean distance for global NSSI functions functions Agglomeration schedule Cluster combined Stage cluster first appears Stage Cluster 1 Cluster 2 Coefficients Cluster 1 Cluster 2 Next stage 1 8 9 127.00 0 0 2 2 8 12 294.67 1 0 5 3 1 5 478.17 0 0 4 4 1 13 727.33 3 0 6 5 6 8 982.17 0 2 8 6 1 10 1326.25 4 0 8 7 3 11 1695.75 0 0 11 8 1 6 2095.13 6 5 12 9 2 4 2521.13 0 0 10 10 2 7 3115.13 9 0 11 11 2 3 4234.03 10 7 12 12 1 2 7871.85 8 11 0 194 The second cluster (agglomeration coefficient = 4234.03, stage 11 of Table 7) contained the following five subscales: affect regulation, self-punishment, anti- dissociation/feeling generation, anti-suicide, and marking distress. Combining these two clusters into one cluster increased the squared agglomeration coefficient from 4234.03 to 7871.85 (see stage 12 of Table 7); the size of this increase supports a two cluster solution. Figure 6. Dendrogram using Ward linkage for functions of participants’ global episodes of NSSI. Peer-bonding (8) Revenge (9) Sensation-seeking (12) Interpersonal Influence (6) Autonomy (1) Interpersonal Boundaries (5) Toughness (13) Self-care (10) Affect regulation (3) Self-punishment (11) Anti-dissociation (2) Anti-suicide (4) Marking distress (7) Interpersonal Intrapersonal Figure 6. Dendrogram using Ward linkage for functions of participants’ global episodes of NSSI. The grouping of variables in the two clusters is consistent with previous research (Klonsky & Glenn, 2009; Nock & Prinstein, 2004) in that the first cluster contains subscales that reflect interpersonal functions, while the second cluster contains subscales that reflect intrapersonal functions. The only subscale that does not fit neatly into this intrapersonal/interpersonal dichotomy is self-care, which should theoretically cluster with the intrapersonal functions, but instead has clustered with the interpersonal functions. 195 3.3.1.2 Cluster analysis of participants’ most recent NSSI episode 3.3.1.2 Cluster analysis of participants’ most recent NSSI episode The ISAS subscales for the functions of participants’ most recent episode of self-injury demonstrated an identical two cluster solution to that found for the global NSSI functions, with the exception of self-care, which moved from the first to the second cluster. Evidence for the two cluster solution is presented in Table 8 and Figure 7. Table 7 The first cluster (agglomeration coefficient = 981.43, stage 6 of Table 8) contained the following seven subscales: peer bonding, sensation-seeking, autonomy, interpersonal boundaries, toughness, interpersonal influence, and revenge. Table 8 Agglomeration schedule, Ward’s method and squared Euclidean distance for functions of participants’ most recent NSSI episode Agglomeration schedule, Ward’s method and squared Euclidean distance for functions of participants’ most recent NSSI episode Agglomeration schedule, Ward’s method and squared Euclidean distance for functions of participants’ most recent NSSI episode Agglomeration schedule Cluster combined Stage cluster first appears Stage Cluster 1 Cluster 2 Coefficients Cluster 1 Cluster 2 Next stage 1 8 12 86.50 0 0 2 2 1 8 198.00 0 1 4 3 6 9 331.00 0 0 6 4 1 5 517.25 2 0 5 5 1 13 717.40 4 0 6 6 1 6 981.43 5 3 12 7 7 10 1314.43 0 0 10 8 3 11 1704.43 0 0 11 9 2 4 2116.43 0 0 10 10 2 7 2676.93 9 7 11 11 2 3 4063.76 10 8 12 12 1 2 6970.15 6 11 0 The second cluster (agglomeration coefficient = 4063.76, stage 11 of Table 8) contained the following six subscales: affect regulation, self-punishment, marking distress, self-care, anti-dissociation/feeling generation, and anti-suicide. Combining these two clusters into one cluster results in a substantial increase in the agglomeration coefficient from 4063.76 to 6970.15 (see stage 12 of Table 8), which supports a two cluster solution. These two clusters are once again consistent with 196 previous research on the distinction between the intrapersonal and interpersonal functions of NSSI. functions of NSSI. Figure 7. Dendrogram using Ward linkage for functions of participants’ most recent NSSI episode. Peer-bonding (8) Sensation-seeking (12) Autonomy (1) Interpersonal Boundaries (5) Toughness (13) Interpersonal Influence (6) Revenge (9) Affect regulation (3) Self-punishment (11) Marking Distress (7) Self-care (10) Anti-dissociation (2) Anti-suicide (4) Interpersonal Intrapersonal Figure 7. Dendrogram using Ward linkage for functions of participants’ most recent NSSI episode. 3.3.2 Comparing intrapersonal and interpersonal functions Guided by previous research (Klonsky & Glenn, 2009; Nock & Prinstein, 2004) and based on the results of the cluster analyses, four new variables were created. Participants’ responses for the subscales within each of the four clusters (i.e., the two clusters from each cluster solution) were summed and then averaged to derive the following four scores: global intrapersonal functions (affect regulation, self- Guided by previous research (Klonsky & Glenn, 2009; Nock & Prinstein, 2004) and based on the results of the cluster analyses, four new variables were created. Participants’ responses for the subscales within each of the four clusters (i.e., the two clusters from each cluster solution) were summed and then averaged to derive the following four scores: global intrapersonal functions (affect regulation, self- punishment, anti-dissociation/feeling generation, anti-suicide, and marking distress), global interpersonal functions (peer bonding, revenge, sensation-seeking, interpersonal influence, autonomy, interpersonal boundaries, toughness, and self-care), most recent episode intrapersonal functions (affect regulation, self-punishment, marking distress, self-care, anti-dissociation/feeling generation, and anti-suicide), and most recent episode interpersonal functions (peer bonding, sensation-seeking, autonomy, punishment, anti-dissociation/feeling generation, anti-suicide, and marking distress), global interpersonal functions (peer bonding, revenge, sensation-seeking, interpersonal influence, autonomy, interpersonal boundaries, toughness, and self-care), most recent episode intrapersonal functions (affect regulation, self-punishment, marking distress, self-care, anti-dissociation/feeling generation, and anti-suicide), and most recent episode interpersonal functions (peer bonding, sensation-seeking, autonomy, 197 interpersonal boundaries, toughness, interpersonal influence, and revenge). Internal consistency was questionable for the global intrapersonal (α = .63) and global interpersonal (α = .68) functions, and acceptable for the most recent episode intrapersonal functions (α = .70) and most recent episode interpersonal functions (α = .79). Before beginning the analyses to determine whether there were significant differences between the intrapersonal and interpersonal functions for global and most recent episodes, I once again examined the histograms and Q-Q plots of the data to check whether the distributions were normal. Both the global intrapersonal functions variable (M = 3.31, SD = 1.23, Median = 3.40) and most recent intrapersonal functions variable (M = 2.79, SD = 1.26, Median = 2.67) were normally distributed. However, both global interpersonal functions variable (M = 0.94, SD = 0.72, Median = 0.75) and most recent interpersonal functions variable (M = 0.68, SD = 0.85, Median = 0.43) were positively skewed. 3.3.2 Comparing intrapersonal and interpersonal functions Normality tests confirmed that these variables were significantly non-normal; for the global interpersonal functions variable, D(163) = .14, p < .001, and for the most recent interpersonal functions variable, D(140) = .22, p < .001. Before beginning the analyses to determine whether there were significant differences between the intrapersonal and interpersonal functions for global and most recent episodes, I once again examined the histograms and Q-Q plots of the data to check whether the distributions were normal. Both the global intrapersonal functions variable (M = 3.31, SD = 1.23, Median = 3.40) and most recent intrapersonal functions variable (M = 2.79, SD = 1.26, Median = 2.67) were normally distributed. Given that two out of the four variables of interest were significantly non- normal, Wilcoxon signed-rank tests were used to analyse whether there was a significant difference between the global intrapersonal and interpersonal functions, and the intrapersonal and interpersonal functions for participants’ most recent episode. Participants’ endorsement of intrapersonal versus interpersonal functions for their global experiences of NSSI was significantly different, T = 118.00, p < .001, r = .60. More specifically, 152 participants endorsed intrapersonal functions more highly than interpersonal functions, nine participants endorsed interpersonal functions more highly than intrapersonal functions, and two participants did not demonstrate any difference in their endorsement of either intrapersonal or interpersonal functions. 198 There was also a significant difference in participants’ endorsement of intrapersonal versus interpersonal functions for their most recent experience of NSSI, T = 263.50, p < .001, r = .58. Out of 140 participants, 130 participants endorsed intrapersonal functions more highly than interpersonal functions, nine participants endorsed interpersonal functions more highly than intrapersonal functions, and one participant did not demonstrate any difference in their endorsement of either intrapersonal or interpersonal functions. Although intrapersonal functions were more commonly endorsed, 111 participants (79.3%) out of the 140 participants reported self-injuring most recently for both intrapersonal and interpersonal reasons. Only 28 participants (20.0%) reported engaging in their most recent episode of NSSI for exclusively intrapersonal reasons and one participant (0.7%) reported self-injuring most recently for exclusively interpersonal reasons. following NSSI? To assess whether participants would retrospectively report decreased negative affect and increased positive affect following self-injury, they were asked to complete the PANAS-X (Watson & Clark, 1994) twice; once in relation to how they felt before their most recent episode of NSSI and once in relation to how they felt after their most recent episode of NSSI. Responses were averaged to create composite scores for each participant for the following 13 PANAS-X subscales: negative affect, positive affect, fear, hostility, guilt, sadness, joviality, self-assurance, attentiveness, shyness, fatigue, serenity, and surprise (Watson & Clark, 1994). Since participants completed the PANAS-X twice, they each had a total of 26 scores. Visual inspection of the histograms and Q-Q plots showed that, apart from negative affect before the episode, all of the scales appeared to violate the normality assumption. Specifically, most of the scales were positively skewed. Note. All D’s(142-149) > .08, p’s < .05. Note. All D’s(142-149) > .08, p’s < .05. following NSSI? Kolmogorov- Smirnov tests with Lilliefors correction confirmed that the distributions of all of the scales were significantly non-normal, D’s(142-149) ≥ .08 (p’s < .05), with the exception Table 9 Descriptive statistics and reliability coefficients for the PANAS-X subscale responses before and after participants’ most recent episode of NSSI Descriptive statistics and reliability coefficients for the PANAS-X subscale responses before and after participants’ most recent episode of NSSI Before the most recent episode After the most recent episode PANAS-X subscale N Mean (SD) Median Cronbach’s α Skewness Kurtosis N Mean (SD) Median Cronbach’s α Skewness Kurtosis Sadness 149 3.49 (1.18) 3.80 .89 -0.52* -0.84 142 3.03 (1.18) 3.20 .90 -0.08* -1.15 Guilt 149 3.14 (1.21) 3.17 .89 -0.22* -1.01 143 3.05 (1.23) 3.00 .92 0.12* -1.12 Hostility 149 2.75 (0.98) 2.67 .78 0.29* -0.61 143 2.27 (0.90) 2.00 .78 0.55* -0.56 Negative affect 149 2.71 (0.87) 2.60 .83 0.12 -0.69 143 2.36 (0.96) 2.20 .90 0.64* -0.48 Fear 149 2.32 (1.08) 2.00 .88 0.59* -0.72 143 2.10 (1.12) 1.67 .92 0.90* -0.22 Fatigue 149 2.13 (0.99) 2.00 .79 0.97* 0.33 142 2.04 (1.10) 1.75 .87 0.99* -0.03 Attentiveness 149 1.76 (0.80) 1.50 .74 1.13* 0.74 142 1.70 (0.76) 1.50 .68 1.07* 0.38 Positive affect 149 1.46 (0.59) 1.20 .86 2.23* 6.39 142 1.47 (0.55) 1.25 .83 1.38* 1.03 Serenity 149 1.43 (0.78) 1.00 .84 2.37* 5.71 142 2.50 (1.15) 2.33 .85 0.39* -0.81 Self-assurance 149 1.43 (0.71) 1.17 .86 2.46* 6.76 142 1.49 (0.70) 1.17 .83 1.65* 1.90 Shyness 149 1.45 (0.65) 1.00 .74 1.46* 1.31 142 1.57 (0.87) 1.00 .83 1.77* 2.49 Joviality 149 1.25 (0.64) 1.00 .95 3.67* 14.31 142 1.30 (0.51) 1.00 .89 2.19* 4.45 Surprise 149 1.25 (0.56) 1.00 .73 2.86* 8.60 142 1.35 (0.64) 1.00 .77 2.09* 4.01 Note. All D’s(142-149) > .08, p’s < .05. 1 After the most recent episode 199 200 of negative affect before the episode. Descriptive statistics and reliability coefficients for the PANAS-X scales are presented in Table 9. Wilcoxon signed-rank tests showed that negative affect (T = 2433.00, p < .001, r = .30), hostility (T = 1750.00, p < .001, r = .35), sadness (T = 1625.50, p < .001, r = .33), and fear (T = 2113.50, p < .001, r = .21) decreased significantly after participants had self-injured, whereas there was no significant change in feelings of guilt (T = 4021.50, p=.17, r = .06). Table 10 anks for PANAS-X subscales before and after participants’ most recent episode of NSSI Ranks for PANAS-X subscales before and after participants’ most recent episode of NSSI Ranks for PANAS-X subscales before and after participants’ most recent episode of NSSI Ranks PANAS-X subscale Negative - decrease after NSSI (% of N) Positive - increase after NSSI (% of N) Tie - no change after NSSI (% of N) Negative Affect 99 (69.23) 40 (27.97) 4 (2.80) Hostility 98 (68.53) 33 (23.08) 12 (8.39) Sadness 89 (62.68) 34 (23.94) 19 (13.38) Fear 78 (54.55) 38 (26.57) 27 (18.88) Guilt 71 (49.65) 62 (43.36) 10 (6.99) Fatigue 66 (46.48) 40 (28.17) 36 (25.35) Attentiveness 56 (39.44) 46 (32.39) 40 (28.17) Positive Affect 52 (36.62) 55 (38.73) 35 (24.65) Self-assurance 37 (26.06) 46 (32.39) 59 (41.55) Joviality 24 (16.90) 53 (37.32) 65 (45.77) Shyness 24 (16.90) 45 (31.69) 73 (51.41) Surprise 17 (11.97) 35 (24.65) 90 (63.38) Serenity 13 (9.15) 107 (75.35) 22 (15.49) Note. All N = 142-143. Modal findings are in bold. Note. All N = 142-143. Modal findings are in bold. Note. All N = 142-143. Modal findings are in bold. following NSSI? In comparison, serenity (T = 715.50, p < .001, r = .45) and joviality (T = 1045.00, p < .05, r = .14) increased significantly after self-injury, but there was no significant change in general positive affect (T = 2755.00, p = .34, r = .02). The results for the remaining five subscales of the PANAS-X should be considered exploratory given that I had no a priori hypotheses as to whether these emotions would change following NSSI. Shyness (T = 765.00, p < .01, r = .16) increased significantly and surprise (T = 388.50, p < .01, r = .16) decreased significantly following NSSI, but there was no significant changes in fatigue (T = 2405.50, p = .17, r = .08), attentiveness (T = 2386.00, p = .42, r = .05), or self- assurance (T = 1507.00, p = .28, r = .06). Although it is useful to know which affect states changed significantly following participants’ engagement in NSSI, these statistics do not provide an indication of how many people experienced such changes. However, the positive, negative, and tie ranks for each of the subscales are in effect change scores, which can be used to determine how many participants experienced increases, decreases, or no change in particular emotional states following their episode of self-injury. The ranks for each of the PANAS-X subscales are presented in Table 10. Although the majority of participants experienced decreased negative affect (69.32%), hostility (68.53%), sadness (62.69%), and fear (54.55%) following NSSI, there were a number of participants whose negative emotions increased and others who experienced no change. For example, 23.08% of people reported feeling more hostile following self-injury and 8.39% reported no change in their levels of hostility. 201 Variable rates of endorsement were also observed for what can be considered neutral or positive affect states. An overwhelming majority of participants (75.35%) reported an increase in serenity after they had self-injured, but some participants’ (9.15%) experience of serenity decreased, while others (15.49%) experienced no change. In contrast, more participants (45.77%) reported that their experience of joviality did not change after self-injuring, than those who reported an increase (37.32%) or decrease (16.90%) in joviality. 3.5 Does the content of people’s cognitions change following NSSI? To examine whether there was any change in participants’ self-reported cognitions following a NSSI episode, I summed and then averaged participants’ scores on the negative and positive/neutral items from the ATQ-R (Kendall et al., 1989). This resulted in four new variables for each participant: negative cognitions 202 before most recent episode, negative cognitions after most recent episode, positive/neutral cognitions before most recent episode, and positive/neutral cognitions after most recent episode. Internal consistency for negative cognitions before (α = .94) and after (α = .95) self-injury was excellent, while internal consistency for positive/neutral cognitions before (α = .80) and after (α = .74) self-injury was acceptable to good. Visual inspection of histograms and Q-Q plots showed that the negative cognitions data was skewed to the left, while the positive/neutral cognitions data was skewed to the right. Kolmogorov-Smirnov tests with Lilliefor’s correction (D’s(140-142) > .12, all p’s < .001) confirmed that all of the subscales were significantly non-normal, necessitating the use of non-parametric tests to determine whether there were significant changes in participants’ self-reported negative or positive/neutral cognitions following engagement in self-injury. Wilcoxon signed-rank tests showed that negative cognitions (T = 1906.00, p <. 001, r = .31) decreased significantly and positive cognitions (T = 777.00, p < .001, r = .31) increased significantly following NSSI. More specifically, 91 (64.54%) participants reported a decrease in negative cognitions following NSSI, 36 (25.53%) reported an increase in negative cognitions, and 14 (9.93%) reported no change. Less conclusive findings were evident with positive cognitions as although 74 (52.86%) participants reported that they experienced an increase and 16 (11.43%) experienced a decrease in positive/neutral cognitions following NSSI, 50 (35.71%) participants reported no change in their positive/neutral cognitions. 3.6 Summary of quantitative findings The results supported the hypotheses in that affect regulation was the most highly endorsed function of both participants’ global and most recent episodes of NSSI; however, there was no significant differences in the level of participants’ endorsement for affect regulation or self-punishment. Consistent with the high endorsement of affect regulation and self-punishment, intrapersonal functions were more highly endorsed than interpersonal functions for both global and most recent 203 episodes of NSSI. Specific negative affect states were reported to decrease significantly following NSSI, but only one positive affect state (i.e., joviality) increased. Although serenity increased significantly, this is classified by Watson & Clark (1994) under the rubric of other affective states. Participants similarly reported a significant decrease in negative cognitions and a significant increase in positive cognitions after they had self-injured most recently. Before discussing the implications of these findings, I present the qualitative analyses of participants’ descriptions of the antecedents and consequences of their most recent episode of self-injury. 4. OPEN-ENDED RESPONSES To add depth and scope to my understanding of the events that establish the conditions for, and potentially reinforce, self-injurious behaviours, participants were given the opportunity to describe, in their own words, what had led to their most recent episode of NSSI and the consequences of that episode. To code the antecedents of the NSSI episodes, a coding system for aversive events, based on the stressful event categories listed in the Unpleasant Events Schedule (Lewinsohn, Mermelstein, Alexander, & MacPhillamy, 1983), was developed for the purposes of this study (see Appendix I). The consequences described by participants were analysed using Thematic Analysis (Braun & Clarke, 2006). Table 11 Table 11 Antecedent event categories with percentage endorsement and qualitative examples Antecedent event categories with percentage endorsement and qualitative examples Event category N (%) Examples of event descriptions Health and well-being 7 (6.60)  I had a lot to drink and was sick of not feeling loved and wanted and a guy was being nice to me for once in my life. Next thing you know me and him are in the parking lot down the road from a club and he completely uses me and it hurt really bad and then ran off afterwards with me bawling my eyes out. (Female, 19)  Binging. (Female, 20) Achievement-academic-job 12 (11.32)  Being pressured at work so when I got home I went out for a smoke and burned myself with it. (Female, 28)  Occurred during exam period. Was punishing myself because I wasn’t studying and because I hoped the adrenaline would keep me awake. (Female, 19) Interpersonal relationships 59 (55.66)  I had had an argument with my mother, and left my parental house very distressed. I wanted to drive to my husband's work so I could talk to him (it was near the end of his shift), but was too upset to drive properly. So I drove to the nearest parking lot and cut my left arm repeatedly with a razor blade that I keep in my car. (Female, 23)  After a messy break up caused by a cheating girlfriend, the (now) ex girlfriend told me that she had power over me, and that she could hurt me more than anything or anyone else could. (Male, 19) Material-financial 2 (1.89)  In debt rang study link who won’t pay any more. (Female, 50) Death-related 3 (2.83)  Close friend died in June. (Female, 19)  It was the around the time of my mother’s death. (Female, 19) Other 2 (1.89)  Had a shower and hated the sight of myself. (Female, 42)  Had a discussion on religion, and I came to realise that I have not been a good believer. (Female, 21) No specific event 14 (13.21)  Built up negative emotions that had come to a point of not being able to deal with them anymore. (Female, 19)  I just felt very misunderstood. (Female, 27) Insufficient information to code the event 7 (6.60)  Abandonment. 4.1 Antecedents of NSSI Out of 153 participants, 96 (58.9%) reported that something specific had led to their most recent episode of self-injury. These events were coded within the following 10 categories: health and wellbeing (focused on physical health); achievement-academic-job; domestic, day-to-day inconveniences; interpersonal relationships; legal; material-financial; death-related; other; no specific event; and insufficient information to code the event. Events could be coded in multiple categories. A Clinical Psychologist and I coded all the events; interrater agreement was 82.86% and the final ratings for any discrepant items were determined following discussion. Table 11 shows the number of participants who reported events in each Table 11 (Female, 32)  Memories of traumatic events that happened on that date years before. (Female, 17) 204 205 category, with accompanying examples. No-one reported that their most recent episode of NSSI was precipitated by domestic, day-to-day inconveniences or legal events. As a result, these two categories are excluded from Table 11. The majority of the antecedents (55.66%) described by participants were categorised as interpersonal relationship events, which typically included fights or arguments with friends or family, relationship break-ups, or being mistreated by others. The second highest category of events was concerned with failure to achieve goals, academic struggles, or job stress. 26 Three responses were discarded because participants had typed in ‚none‛ or ‚nothing at all‛ and then rated these responses as neutral. 4.2 Thematic Analysis of the consequences of NSSI When participants were asked to describe up to five consequences of their most recent episode, 122 people identified experiencing at least one consequence, with an average number of 2.59 (SD = 1.38) consequences per person.26 Three people identified consequences but did not evaluate whether these were positive, negative, or neutral; as a result, these responses could not be further analysed. Of the 313 consequences that were included in the thematic analysis, 101 (32.27%) were evaluated by participants as positive, 170 (54.31%) as negative, and 42 (13.42%) as neutral. For the thematic analysis, I analysed the consequences that were positively or negatively evaluated, rather than consequences that were rated as neutral. Two distinct themes were identified within this data—self becomes transgressor and self becomes helper—each of which had a number of sub-themes (see Figure 8). Becoming a transgressor necessitated concealing the transgressions from others, being judged by others for the transgressions, and causing others to suffer, while becoming a helper was enacted through regulating emotions, accessing support and/or treatment, and the physical wound. 206 Figure 8. Thematic Map. Concealing transgressions from others Being judged by others for transgressions Regulating emotions Transgressions cause others to suffer Physical wound Accessing support and/or treatment Self becomes transgressor Self becomes helper Regulating emotions Physical wound Accessing support and/or treatment Self becomes helper Figure 8. Thematic Map. Concealing transgressions from others Being judged by others for transgressions Transgressions cause others to suffer Self becomes transgressor Self becomes helper Self becomes transgressor Concealing transgressions from others Being judged by others for transgressions Regulating emotions Transgressions cause others to suffer Accessing support and/or treatment Figure 8. Thematic Map. 27 Any spelling or major grammatical errors within responses have been corrected to facilitate readability. Furthermore, the way in which the quotes are presented differs from how I presented the interview quotes (see Chapter 4) as the responses in this study were anonymous. 4.2.1 Self becomes transgressor Self-injury is an anathema to many people, who struggle to understand why someone would purposefully cut, burn, or otherwise damage their own skin (Strong, 2000). As identified by two participants27, injuring oneself in such a way contravenes pervasive Western cultural discourses of protecting, preserving, and extending the life of one’s body and clearly transgresses social mores: I felt guilty at what I had done because I had damaged my body. (Female, 18) I felt guilty at what I had done because I had damaged my body. (Female, 18) I felt disgusted with myself, that I was harming my body which should be something precious, was burdened with this guilt for quite some time. (Female, 19) In contrast to non-Western cultures where self-injury can signify socially desirable psychological and physical transitions (e.g., initiation rites) (Favazza, 1996), amongst Pākehā living in Aotearoa New Zealand self-injury typically signifies psychopathology and an attendant inability to cope with problems, in the words of one participant, ‚like a ‘normal’ person‛. Participants readily judged themselves as abnormal—that is, as transgressors of social norms—because they had self-injured: Participants readily judged themselves as abnormal—that is, as transgressors of social norms—because they had self-injured: I felt like everyone would notice and think I was crazy. (Female, 18) I felt like everyone would notice and think I was crazy. (Female, 18) I felt like everyone would notice and think I was crazy. (Female, 18) 27 Any spelling or major grammatical errors within responses have been corrected to facilitate readability. Furthermore, the way in which the quotes are presented differs from how I presented the interview quotes (see Chapter 4) as the responses in this study were anonymous. 27 Any spelling or major grammatical errors within responses have been corrected to facilitate readability. Furthermore, the way in which the quotes are presented differs from how I presented the interview quotes (see Chapter 4) as the responses in this study were anonymous. 207 What's wrong with me<why did I slip? It had been awhile since I self harmed, what does it mean that I have done this again? (Female, 22) What's wrong with me<why did I slip? It had been awhile since I self harmed, what does it mean that I have done this again? 4.2.1 Self becomes transgressor (Female, 22) That such judgements occurred in the context of particular self-referential emotions (e.g., guilt, shame) is unsurprising given that experiencing these emotions necessitates processes of self-representation and -evaluation, both of which are strongly influenced by prevailing community norms (Zinck, 2008): These emotions specifically contribute to highlighting the difference between one’s own and another’s perspective, coordinate the subject’s behavior in a social environment, promote her integration with the social group and support the mediation between specific individual and social goals. They further promote an adjustment of the self-concept in relation to the feedback of a social environment and to an internal evaluation of behavior and thoughts according to the subject’s own standard. (Zinck, 2008, p. 498) In this way, self-referential emotions function to communicate and regulate individuals’ identities and self-concepts (Zinck, 2008). Given that self-referential emotions cannot occur in the absence of self- evaluation, the reporting of these emotions as negative consequences of self-injury demonstrates that participants have judged themselves as transgressors. However, for those participants who simply listed negative emotions—guilt, shame, embarrassment—as consequences, it is impossible to determine why the act of self- injury had made them feel guilty or ashamed, or more specifically, exactly what personal and social values they believed they had broken. Other people, however, did provide explanations that revealed the self- evaluative component of these emotions in more detail. For several participants, the feelings of guilt and shame stemmed from the fact that they had hurt themselves again: I felt bad that I could not resist the compulsion to do it. (Female, 29) Guilt for self-injuring when I told myself I wouldn't. (Female, 18) Guilt for self-injuring when I told myself I wouldn't. (Female, 18) I felt like a failure for a long time because I let myself down by relapsing into self harming again. (Female, 19) Incredible amount of shame at resorting to old coping strategies to cope. (Female, 44) Feeling extremely disappointed in myself because I was supposed to have stopped SI-ing. (Female, 17) 208 The inability to resist self-injury and utilise healthier coping strategies was evaluated by these participants as a personal failing, which impacted on the way that they perceived themselves and had the potential to impact on the way that they were perceived by others. 4.2.1 Self becomes transgressor (Female, 21) Couldn't wear short sleeves so I was boiling hot all summer. (Female, 21) I couldn't wear shorts. (Female, 35) I couldn't wear shorts. (Female, 35) On a psychological level, having to guard against being found out impacted negatively on participants: On a psychological level, having to guard against being found out impacted negatively on participants: The constant fear of it being seen or someone realising what it was. Puts you more on edge than you were beforehand. (Female, 19) One person was unable to hide his wounds in his work environment, which led him to resign: Quit my job as a preschool-school age swimming teacher. Because I was too depressed and embarrassed of my cuts especially in front of little children who I am a role model to. (Male, 20) The psychological toll of hiding the evidence of self-injury to maintain a facade of The psychological toll of hiding the evidence of self-injury to maintain a facade of success and well-being was particularly well-articulated by one woman who wrote: p y g g j y success and well-being was particularly well-articulated by one woman who wrote: I felt detached from everyone around me, like I was fake and no one knew the real me. It was as though there was another side to me that no one knew and they could never know. On the outside I was picture perfect, or should have been, to them I had everything going for me. (Female, 19) While it is understandable that participants hid their scars to prevent others from finding out about their self-injury, it is unclear from most of the responses what type of reactions these participants expected to receive. However, a few participants did specify that they hid their wounds or scars to avoid being judged: Having to hide what I have done because it embarrasses me and I don’t want anyone to see how pathetic I am. (Female, 33) Having to deal with the scars that takes a very long time to heal, and having to hide them from others so I don't get judged. 4.2.1 Self becomes transgressor Indeed, having others find out about the self-injury was explicitly identified by some participants as a source of shame: Indeed, having others find out about the self-injury was explicitly identified by some participants as a source of shame: Shame in having my flatmates see the cuts on my arms. (Female, 22) I had to mention it to two of my friends, which was hard. I felt embarrassed because I had not cut myself in at least a year. (Transgender, 26) The shame of my parents finding out what I had done. (Female, 41) Given that self-injury is perceived as shameful, having to prevent others from finding out about their transgressions was one of the most commonly reported negative consequences of self-injury. Given that self-injury is perceived as shameful, having to prevent others from finding out about their transgressions was one of the most commonly reported negative consequences of self-injury. 4.2.1.1 Concealing transgressions from others 4.2.1.1 Concealing transgressions from others Many people described that having to hide the physical evidence of their self- injury from others was a negative consequence of their behaviour: More scars to hide. (Female, 39) More scars to hide. (Female, 39) Having to hide new cuts. (Female, 17) Having to hide new cuts. (Female, 17) Had to cover it up. (Female, 17) Had to cover it up. (Female, 17) Having to hide wounds from my boyfriend. (Female, 24) I had to be sure not to let the wound/scar show. (Female, 27) Ensuring those around them did not find out that they had self-injured had both practical and psychological implications. On a practical level, participants’ clothing choices were restricted because they had to keep their wounds or scars covered: The wound was on a place on my arm which could be seen by others if I wore a T-shirt. I had to wear long sleeves while it healed even when it was hot to cover it up. I don't want my flat- mates or friends to know I've been struggling. I now have to continue wearing long sleeves or use concealer if I want to wear short sleeves. (Female, 27) My arm had to be covered at all times to hide what I had done while it healed. (Female, 20) 209 Couldn't wear short sleeves so I was boiling hot all summer. (Female, 21) Couldn't wear short sleeves so I was boiling hot all summer. 4.2.1.3 Transgressions cause others to suffer The effect of self-injury on family and friends was another sub-theme identified in the overarching theme of the self becoming a transgressor: It upset the person closest to me to see me in that state. (Female, 19) Hurting my husband by him knowing the extent of my emotional distress was so much that hurt myself. (Female, 26) Hurting my husband by him knowing the extent of my emotional distress was so much that I hurt myself. (Female, 26) I frightened those I love. (Female, 30) I frightened those I love. (Female, 30) My family and people found out and got really scared and hurt. I felt so guilty and even worse for making them feel that way. (Female, 16) This may have been an emotional release to me, but it seems that it was an uncomfortable display to other people around me; this distressed them deeply. (Female, 20) This may have been an emotional release to me, but it seems that it was an uncomfortable display to other people around me; this distressed them deeply. (Female, 20) In one case, the participant’s episode of self-injury actually led to the her boyfriend relapsing himself: ‚My boyfriend got upset with me self harming as he had issues with it also and we were trying to stop together and then he was upset and self harmed also‛ (Female, 18). 4.2.1 Self becomes transgressor Although people tended to report being judged for failing to live up to familial, peer, and societal expectations because they had self-injured, one woman 211 described a situation where the opposite occurred and her self-injury was normalised: described a situation where the opposite occurred and her self-injury was normalised: Made it more likely that Student Health people might actually bloody well take seriously when I said I was depressed and needed help. Therefore made me feel ten million times more unloved when they still didn't. Apparently self-harm is a 'perfectly natural coping mechanism' and means nothing. What a load of bollocks. Did these people even go to med school? (Female, 18) Made it more likely that Student Health people might actually bloody well take seriously when I said I was depressed and needed help. Therefore made me feel ten million times more unloved when they still didn't. Apparently self-harm is a 'perfectly natural coping mechanism' and means nothing. What a load of bollocks. Did these people even go to med school? (Female, 18) In this instance, she had tried to use self-injury—which she perceived as an abnormal coping mechanism—to signal her need for mental health support. Other participants’ self-injurious behaviours were viewed as pathological, but her behaviour was dismissed as an acceptable coping strategy, leading her to feel rejected and frustrated. 4.2.1.3 Transgressions cause others to suffer 4.2.1 Self becomes transgressor (Female, 19) For one adolescent, being found out would have resulted in the loss of a position of responsibility: ‚I am a school leader, and one of the reasons I got in was because I For one adolescent, being found out would have resulted in the loss of a position of responsibility: ‚I am a school leader, and one of the reasons I got in was because I 210 had stopped self harming, and if it was found out that I did slip up my role would be taken from me‛(Female, 16). It is apparent from the majority of examples within this sub-theme that the anticipation, rather than the actual experience, of being judged for transgressing social norms through self-injuring drove participants to conceal the evidence of their self-injurious behaviours. Some participants, however, did report actually being judged by others for engaging in NSSI. 4.2.1.2 Being judged by others for transgressions Despite the considerable effort undertaken by people to protect themselves from being found out as transgressors, at times the scars or wounds were seen by others. Some participants lied to avoid being judged, although this was not always sufficient: Embarrassment when someone saw the wounds and I had to lie about them to avoid being judged negatively. (Female, 24) Having to lie to people that are close to you, particularly the ones that know you self-injure. Often they won't believe you anyway and you feel like you have angered or disappointed them yet again. (Female, 19) Indeed, several participants mentioned being judged by others as a negative consequence of their self-injury: Made to feel even worse at ED by their judgements and treatment. (Female, 33) The internet friend I told scolded me and got very upset about it. (Female, 16) People telling me I overreacted (not understanding). (Female, 24) People telling me I overreacted (not understanding). (Female, 24) My parents and friends were disappointed in me. (Female, 16) One woman stated that her self-injury had resulted in her girlfriend leaving her and her family sending her to therapy, thus providing evidence that self-injurious behaviour within her social network is perceived as an unacceptable manifestation of psychopathology, which needs to be fixed. 4.2.2 Self becomes helper The second theme—self becomes a helper—was identified from the positive consequences reported by the participants. This theme comprised of ways in which participants had succeeded in helping themselves; in particular, three sub-themes of 212 emotion regulation, accessing support/treatment, and the presence of physical wounds were identified. 4.2.2.1 Regulating emotions 4.2.2.1 Regulating emotions 4.2.2.1 Regulating emotions The most common positive emotional experiences reported by participants following their most recent episode of NSSI were calmness, relief, and release: Calming down, stilling my emotions which had been in turmoil. (Female, 26) I gained some relief from the overwhelming feelings of despair I was experiencing. (Female, 52) I felt a break from all the pain I was feeling. It was a huge release. Almost euphoric. (Female, 18) Calm, more relaxed, almost a meditation. (Female, 35) Felt calmer, more grounded and in control. (Female, 42) By regulating their overwhelming emotions, people were able to move forward, whether this involved getting some sleep, going to work, or generally managing the situation they were in: Felt calmer, released 'stuck' emotions so I could cry, sleep, soothe myself, care for myself. (Female, 24). It worked to calm me down and get me out of the house and to work on time. (Female, 23) I calmed down enough to finish my day without too much trouble or time spent stressing. (Female, 20) I felt like I had released all the built up internal emotion and better able to cope with the situation without my feelings getting the best of me. (Female, 20) I sat down and was able to get on with my study without feeling stressed or anxious. (Male, 18) By regulating their overwhelming emotions, people were able to move forward, whether this involved getting some sleep, going to work, or generally managing the situation they were in: Felt calmer, released 'stuck' emotions so I could cry, sleep, soothe myself, care for myself. (Female, 24). It worked to calm me down and get me out of the house and to work on time. (Female, 23) I calmed down enough to finish my day without too much trouble or time spent stressing. (Female, 20) I felt like I had released all the built up internal emotion and better able to cope with the situation without my feelings getting the best of me. (Female, 20) I sat down and was able to get on with my study without feeling stressed or anxious. (Male, 18) By regulating their overwhelming emotions, people were able to move forward, whether this involved getting some sleep, going to work, or generally managing the situation they were in: Felt calmer, released 'stuck' emotions so I could cry, sleep, soothe myself, care for myself. (Female, 24). 4.2.2.1 Regulating emotions It worked to calm me down and get me out of the house and to work on time. (Female, 23) I calmed down enough to finish my day without too much trouble or time spent stressing. (Female, 20) I sat down and was able to get on with my study without feeling stressed or anxious. (Male, 18) Some participants commented on the temporary nature of the relief noting that it was ‚momentary‛, ‚lasted for a few hours‛, or that they felt ‚better in the short term‛. However, for one person, the memory of self-injury continued to induce a calming effect: ‚It was a special secret that only I knew about. The memory creates a small feeling of calm‛ (Female, 27). 213 Self-injury not only allowed people to reduce particular negative emotions, such as despair and anxiety, but also to induce positive emotions, such as pride, hope, and happiness: Feeling stronger, feeling happier. (Female, 20) Feeling stronger, feeling happier. (Female, 20) Discover hope. (Female, 21) Discover hope. (Female, 21) I felt proud because I felt like injuring myself was a good way of dealing with my emotions because I could think of nothing else to do to make it better - as if I had found an effective solution. (Female, 17) Furthermore, successfully regulating their emotions appeared to engender a sense of agency for people, allowing them to feel more in control. In this way, self-injury was an active solution to feeling emotionally overwhelmed: ‚I felt calmer, and that I had achieved something‛ (Female, 17). Importantly, a few participants reported decreased suicidality following NSSI, which they evaluated as a positive consequence: Importantly, a few participants reported decreased suicidality following NSSI, which they evaluated as a positive consequence: NSSI, which they evaluated as a positive consequence: Overwhelming impulse to die was reduced. (Female, 42) Overwhelming impulse to die was reduced. (Female, 42) Decrease of suicidal thoughts. (Male, 20) Easing of stress and the feeling of wanting to die. (Female, 21) I didn't try and kill myself. (Female, 39) It is worth noting that any reductions in suicidal ideation would have involved cognitive as well as emotion regulation, as one of the above responses explains. Aside from decreased suicidal thoughts, only a couple of participants identified positive cognitive consequences following their most recent episode of self-injury: Able to think more clearly. (Female, 18) Able to think more clearly. (Female, 18) Increased cognitive clarity. 4.2.2.1 Regulating emotions (Male, 32) Increased cognitive clarity. (Male, 32) However, the lack of cognitive consequences reported may have resulted from the open-ended structure of the questions, particularly if emotional consequences are more salient. 214 4.2.2.2 Accessing support and/or treatment 4.2.2.2 Accessing support and/or treatment Several participants received help or support from others as a positive consequence of their self-injury: My husband looked after me and was very gentle with me after I hurt myself. (Female, 23) That my friend was made aware of how I was feeling and there for me. (Female, 20) My husband looked after me and was very gentle with me after I hurt myself. (Female, 23) That my friend was made aware of how I was feeling and there for me. (Female, 20) I had a discussion with my flatmate as he noticed the wounds a few days later - I discovered that he had been through similar things and we were able to talk about it openly - he has since been very supportive. (Female, 27) I had a discussion with my flatmate as he noticed the wounds a few days later - I discovered that he had been through similar things and we were able to talk about it openly - he has since been very supportive. (Female, 27) People attended to me with more interest and put in genuine effort to help me. (Female, 20) My boyfriend and I had a long talk about my inability to cope with stress and anger and self loathing. I agreed whenever I was feeling in too deep or like I wanted to hurt myself again that I would call him any time. (Female, 20) For these participants, self-injury facilitated access to support by signalling the intensity of their distress to concerned friends and family members. Some people were prompted to actively seek treatment or support from others following their self-injury: Some people were prompted to actively seek treatment or support from others following their self-injury: I eventually got help from a counsellor. (Female, 19) I spoke to my counsellor who referred me to mental health services after hurting myself 3 times in a month. (Female, 28) Allowed me to realise just how badly I was distressed, meaning I went to ask for help from others. (Female, 27) Finding a great new GP who described the process going on and who identified what I had experienced ever since I can remember. 4.2.2.2 Accessing support and/or treatment (Female, 35) Even when actively seeking support from friends proved to be ineffective for one woman, she felt compelled to continue to search for the support she needed: woman, she felt compelled to continue to search for the support she needed: Realised how serious the situation was, that I absolutely didn't have the strength to deal with this by myself. Had already asked for help from friends/ex-boyfriend, but now realised that since they wouldn't help, MUST get help by other means. (Female, 18) Realised how serious the situation was, that I absolutely didn't have the strength to deal with this by myself. Had already asked for help from friends/ex-boyfriend, but now realised that since they wouldn't help, MUST get help by other means. (Female, 18) For her, this realisation was a positive consequence of her self-injury episode. Two people reported being hospitalised as a positive consequence of their self-injury with one participant stating: ‚Got a bed in psych unit quicker and 215 therefore felt safer and cared for‛ (Female, 41). Another woman went back on mood stabilisers as a result of her self-injury and also began taking sleeping pills, which she found helpful. Although help-seeking typically resulted in some form of attention from others, only three people specifically identified receiving attention or sympathy from others as a positive consequence of self-injury: Attention from others. (Female, 19) Opportunity for attention. (Female, 18) Felt like now that I have a deepish cut, people who may happen to see it -even though I try to hide it- will feel sympathy for me and like me more. (Female, 19) The last response is particularly intriguing as this woman’s expectation of how people will react to her cut is antithetical to the responses given by other participants within the self becomes transgressor theme. However, at the same time, she acknowledges trying to hide the injury, implying that she is aware of the status of self-injury as a socially transgressive behaviour. It may be that people within this woman’s community judge those who attempt to conceal self-inflicted wounds more favourably than those who display them openly. 4.2.2.3 The physical wound Contrary to the negative evaluation of wounds or scarring in the self becomes transgressor theme, the physical evidence of self-injury within the self becomes helper theme was identified as a positive consequence. 4.3 Summary of the findings from the open-ended responses The open-ended responses highlight the important influence of negative social interactions (especially interpersonal conflict) and community norms on the incidence and maintenance of NSSI. The paradox inherent in self-injury functioning simultaneously as an act of transgression and an act of self-help was evident in the consequences reported by participants. Negotiating the dissonance that results from this paradox has the potential to maintain NSSI if people attempt to avoid, or escape from, the painful, self-referent emotions that occur following self-injury through further self-injurious behaviours. 5. DISCUSSION In this study, I was primarily interested in examining three hypotheses informed by the EAM (Gratz et al., 2006), extant literature on why people engage in NSSI, and the results of the Interpretative Functional Analysis that I presented in the previous chapter. Specifically, I hypothesised that (1) affect regulation would be endorsed as the primary function of NSSI, (2) intrapersonal functions would be more highly endorsed than interpersonal functions, and (3) negative affect would decrease following self-injury but positive affect would increase. Finally, I was also interested in exploring whether people reported shifts in negative, self-referent thoughts following episodes of NSSI. 4.2.2.2 Accessing support and/or treatment It is worth noting, however, that many more people evaluated wounds and scarring as a negative consequence than as a positive consequence. It was not readily apparent from the quotes why people reported blood, scars, and bruises as positive consequences. It is likely that these physical manifestations of self-injury were interpreted positively for different reasons, as is evident from the following responses: I had a large burn mark on my arm. It looked pretty flash, eh. (Male, 19) I saw blood. (Female, 27) I saw blood. (Female, 27) 216 Needing to attend to the cut on my leg. Stop the bleeding, apply a plaster and anti-septic. (Female, 35) Unfortunately, there is not enough detail provided in these responses to determine why participants experienced the sight of their blood or wounds as positive. Unfortunately, there is not enough detail provided in these responses to determine why participants experienced the sight of their blood or wounds as positive. 5.1 Affect regulation and self-punishment are the primary functions of NSSI Although the average ratings and mean ranks for affect regulation were slightly higher than self-punishment for participants’ global and most recent episodes of NSSI, these functions had the same median and were not significantly different from one another. However, for both global and most recent episodes, 217 affect regulation and self-punishment were rated significantly higher than any of the other functions. This is consistent with Klonsky and Glenn’s (2009) finding that affect regulation and self-punishment respectively were the first and second most commonly reported functions of NSSI among university students. As I discussed in Chapter 3, it is likely that punishing oneself through self- injury is a specific form of affect regulation because in such instances the self- injurious act is carried out to regulate self-directed anger and self-hatred (Chapman et al., 2006; Klonsky, 2007, 2009). Other functions (i.e., marking distress, anti- dissociation/feeling generation, and anti-suicide) that could conceivably be incorporated within a broader conceptualisation of affect regulation than that which is represented in the ISAS (Klonsky & Glenn, 2009) were also highly endorsed by the participants in this study. Although these three functions differed significantly to affect regulation, self-punishment, and all the other subscales, they did not differ significantly from one another. The five most highly endorsed functions across both the global and most recent episodes of NSSI—affect regulation, self-punishment, marking distress, anti- dissociation/feeling generation, and anti-suicide—can all be understood as forms of experiential avoidance, thus supporting the EAM’s premise, which is that self-injury primarily functions as an experientially avoidant behaviour (Chapman et al., 2006). Although people did endorse access functions (e.g., interpersonal influence, revenge, peer-bonding) of self-injury along with the avoidant functions, these were rated significantly lower. Furthermore, the role of self-injury in facilitating affect regulation was exemplified in the open-ended responses from participants about the consequences of their self-injury. Most notably, self-injury functioned to calm participants down and to help them relax. However, it was also evident that transgressing social norms through self-injury led participants to feel ashamed, guilty, and fearful of being judged by others. These responses provide some insight into how the cycle of self- injury may be maintained through self-punishment. 218 Given that self-injury functions as a way to avoid or escape negative affect, painful self-referential emotions, such as shame and guilt, may be particularly powerful antecedents for future NSSI. 5.1 Affect regulation and self-punishment are the primary functions of NSSI The experience of these emotions could lead people to conclude that they deserve to be punished for their transgressive behaviour, which in turn may result in further self-injury. Indeed, women with BPD who demonstrated greater non-verbal shame behaviours were more likely to self- injure than women who did not show these behaviours (Brown, Linehan, Comtois, Murray, & Chapman, 2009). 5.2 Intrapersonal functions are more highly endorsed than interpersonal functions It is apparent from the ISAS results that people endorsed items from across all of the single-function subscales. This range of responses calls into question the clinical utility and validity of single-function models and emphasises the importance of multi-function conceptualisations of NSSI, such as the EAM (Chapman et al., 2006) and the FFM (Nock & Prinstein, 2004, 2005). For participants’ global and most recent episodes of NSSI, the most appropriate solution in both cases comprised of two clusters, which was consistent with previous theoretical and empirical demarcations between intrapersonal and interpersonal motivations for self-injury (Klonsky & Glenn, 2009; Nock & Prinstein, 2004). The only anomaly in the cluster solution for participants’ general episodes was the inclusion of self-care within the interpersonal cluster when theoretically it should have clustered with intrapersonal functions. On inspection, the self-care items demonstrated good face validity (e.g., ‚creating a physical injury that is easier to care for than my emotional distress‛) making it unlikely that these items actually reflect interpersonal motivations for self- injury. In Klonsky and Glenn’s (2009) study, self-care also was more closely aligned with interpersonal, rather than intrapersonal, functions, but the loadings of this subscale on the intrapersonal and interpersonal factors were very similar. In the current study, self-care was the last function from the interpersonal group to enter the solution, which is comparable to Klonsky and Glenn’s (2009) self-care factor 219 loadings. The status of self-care as a liminal function is supported by the cluster solution that was identified for participants’ most recent episode. In this analysis, self-care moved from the interpersonal into the intrapersonal cluster. Comparisons of the intrapersonal versus interpersonal functions demonstrated that participants were significantly more likely to report self-injuring for intrapersonal than interpersonal reasons, in relation to both a single episode of self-injury and their global episodes of NSSI. Indeed, only nine people endorsed interpersonal functions more highly than intrapersonal functions. This is consistent with the EAM’s (Gratz et al., 2006) exclusion of interpersonal reasons as primary to the maintenance of NSSI. Considering that difficulties in interpersonal relationships were the most commonly reported events to occur prior to participants’ most recent episode of self- injury, it is noteworthy that self-injuring for interpersonal functions was infrequently endorsed. One possibility for this incongruence is that people who self-injure may blame themselves for interpersonal conflict, leading them to consider themselves unworthy of the support or care of others. 5.2 Intrapersonal functions are more highly endorsed than interpersonal functions As such, they may be more likely to self- injure as a form of punishment rather than as a way to access support from others. 5.3 Negative affect decreased and positive affect increased following NSSI Self-reported negative affect, hostility, sadness, and fear all decreased significantly following self-injury, but there were no significant changes in guilt. This is surprising in light of my qualitative analysis where I proposed that people become transgressors because of their self-injurious behaviour, which would seem to suggest that participants’ self-reported guilt should increase following NSSI. Individual ranks show that 71 people experienced a decrease in guilt following self-injury but 62 people experienced an increase. It may be that the qualitative findings are predominantly reflecting the latter group of participants. Although there was no significant difference in participants’ ratings of general positive affect before and after the episode, there was a significant increase in joviality (e.g., happy, joyful, energetic). Furthermore, while not identified by 220 Watson and Clark (1994) as a positive emotion, serenity (i.e., calm, relaxed, at ease) also increased significantly. More specifically, three quarters of the participants reported experiencing increased serenity after they self-injured. This aligns with the positive, open-ended consequences described by participants as one of the most frequently recalled emotion words within the self becomes helper theme was calm. The effectiveness of self-injury as an emotion regulation strategy is supported by other research (e.g., Klonsky, 2007) and forms the basis of the EAM (Chapman et al., 2006). However, it is important to keep in mind that not all participants managed to regulate their emotions through self-injury; indeed, several participants reported feeling worse following their most recent episode. However, it should be noted that participants were not asked to refer to a specific timeframe when reporting the emotions that followed their episode of NSSI. As a result, participants who endorsed improved affect may have been reflecting on how they felt immediately after self- injuring, whereas participants who endorsed worse affect may have been focusing on a more temporally distant time period (i.e., days after having self-injured). 5 4 Negative cognitions decrease following NSSI 5.4 Negative cognitions decrease following NSSI Studying shifts in cognitions following self-injury is a recent development in the NSSI literature (see Nock et al., 2009), but research suggests that attempts to regulate unwanted thoughts may play an important role in the maintenance of self- injury (Najmi et al., 2007). In the current study, negative automatic thoughts reportedly decreased significantly following self-injury, while positive/neutral thoughts increased significantly. However, caution is required when interpreting these results given that the ATQ-R (Kendall et al., 1989) has not been validated for use in reference to a particular episode of behaviour. The high internal consistency scores obtained in the current study for this measure, however, provide some reassurance that the scale is a reliable measure of self-referent thoughts in this context. Similarly to the reports of affect changes following self-injury, there was variability in the number of participants who experienced shifts in cognitions. Some 221 people reported that their negative cognitions increased following NSSI, while others experienced no change in their negative thoughts. A substantial number of people also reported that their positive/neutral cognitions did not change after they self-injured. Although cognitive regulation is minimised within the EAM (Chapman et al., 2006), experiential avoidance theory more generally (cf., Hayes et al., 1996) does not privilege emotion escape/avoidance over cognitive escape/avoidance. It is unclear from this study whether aversive emotions are triggered by unwanted thoughts or whether these thoughts occur in the context of aversive emotions. It is likely a combination of both, although some people’s experience of self-injury may be primarily driven by either cognitive or emotional avoidance. For example, someone with OCD may self-injure specifically as a strategy to cope with intrusive thoughts. Furthermore, the relative absence of cognitive consequences in the open-ended survey responses suggests that self-injury is primarily used for emotion, rather than cognitive, regulation, or that emotional consequences are much more salient and therefore easier to recall. 5.6 Limitations The data collected in this study focused on historical episodes of self-injury and therefore is subject to limitations associated with retrospective self-report (Tourangeau, 2000). By restricting inclusion to people who had self-injured within the past 12 months, I attempted to minimise the impact of retrospection on the data. However, the average amount of time that had lapsed between participants’ most recent episode of self-injury and their participation in the survey was 81 days and this length of time varied considerably between participants. Sophisticated ecological momentary assessment studies are needed to examine the functions of NSSI in more depth without retrospective bias. Another limitation of this study was that I was unable to explicitly assess the role of positive or negative reinforcement in participants’ episodes of self-injury because the ISAS does not categorise the function subscales according to reinforcement value. However, it is possible to deduce from the items whether they reflect escape/avoidance or access motivations. For example, the affect regulation subscale contains items about reducing aversive emotion and it is therefore likely that people who endorse these items engage in NSSI to escape or avoid emotional experiences. 5.5 Strengths Given that it is somewhat atypical for phenomenological studies of self-injury to include previously validated and reliable measures, one of the strengths of this study was the measures chosen to assess the frequency, functions, and emotional antecedents and consequences of NSSI. Additionally, comparing the behavioural trajectories of participants’ general and most recent episodes of self-injury provided further insight into the functional complexity of individual episodes of NSSI. Another strength of the current study and a novel approach to testing whether self- injury functions primarily as a form of experiential avoidance was examining whether the content of participants’ automatic thoughts shifted following self-injury. Finally, the diversity of the sample was also a strength as much of the self-injury research with community populations has focused exclusively on university students. Although the majority of participants in this study were at university, approximately 40% were engaged in other occupations. Given that it is somewhat atypical for phenomenological studies of self-injury to include previously validated and reliable measures, one of the strengths of this study was the measures chosen to assess the frequency, functions, and emotional antecedents and consequences of NSSI. Additionally, comparing the behavioural trajectories of participants’ general and most recent episodes of self-injury provided further insight into the functional complexity of individual episodes of NSSI. Another strength of the current study and a novel approach to testing whether self- injury functions primarily as a form of experiential avoidance was examining whether the content of participants’ automatic thoughts shifted following self-injury. Finally, the diversity of the sample was also a strength as much of the self-injury research with community populations has focused exclusively on university students. Although the majority of participants in this study were at university, approximately 40% were engaged in other occupations. 222 5.7 Conclusion The behavioural trajectory of self-injury described in EAM (Chapman et al., 2006) is broadly consistent with the results of this study, which suggests that the EAM can be usefully applied to New Zealanders’ experiences of self-injury. While this model is not representative of every episode of self-injury, experiential The behavioural trajectory of self-injury described in EAM (Chapman et al., 2006) is broadly consistent with the results of this study, which suggests that the EAM can be usefully applied to New Zealanders’ experiences of self-injury. While this model is not representative of every episode of self-injury, experiential avoidance did appear to be the primary function of NSSI in this sample. Given that self-injury is commonly used to escape or avoid negative emotions and/or thoughts, in the next chapter I focus on assessing whether people who self-injure can be differentiated from people who do not hurt themselves on purpose on the basis of their general intrapersonal experiences and coping styles. 223 1. INTRODUCTION In the interview and survey studies I presented in Chapters 4 and 5, I focused on clarifying the antecedents and consequences of NSSI episodes to determine whether self-injury functions primarily as a form of experiential avoidance within Aotearoa New Zealand. In the current chapter, I take a step back from the immediate behavioural contingencies of self-injury to compare the global intrapersonal experiences (i.e., thoughts and emotions) and coping styles of people who have self- injured with those who have never self-injured. Research on the emotional and cognitive experiences and coping styles of people who self-injure complements functional analyses of NSSI in a number of ways. First, a wealth of studies support the affect-regulation function of NSSI (Klonsky, 2007), making it important to understand more about the general affective experiences of people who self-injure. Given that the complex relationship between affect and cognition is difficult to disentangle (Hayes et al., 1996), it is equally important to consider whether specific types of thoughts differentiate people with a history of NSSI from those without such a history. Indeed, experiencing negative thoughts and feelings about oneself has been found to predict engagement in NSSI among university students (Armey & Crowther, 2008), while the temperament dimension of negative affectivity was shown to predict NSSI among community-based adolescents (Baetens et al., 2011). People with a history of NSSI have also been shown to experience higher levels of negative emotion than people who have never hurt themselves on purpose (Andover et al., 2007; Brown et al., 2007). These findings are useful because they advance our understanding of the broader emotional and cognitive context in which individual episodes of NSSI are situated. 224 Second, although NSSI is frequently referred to as a coping strategy in the literature (e.g., Claes & Vandereycken, 2007; Nock, 2010; Swenson et al., 2008) and functional analyses highlight that self-injury is an adaptive process for managing distress, the taxonomic parameters of this coping response and its relationship to other ways of coping are seldom addressed. One of the few exceptions is the EAM (Chapman et al., 2006) because it proposes that self-injury is an avoidant coping strategy, which belongs in a functional response class with other avoidant behaviours. 1. INTRODUCTION Support for this contention comes from studies showing that people with a history of NSSI use more avoidant coping strategies to deal with stressful events than people who have never self-injured (Andover et al., 2007; Brown et al., 2007). Furthermore, the use of avoidant coping by people who have self-injured has been shown to vary as a function of self-injury severity (Hasking et al., 2008). Given that the intrapersonal experiences of people who self-injure appear to be more negative in general than people who do not self-injure (Brown et al., 2007; Hasking et al., 2008), it seems hardly surprising that they are more likely to utilise avoidant coping strategies (including self-injury) to escape from their distressing emotions and thoughts. Such an assertion, however, assumes a causal direction from negative affect and cognitions to self-injury. It is possible, of course, that self-injury causes negative affect and cognitions. In one of the few prospective studies of NSSI, Hankin and Abela (2011) identified that having a more negative cognitive style predicted future self-injury among adolescents. However, this research is an exception as most of the studies examining the relationships between global intrapersonal experiences, avoidant coping strategies, and NSSI have been cross- sectional, which precludes causal inferences in either direction. Although additional over-time studies are necessary to determine whether negative affect and cognitions cause NSSI, research on the mechanisms underlying NSSI is also sorely needed. To this end, researchers have begun to investigate whether people with a history of NSSI have a general tendency towards avoidant coping. Certainly, it makes intuitive sense that a propensity towards avoidance may 225 underlie people’s continued use of NSSI and evidence in support of this hypothesis would add weight to the contention that self-injury functions as an avoidant coping strategy. A tendency towards thought suppression, the process of attempting to prevent oneself from thinking about certain topics (Wegner & Zanakos, 1994), is a specific form of experiential avoidance that has been implicated as a causal mechanism underlying NSSI (Najmi et al., 2007). In one cross-sectional study, the relationship between adolescents’ self-reported reactivity to emotions and the frequency of NSSI was mediated by thought suppression (Najmi et al., 2007); in another study, the relationship between past experience of childhood sexual abuse and NSSI frequency was mediated by avoidance symptoms of PTSD (Weierich & Nock, 2008). 1. INTRODUCTION While these findings suggest that the desire to avoid unwanted thoughts may play a key role in perpetuating self-injurious behaviours, these results need to be replicated longitudinally to confirm the hypothesised causal pathways (Najmi et al., 2007; Weierich & Nock, 2008). In the current study, I attempted to expand on my previous findings—that NSSI functions as both emotional and cognitive avoidance—by examining how people who have self-injured think, feel, and cope, compared with people who have never self-injured. More specifically, I hypothesised that people with a history of self-injury would report experiencing higher levels of general negative affect (Brown et al., 2007; Hasking et al., 2008), more negative automatic thoughts (Hankin & Abela, 2011), and have a greater tendency towards avoidant coping (Andover et al., 2007; Brown et al., 2007) than people without a history of self-injury. If people do use NSSI as a strategy to avoid experiencing negative emotions and thoughts, it seems probable that they would report higher levels of general negative affect and aversive thoughts than people who do not self-injure. Furthermore, in light of the dearth of over-time studies, I chose to survey participants about their affect, cognitions, and coping styles at two time-points, two months apart, to test whether negative affect and cognitions would predict new 226 incidents of NSSI. Certainly, the EAM (Chapman et al., 2006) and previous empirical findings (Hankin & Abela, 2011) suggest that negative intrapersonal experiences should place people at risk of engaging in future episodes of self-injury. Consequently, I hypothesised that negative intrapersonal experiences reported at Time 1 (T1) would be causally related to new episodes of NSSI reported at Time 2 (T2). Finally, if a predictive relationship between negative intrapersonal experiences and NSSI was identified, I hypothesised that this relationship would be mediated by a tendency towards experiential avoidance (Najmi et al., 2007). 2 METHOD incidents of NSSI. Certainly, the EAM (Chapman et al., 2006) and previous empirical findings (Hankin & Abela, 2011) suggest that negative intrapersonal experiences should place people at risk of engaging in future episodes of self-injury. p p p g g g p j y Consequently, I hypothesised that negative intrapersonal experiences reported at Time 1 (T1) would be causally related to new episodes of NSSI reported at Time 2 (T2). 1. INTRODUCTION Finally, if a predictive relationship between negative intrapersonal experiences and NSSI was identified, I hypothesised that this relationship would be mediated by a tendency towards experiential avoidance (Najmi et al., 2007). 2 METHOD Consequently, I hypothesised that negative intrapersonal experiences reported at Time 1 (T1) would be causally related to new episodes of NSSI reported at Time 2 (T2). Finally, if a predictive relationship between negative intrapersonal experiences and NSSI was identified, I hypothesised that this relationship would be mediated by a tendency towards experiential avoidance (Najmi et al., 2007). 2 METHOD 2.1 Participants A total of 443 (excluding repeat entries) first year Psychology students from Victoria University of Wellington consented to participate in the T1 phase of this study, but only 408 (92%) surveys were retained for analysis (see Figure 9 for rationale). Almost three quarters of the participants were female (72.1%); 113 (27.7%) were male, and 1 (0.2%) was male-to-female transsexual. The average age of participants was 19.46 years (SD = 3.43), with a range of 17 to 44 years. Although the majority of participants identified as New Zealand European (83.3%), a variety of ethnicities were reported: 34 (8.3%) Māori, 14 (3.4%) Chinese, 9 (2.2%) Indian, 7 (1.7%) Samoan, 4 (1.0%) Cook Islands Maori, and 43 (10.5%) other (e.g., South African, Japanese). One or more mental health diagnoses were reported by 67 (16.4%) of the participants, with an average of 1.46 (SD = 0.75) diagnoses per person (range: 1-5). Out of the participants who reported receiving a mental health diagnosis in their lifetimes, 58 (87.9%) had been diagnosed with Depression, 24 (36.4%) with Anxiety, 6 (9.1%) with Anorexia, 4 (6.1%) with BPD, 3 (4.5%) with Bulimia, and 1 (1.5%) each with Bipolar Disorder, Substance Use Disorder and Drug-induced Psychosis. More than one third of the participants (37.5%) had received therapy or counselling in the past, but only 19 (4.7%) were currently engaged in therapy or counselling. 227 \ Excluded (N = 82)  Repeat entries (N = 47)  ≥ 62.5% of survey sections incomplete (N = 6)  Asperger Disorder diagnosis (N = 1)  No T1 NSSI, but T2 NSSI (N = 6)  T1 NSSI, but no T2 NSSI (N = 12) Included in T1 analyses (N = 408) Excluded (N = 42)  Repeat entries (N = 4)  ≥ 62.5% of survey sections incomplete (N = 9)  Asperger Disorder diagnosis (N = 1)  No T1 NSSI, but NSSI at T2 (N = 16)  NSSI at T1, but T2 NSSI (N = 12) Included in T2 analyses (N = 224) TIME 1 TIME 2 Consented to participate in T1 survey (N = 490) Consented to participate in T2 survey (N = 266) TIME 1 Figure 9. Flow of participants through the study at Times 1 and 2. Figure 9. Flow of participants through the study at Times 1 and 2. Out of the 262 (excluding repeat entries) surveys completed at T2, 224 were retained for analysis (see Figure 9 for rationale). Prior to deleting cases, the response rate for T2 was 59.1%. Chi-square tests of independence at an alpha level of .05 were used to determine whether there were any significant differences between the participants who only completed the T1 survey and those who completed both surveys. Although participants who completed the T1, but not the T2 survey, were 228 significantly more like to be male28 (Χ2(1, N = 407) = 17.30, p < .001), they were just as likely to report a history of mental illness (Χ2(1, N = 405) = 1.65, p = .20) and/or self- injury (Χ2(1, N = 408) = .107, p = .74). The one transsexual student was omitted from this analysis. 29 One student completed the second survey three weeks after the initial reminder was sent. Th survey was included in the analyses. 2.2 Procedure First year Psychology students at Victoria University of Wellington are required to participate in four hours of research each trimester to fulfill their course requirements. The current study was listed on the Psychology Department’s Experimetrix.com website, where students are presented with a range of study choices. Students who do not want to take part in research studies are able to summarise published journal articles instead. Prospective participants were advised prior to sign-up that participation would involve completing two online surveys, both of which included questions about NSSI. Students who had a history of NSSI and those who had never self-injured were invited to participate. Once they had signed up, they were required to click ‚yes‛ at the end of the information page to indicate that they consented to participate in the study before they were directed through to the first survey. Those who did not consent to participate were directed to a page that contained the following message: ‚You did not consent to participate in this survey. Please close your browser window to exit‛. The study was conducted across two time-points: (1) participants completed an online survey (see Appendix K) via Surveymonkey and (2) two months later, they were emailed a link to the same survey (excluding the demographic questions) and invited to complete it a second time. Each participant was given two weeks to complete the second survey29 and was sent up to two reminders. Surveys were matched using the student ID numbers that participants provided at each time point. The two-month time period between survey completions was chosen for pragmatic reasons to ensure that the study could be completed within one university trimester. 229 As with the online survey described in the previous chapter, anyone who started either of the surveys for the current study was emailed a copy of the contact details for various support organisations (see Appendix L). This was done to ensure that any participants who did not finish the survey would still have access to relevant debriefing information. The options for support were also presented on the final pages of both the T1 and T2 surveys. Finally, it was suggested to participants on the survey information page that they ask a support person to be available while they complete the survey if appropriate. 2.3.1 The Deliberate Self-harm Inventory The modified version of the DSHI (Gratz, 2001) used in the second study to measure the prevalence and frequency of 14 different types of NSSI was also used in the current study. These modifications are described in detail in Chapter 5 (see p. 179) and the psychometric properties of this measure are described in Chapter 4 (see p. 101). 30 I intended to complete an ecological momentary assessment study, but a change in ethics policy at Victoria University meant that the ethics committee could no longer consider my application. As a result of the significant delays I experienced following this policy change, I was unable to follow through with my original study and had to amend my research design. 2.2 Procedure Each student received half an hour of research credits for completing the T1 survey and a $10 Motor Trade Association voucher for completing the T2 survey. The study30 was approved by the Multi- region Ethics Committee, a New Zealand Health and Disability Ethics Committee administered by the Ministry of Health. 2.3.2 The Acceptance and Action Questionnaire The Acceptance and Action Questionnaire (AAQ; Hayes et al., 2004) is a 9- item, self-report questionnaire designed to measure experiential avoidance. Each item (e.g., ‚If I could magically remove all the painful experiences I've had in my life, I would do so‛) is rated on a seven point likert scale from 1 (never true) to 7 (always true); four items are reverse scored. Responses are summed to give a maximum score 30 I intended to complete an ecological momentary assessment study, but a change in ethics policy at Victoria University meant that the ethics committee could no longer consider my application. As a result of the significant delays I experienced following this policy change, I was unable to follow through with my original study and had to amend my research design. 230 of 63 with higher scores indicating a stronger tendency towards experiential avoidance. of 63 with higher scores indicating a stronger tendency towards experiential avoidance. The AAQ has demonstrated adequate internal consistency (α = .70) and test- retest reliability (r = .65); fairly low test-retest reliability is to be expected given that experiential avoidance is conceptualised as a behavioural process, which in all likelihood is amenable to contextual influences (Hayes et al., 2004). Convergent validity with thought suppression and psychopathology measures has also been established (Hayes et al., 2004). The only time this measure has been used in a self- injury study, the authors did not report any study-specific psychometric statistics (Chapman, Specht, & Cellucci, 2005). 2.3.3 The Positive and Negative Affect Schedule The PANAS-X (Watson & Clark, 1994) is a 60-item, self-report measure that contains the following 13 scales: negative affect, positive affect, fear, hostility, guilt, sadness, joviality, self-assurance, attentiveness, shyness, fatigue, serenity, and surprise. The emotions included in each of these scales are listed in Chapter 5 (see p. 181). Each emotion word (e.g., angry, joyful) is rated on a five point scale from 1 (very slightly or not at all) to 5 (extremely). Mean scores are calculated for each of the 13 scales. In the current study, the PANAS-X was used to measure affect experienced by participants in general; accordingly, the general timeframe instructions were chosen (i.e., ‚Indicate to what extent you feel this way in general, that is, on the average‛). As stated in Chapter 5, the excellent psychometric properties of the PANAS-X are well-established (Watson & Clark, 1994). For example, internal consistency rates across six samples for the negative affect scale when completed according to the general timeframe instructions ranged from .85 to .93 (Watson & Clark, 1994). 2 3 4 The White Bear Suppression Inventory 2.3.4 The White Bear Suppression Inventory The White Bear Suppression Inventory (WBSI; Wegner & Zanakos, 1994) is a 15-item, self-report measure of people’s propensity to suppress unwanted thoughts. Each item (e.g., ‚There are things that I try not to think about‛) is rated from 1 231 (strongly disagree) to 5 (strongly agree). Responses are summed to give a maximum score of 75; higher scores indicate a greater tendency towards thought suppression. The WBSI has demonstrated good internal consistency (α = .85 - .89) across a number of studies (Blumberg, 2000; Rassin, 2003; Wegner & Zanakos, 1994). Scores on the WBSI have been found to converge with scores on well-validated depression and anxiety measures (Wegner & Zanakos, 1994), and NSSI frequency (Chapman et al., 2005; Najmi et al., 2007). 2.3.5 The Depression Anxiety Stress Scales The short version of the Depression Anxiety Stress Scales (DASS-21; Lovibond & Lovibond, 1995) is a 21-item, self-report measure developed to test the occurrence of three connected constructs—depression, anxiety, and tension/stress—within the past week. Each construct is operationalised in a 7-item subscale; item examples include: ‚I felt that I had nothing to look forward to‛ (depression), ‚I felt scared without any good reason‛ (anxiety), and ‚I found it hard to wind down‛ (tension/stress). Items are rated on a four point scale from 0 (did not apply to me at all) to 3 (applied to me very much, or most of the time). Scores are summed to give a maximum of 21 for each subscale and 63 for the total score. Cronbach alphas for the DASS-21 subscales range from .82 to .94, which suggest it is a highly reliable measure (Antony, Bieling, Cox, Enns, & Swinson, 1998; Henry & Crawford, 2005). It has also demonstrated adequate construct validity (Henry & Crawford, 2005). 2 3 6 The Automatic Thoughts Questionnaire – short version 2.3.6 The Automatic Thoughts Questionnaire – short version Netemeyer and colleagues (2002) constructed a 15-item version of the original 30-item Automatic Thoughts Questionnaire (ATQ; Hollon & Kendall, 1980) by factor-analysing the ATQ and inspecting the content of individual items. Although the psychometric properties of the shortened version have not been extensively tested, it demonstrated excellent internal consistencies (α = .92 - .96) across three samples (Netemeyer et al., 2002). The frequencies of specific, negative automatic thoughts (e.g., ‚I’m no good‛, ‚It’s just not worth it‛) experienced within the past week are rated on a five point scale from 1 (not at all) to 5 (all the time). Scores are 232 summed to total a maximum of 75, with higher scores signifying more pervasive negative thoughts. 3.1 Prevalence and frequencies of self-injury types The prevalence and frequencies of NSSI types for both time-points are presented in Table 12. At T1, almost half the participants (46.8%) reported having engaged in NSSI in their lifetimes. The average age of onset for NSSI was 13.17 years (SD = 2.84; range: 3-19), with cutting (24.8%), severe scratching (21.8%), and sticking sharp objects into the skin (19.9%) the most commonly reported types of self-injury. The least common types of NSSI were rubbing sandpaper on the body (2.7%), dripping acid onto the skin (2.0%), and using bleach or oven cleaner to scrub the skin (0.2%). No participants reported breaking their own bones. Additionally, 16 (3.9%) participants endorsed engaging in at least one other type of NSSI that was not listed in the DSHI (e.g., ‚rubbed steel wool against skin‛, ‚scalded self with boiling water‛). The 191 participants who reported self-injuring had engaged in an average of 3.12 (SD = 2.26) types of NSSI, with a range of 1 to 13. The prevalence and frequencies of NSSI types for both time-points are presented in Table 12. At T1, almost half the participants (46.8%) reported having engaged in NSSI in their lifetimes. The average age of onset for NSSI was 13.17 years (SD = 2.84; range: 3-19), with cutting (24.8%), severe scratching (21.8%), and sticking sharp objects into the skin (19.9%) the most commonly reported types of self-injury. The least common types of NSSI were rubbing sandpaper on the body (2.7%), Despite the high lifetime prevalence of self-injury at T1, only 56 (32.0%) of the participants who endorsed a history of NSSI reported having injured themselves on purpose within the past 12 months. In the two months prior to T1 survey completion, a total of 42 participants had injured themselves on average 7.26 times (SD = 17.76; range: 1-100). A more detailed breakdown of self-injury recency for both time points is presented in Table 13. Despite the high lifetime prevalence of self-injury at T1, only 56 (32.0%) of the participants who endorsed a history of NSSI reported having injured themselves on purpose within the past 12 months. In the two months prior to T1 survey completion, a total of 42 participants had injured themselves on average 7.26 times (SD = 17.76; range: 1-100). A more detailed breakdown of self-injury recency for both time points is presented in Table 13. 3. RESULTS 3. RESULTS 2.3.7 The Brief COPE The Brief COPE was developed by Carver (1997) to reduce the participant response burden associated with the 60-item COPE inventory (Carver, Scheier, & Weintraub, 1989). Coping styles, rather than situational responses, were assessed in the current study and, as such, the instructions and items were presented in a dispositional format (Carver et al., 1989). All but two of the COPE scales are present in the Brief COPE, but there are only two, rather than four, items per scale. Carver (1997) also added a new two item scale, self-blame, to the Brief COPE. In total, the Brief COPE is comprised of the following 14 scales: self- distraction (e.g., ‚I turn to work or other activities to take my mind off things‛), active coping (e.g., ‚I take action to try to make the situation better‛), denial (e.g., ‚I refuse to believe that it has happened‛), substance use (e.g., ‚I use alcohol or other drugs to make myself feel better‛), use of emotional support (e.g., ‚I get emotional support from others‛), use of instrumental support (e.g., ‚I get help and advice from other people‛), behavioural disengagement (e.g., ‚I give up trying to deal with it‛), venting (e.g., ‚I express my negative feelings‛), positive reframing ( e.g., ‚I look for something good in what is happening‛), planning (e.g., ‚I think hard about what steps to take‛), humour (e.g., ‚I make jokes about it‛), acceptance (e.g., ‚I learn to live with it‛), religion (e.g., ‚I pray or meditate‛), and self-blame (e.g., ‚I blame myself for things that happened‛). Each item is rated on a four point scale from 1 (I usually don’t do this at all) to 4 (I usually do this a lot). Item responses are summed to give a maximum score of eight per scale. Averaged scale reliabilities have been found to vary considerably from .50 (venting) to .90 (substance use) but caution is required when interpreting internal consistency values in measures with only two items per scale (Carver, 1997). 233 3.1 Prevalence and frequencies of self-injury types As is to be expected given the prevalence of NSSI at T1, almost half (47.8%) of the participants endorsed a lifetime history of NSSI at T2. The most frequently endorsed types of NSSI at T2 were the same as T1: cutting (29.5%), severe scratching (20.5%), and sticking sharp objects into the skin (20.5%). The least frequently endorsed types of NSSI at T2 were also the same as T1: rubbing sandpaper on the body (2.2%), dripping acid (2.2%) onto the skin, and using bleach or oven cleaner (0.9%) to scrub the skin. Once again, no participants reported breaking bones. 3.1 Prevalence and frequencies of self-injury types Comparable to T1 reports of NSSI, the participants who reported a history of NSSI at Comparable to T1 reports of NSSI, the participants who reported a history of NSSI at Table 12 Frequencies of NSSI types reported at Times 1 and 2 Frequencies of NSSI types reported at Times 1 and 2 NSSI type Time 1 (N = 408) Time 2 (N = 224) N (%) endorsing NSSI type Frequency of NSSI types N (%) endorsing NSSI type Frequency of NSSI types Never 1 time 2-10 times 11-50 times >50 times Never 1 time 2-10 times 11-50 times >50 times Cutting wrists, arms, or other areas of body 101 (24.8) 307 (75.2) 14 (3.4) 50 (12.3) 25 (6.1) 11 (2.7) 66 (29.5) 157 (70.1) 8 (3.6) 34 (15.2) 15 (6.7) 6 (2.7) Severe scratching to extent of bleeding/ scarring 89 (21.8) 315 (77.2) 16 (3.9) 44 (10.8) 20 (4.9) 5 (1.2) 46 (20.5) 172 (76.8) 4 (1.8) 26 (11.6) 8 (3.6) 3 (1.3) Sticking sharp objects into skin 81 (19.9) 325 (79.7) 12 (2.9) 54 (13.2) 9 (2.2) 0 (0.0) 46 (20.5) 175 (78.1) 2 (0.9) 33 (14.7) 5 (2.2) 0 (0.0) Carving words/pictures/ designs/marks into skin 70 (17.2) 337 (82.6) 33 (8.1) 30 (7.4) 3 (0.7) 1 (0.2) 44 (19.6) 180 (80.4) 16 (7.1) 23 (10.3) 1 (0.4) 0 (0.0) Burning with cigarette/ lighter/match 54 (13.2) 350 (85.8) 15 (3.7) 33 (8.1) 5 (1.2) 0 (0.0) 31 (13.8) 192 (85.7) 6 (2.7) 22 (9.8) 0 (0.0) 0 (0.0) Preventing wounds from healing 50 (12.3) 355 (87.0) 2 (0.5) 23 (5.6) 15 (3.7) 8 (2.0) 34 (15.2) 189 (84.4) 4 (1.8) 14 (6.3) 4 (1.8) 6 (2.7) Banging head to extent of bruising 50 (12.3) 357 (87.5) 10 (2.5) 27 (6.6) 7 (1.7) 1 (0.2) 23 (10.3) 195 (87.1) 1 (0.4) 16 (7.1) 1 (0.4) 1 (0.4) Punching to extent of bruising 40 (9.8) 367 (90.0) 9 (2.2) 28 (6.9) 1 (0.2) 0 (0.0) 19 (8.5) 203 (90.6) 1 (0.4) 11 (4.9) 3 (1.3) 0 (0.0) Biting to extent of breaking skin 24 (5.9) 384 (94.1) 6 (1.5) 15 (3.7) 3 (0.7) 0 (0.0) 16 (7.1) 201 (89.7) 5 (2.2) 8 (3.6) 1 (0.4) 1 (0.4) Rubbing glass into skin 16 (3.9) 386 (94.6) 6 (1.5) 9 (2.2) 1 (0.2) 0 (0.0) 7 (3.1) 212 (94.6) 2 (0.9) 4 (1.8) 0 (0.0) 0 (0.0) Rubbing sandpaper on body 11 (2.7) 396 (97.1) 9 (2.2) 2 (0.5) 0 (0.0) 0 (0.0) 5 (2.2) 216 (96.4) 4 (1.8) 0 (0.0) 0 (0.0) 0 (0.0) Dripping acid onto skin 8 (2.0) 395 (96.8) 3 (0.7) 5 (1.2) 0 (0.0) 0 (0.0) 5 (2.2) 214 (95.5) 4 (1.8) 1 (0.4) 0 (0.0) 0 (0.0) Using bleach/oven cleaner to scrub skin 1 (0.2) 406 (99.5) 1 (0.2) 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.9) 221 (98.7) 0 (0.0) 1 (0.4) 0 (0.0) 0 (0.0) Breaking own bones 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Note. 3.1 Prevalence and frequencies of self-injury types N endorsing NSSI type may not equal frequencies of engagement as some participants endorsed type of NSSI but did not report frequency. Frequencies may not add up to 100% because of missing data. Time 1 (N = 408) Time 2 (N = 224) Time 2 (N = 224) 234 235 T2 had engaged in an average of 3.27 (SD = 2.27) types of self-injury with a range of 1 to 10. NSSI recency at Times 1 and 2 NSSI recency at Times 1 and 2 NSSI recency at Times 1 and 2 Recency of last NSSI episode Time 1 (N = 172) Time 2 (N = 106) N (%) N (%) < 1 week 13 (3.2) 7 (6.6) 1 week > 1 month 11 (2.7) 6 (5.7) 1 month > 6 months 21 (5.1) 16 (15.1) 6 months > 1 year 11 (2.7) 16 (15.1) 1 year > 2 years 18 (15.1) 9 (14.8) 2 years > 3 years 19 (16.0) 10 (16.4) 3 years > 4 years 26 (21.8) 12 (19.7) 4 year > 5 years 15 (12.6) 10 (16.4) > 5 years 38 (31.9) 20 (32.8) The order of frequency for the different types of NSSI was identical at T2 to T1, with one exception; burning oneself with a cigarette, lighter, or match was more frequently endorsed than preventing wounds from healing at T1 than T2. At T2, other forms of NSSI (e.g., ‚hit myself on the head with a rock’, ‚flagellation‛) were endorsed by 9 (4.0%) participants. A total of 23 people reported self-injuring on average 6.43 times (SD = 12.72; range: 1-60) in the two months between completing the T1 and T2 surveys. Out of the 23 people who reported self-injuring between T1 and T2, a total of 11 also reported self-injuring in the two months prior to T1. 3.2 Do people who have self-injured experience more negative emotions and thoughts? 31 Two of the scores were from the ATQ and DASS anxiety scales, while three scores were from the DASS stress scale. 31 Two of the scores were from the ATQ and DASS anxiety scales, while three scores were from the DASS stress scale. Note. 1 N’s range from 213 to 217, 2 N’s range from 185 to 191 due to missing values. p < 001 m 213 to 217, 2 N’s range from 185 to 191 due to missing values. nge from 185 to 191 due to missing values. thoughts? To examine whether people who have self-injured generally experience higher levels of negative emotions and thoughts than people who have never self- injured, I planned to conduct a multivariate analysis of variance (MANOVA) using the T1 negative affect and emotion (i.e., fear, hostility, guilt, and sadness) scores 236 from the PANAS-X, the T1 depression, anxiety, and stress scores from the DASS-21, and the T1 ATQ-R total scores. All of these scales demonstrated good to excellent internal consistency (α = .82 - .96) as is evident in Table 14. Prior to running the MANOVA, visual inspection of the histograms and Q-Q plots for these nine variables showed that their distributions were positively skewed with varying degrees of kurtosis (see Table 14). Kolmogorov-Smirnov tests with Lilliefors Correction (Field, 2009) confirmed that the distributions for all of the variables for both groups were significantly non-normal, D’s(185-217) ≥.10 (all p’s < .001). To test for univariate outliers, I converted all of the scores for these nine variables into z-scores to check whether any were less than -3.29 or greater than 3.29 as recommended by Tabachnik and Fidell (2007). In total, seven scores31 from different participants were identified as possible outliers. Removing these scores did not alter the significance of subsequent normality tests and, as a result, they were retained in the analyses. Despite the significantly non-normal distributions of these and other variables analysed for this chapter, I chose to continue with MANOVA tests for the following reasons. First, the sample size was large enough to reduce the impact of skewness and kurtosis on the analyses (Tabachnick & Fidell, 2007). Although positive and negative kurtosis can lead to underestimates of variance, this typically only applies to samples that consist of less than 100 or 200 cases respectively (Tabachnick & Fidell, 2007; Waternaux, 1976). Second, MANOVAs are thought to be robust to deviations from normality when Pillai’s trace is used (Olson, 1974). Transforming the data was considered unnecessary given these guidelines and undesirable since transformation alters the constructs under investigation (Grayson, 2004). Finally, the most pragmatic reason to use MANOVA is simply that there are no commonly used non-parametric alternatives (Field, 2009). p < .001 All df’s = 1, 384-406. thoughts? Table 14 Table 14 Time 1 reliability coefficients, descriptive statistics, and ANOVAs for the No NSSI versus Lifetime NSSI groups descriptive statistics, and ANOVAs for the No NSSI versus Lifetime NSSI groups Time 1 reliability coefficients, descriptive statistics, and ANOVAs for the No NSSI ve Time 1 reliability coefficients, descriptive statistics, and ANOVAs for the No NSSI versus Lifetime NSSI groups No history of NSSI1 Lifetime history of NSSI2 ANOVAs Variable Cronbach’s α M (SD) Skewness Kurtosis M (SD) Skewness Kurtosis F ηp2 PANAS-X Negative affect .89 2.00 (0.61) 0.51 -0.45 2.39 (0.72) 0.52 -0.40 35.40* .08 Fear .86 1.97 (0.67) 0.54 -0.51 2.27 (0.76) 0.65 -0.26 17.80* .04 Hostility .83 1.87 (0.62) 0.74 0.21 2.25 (0.72) 0.47 -0.40 32.00* .08 Guilt .91 1.78 (0.73) 0.83 -0.09 2.32 (0.96) 0.78 -0.10 37.66* .09 Sadness .90 2.09 (0.79) 0.73 -0.22 2.57 (0.95) 0.38 -0.70 29.50* .07 ATQ-R .96 22.70 (9.61) 1.94 4.20 28.70 (13.78) 1.28 0.79 22.05* .05 DASS-21 Depression .89 3.16 (3.43) 1.44 1.80 5.28 (4.73) 0.95 -0.06 23.72* .06 Anxiety .82 3.21 (3.23) 1.22 1.09 4.68 (4.21) 1.01 0.16 13.79* .04 Stress .85 4.74 (3.70) 0.70 -0.16 6.68 (4.66) 0.77 -0.13 18.71* .05 WBSI .92 45.60 (11.92) -0.35 -0.18 51.59 (11.61) -0.45 0.05 15.14* .04 AAQ .73 32.92 (7.26) 0.18 -0.29 35.65 (7.44) 0.28 -0.22 29.30* .07 COPE Self-distraction .57 4.88 (1.43) 0.21 -0.14 5.25 (1.60) 0.03 -0.73 5.35 .01 Denial .69 2.71 (1.10) 1.69 2.53 2.67 (1.14) 1.98 6.57 0.10 .00 Substance use .91 2.79 (1.30) 1.85 3.33 3.49 (1.90) 1.16 0.27 19.97* .05 Behavioural disengagement .70 2.71 (1.03) 1.44 1.33 3.13 (1.33) 1.27 1.16 13.33* .03 Note 1 N’s range from 213 to 217 2 N’s range from 185 to 191 due to missing values 237 238 Non-parametric bivariate correlations revealed that significant positive relationships existed between all of the variables under investigation. Given that multicollinearity can reduce the power of MANOVA tests, Tabachnick and Fidell (2007) recommend carefully considering the inclusion of any variables that are correlated at or above .70, and excluding those at or above .90. As to be expected, the highest correlations (rs = .78 - .90, all p’s ≥ .001) were observed between the negative affect and four negative emotion scales (fear, hostility, sadness, and guilt) from the PANAS-X. 32 Removing three multivariate outliers (p-values of Mahalanobis D2 < .001) increased the value of the F-statistic to 6.09. Since the test was significant regardless of whether these cases were included or not, they were retained in the analysis. in the analysis. 33 Non-parametric Kruskal-Wallis tests yielded comparable significance values to the ANOVAs. thoughts? To avoid multicollinearity, the negative affect scale was excluded from the MANOVA; instead, the impact of self-injury history on global negative affect was tested using a one-way independent analysis of variance (ANOVA). A MANOVA32 using Pillai’s trace showed a significant effect of NSSI history on the intensity and/or frequency of negative emotions and thoughts experienced by participants, V = 0.11, F(8, 377) = 5.58, p < .001, partial eta squared = .11. The possibility of Type I error was inflated for this and all other MANOVAs presented in this chapter because the covariance matrices were significantly different (Box’s M = 124.74, F (36, 472353) = 3.39, p < .001). However, Box’s M needs to be interpreted with caution given that it is a sensitive test (Tabachnik & Fidell, 2007) and Type I error is less likely if the test statistic is still significant at a reduced alpha of .001. Follow-up ANOVAs33, using a Bonferroni corrected alpha of .006 (Field, 2009), showed that participants who had self-injured reported significantly higher levels of fear, hostility, guilt, sadness, depression, anxiety, and stress, as well as significantly more negative automatic thoughts than participants who had never self-injured (see Table 13). An ANOVA conducted to test whether global negative affect varied as a function of self-injury history also showed a significant effect, F(1,406) = 35.40, p < .001, partial eta squared = .08, with participants who had self-injured reporting higher negative affect. 239 Given that the extent and/or intensity of negative emotions and thoughts varied significantly as a function of self-injury history, further analyses were carried out to determine whether recency of self-injurious behaviours would impact on these differences. To achieve this, participants with a history of self-injury were divided into two groups on the basis of whether or not they reported self-injuring in the previous 12 months. This resulted in a total of three groups for the subsequent analyses: No NSSI, Lifetime NSSI (i.e., had self-injured but not in the past 12 months), and 12-month NSSI (i.e., had self-injured in the past 12 months). Exploratory analyses (i.e., histograms and Q-Q plots) showed that although all of the variables in the three groups exhibited skewness and kurtosis, not all of these distributions were significantly non-normal. 34 Removing three multivariate outliers (p-values of Mahalanobis D2 < .001) increased the value of the F-statistic to 4.91. Since the test was significant regardless of whether these cases were included or not, they were retained in the analysis. in the analysis. 35 Kruskal-Wallis tests yielded comparable significance values to the ANOVAs. thoughts? As presented in Table 14, Kolmogorov-Smirnov tests with Lilliefors correction indicated that the distributions for all the variables in the No NSSI and Lifetime NSSI groups were significantly non- normal (D’s(115-217) ≥ .10, p’s < .001), but that only the ATQR and DASS subscales in the 12-month NSSI group were significantly non-normal D’s(54-56) ≥ .15, p’s ≤ .002. Multivariate tests34 using Pillai’s trace showed that there was a significant effect of recency of NSSI on the intensity of negative emotions and thoughts experienced by participants, V = .19, F(16, 724) = 4.63, p < .001, partial eta squared = .09. The covariance matrices were significantly different (Box’s M = 175.55, F(72, 76924) = 2.33, p < .001). Follow-up ANOVAs35 with Bonferroni corrected alpha of .006 demonstrated that all of the variables of interest varied significantly as a function of NSSI recency (see Table 14). Furthermore, negative affect was also found to vary significantly as a function of NSSI recency, F(2, 389) = 22.62, p < .001, partial eta squared = .10. a, b, c Means with superscripts are significantly different to other group means. Note. 1 N’s range from 213-217, 2 N’s range from 115-119 and 3 N’s range from 54-56 due to missing values. * p < .001 p All df’s = 2, 368 except # df’s = 2, 379. p b, c Means with superscripts are significantly different to other group means. Note. 1 N’s range from 213-217, 2 N’s range from 115-119 and 3 N’s range from 54-56 due to missing values. * p < .001 2, 368 except # df’s = 2, 379. All df’s = 2, 368 except # df’s = 2, 379. All df s = 2, 368 except # df s = 2, 379. a, b, c Means with superscripts are significantly different to other group means. 240 s range from 213-217, 2 N’s range from 115-119 and 3 N’s range from 54-56 due to missing values. xcept # df s 2, 379. uperscripts are significantly different to other group means. N’s range from 54-56 due to missing values. thoughts? Table 14 Table 14 Time 1 descriptive statistics and ANOVAs for the No NSSI, Lifetime NSSI, and 12-month NSSI groups Time 1 descriptive statistics and ANOVAs for the No NSSI, Lifetime NSSI, and 12-month NSSI groups No NSSI1 a Lifetime NSSI2 b 12-month NSSI3 c ANOVAs Variable M (SD) Skewness Kurtosis M (SD) Skewness Kurtosis M (SD) Skewness Kurtosis F ηp2 PANAS-X Negative affect 2.00 (0.61) b, c 0.51 -0.45 2.26 (0.65) a, c 0.68 -0.01 2.63 (0.79) a, b 0.12 -0.90 22.62* .10 Fear 1.97 (0.67) c 0.54 -0.51 2.15 (0.70) c 0.94 0.59 2.50 (0.85) a, b 0.22 -0.79 13.53* .07 Hostility 1.87 (0.62) b, c 0.74 0.21 2.13 (0.70) a, c 0.89 0.46 2.46 (0.69) a, b -0.15 -0.41 20.31* .10 Guilt 1.78 (0.73) b, c 0.83 -0.09 2.12 (0.87) a, c 1.05 0.80 2.77 (1.04) a, b 0.13 -0.96 30.34* .14 Sadness 2.09 (0.79) b, c 0.73 -0.22 2.40 (0.90) a, c 0.78 -0.03 2.98 (0.93) a, b -0.38 -0.47 26.87* .13 ATQ-R 22.70 (9.61) c 1.94 4.20 25.91 (11.78) c 1.66 2.79 35.44 (15.21) a, b 0.74 -0.75 26.19* .13 DASS-21 Depression 3.16 (3.43) c 1.44 1.80 4.30 (4.09) c 1.31 1.38 7.57 (5.07) a, b 0.33 -1.21 28.31* .13 Anxiety 3.21 (3.23) c 1.22 1.09 4.10 (3.93) c 1.25 0.79 6.07 (4.50) a, b 0.69 -0.36 12.41* .06 Stress 4.74 (3.70) c 0.70 -0.16 5.88 (4.31) c 0.69 -0.46 8.28 (4.67) a, b 0.80 -0.11 15.25* .08 WBSI # 45.60 (11.92) b, c -0.35 -0.18 49.64 (11.43) a, c -0.41 0.12 55.64 (11.25) a, b -0.93 0.85 18.12* .09 AAQ # 32.92 (7.26) c 0.18 -0.29 34.13 (6.91) c 0.26 -0.26 39.29 (7.19) a, b 0.17 -0.43 18.87* .09 COPE # Self-distraction 4.88 (1.43) c 0.21 -0.14 5.12 (1.61) 0.16 -0.71 5.54 (1.54) a -0.04 -0.88 4.19 .02 Denial 2.71 (1.10) 1.69 2.53 2.61 (1.13) 2.15 4.27 2.75(1.18) 1.82 3.17 0.36 .00 Substance use 2.79 (1.30) c 1.85 3.33 3.14 (1.65) c 1.43 1.26 3.98 (2.00) a, b 0.87 -0.33 14.07* .07 Behavioural disengagement 2.71 (1.03) c 1.44 1.33 2.99 (1.26) 1.52 2.32 3.39 (1.40) a 0.96 0.16 8.69* .04 Note 1 N’s range from 213-217 2 N’s range from 115-119 and 3 N’s range from 54-56 due to missing values 240 241 Games-Howell post-hoc comparisons conducted at an alpha level of .05 revealed several significant differences in the intensity and/or presence of negative emotions and thoughts between groups. thoughts? Participants who had self-injured in the past 12 months reported significantly higher mean levels of negative affect, hostility, guilt, and sadness than participants who had self-injured more than 12 months ago and those who had never self-injured. Participants who had self-injured more than 12 months ago in turn reported significantly higher mean levels of negative affect, hostility, guilt, and sadness than participants who had never self-injured. Self-reported mean levels of stress, depression, anxiety, fear, and frequency of negative automatic thoughts were significantly greater for participants who had self- injured in the past 12 months compared to those who had self-injured in their lifetimes or who had never engaged in NSSI. Participants who had self-injured more than 12 months ago did not differ significantly from participants who had never self- injured on stress, depression, anxiety, fear, and frequency of negative automatic thoughts. In sum, the intensity of aversive emotions and frequency of negative thoughts experienced by participants varied significantly as a function of how recently they had self-injured. 3.3 Do people who have self-injured have a greater tendency towards avoidant coping? 3.3 Do people who have self-injured have a greater tendency towards avoidant y 37 Kruskall-Wallis tests resulted in comparable significance levels for the AAQ, WBSI, and substance use scale (p < .001), but significance levels for the behavioural disengagement scale decreased slightly to p = .001. Denial was still non-significant (p = .46) but self-distraction (p = .02) was significant at an alpha level of .05. 36 Removing three multivariate outliers (p-values of Mahalanobis D2 < .001) increased the value of the F- statistic to 8.97. Since the test was significant regardless of whether these cases were included or not, they were retained in the analysis. 36 Removing three multivariate outliers (p-values of Mahalanobis D2 < .001) increased the value of the F- statistic to 8.97. Since the test was significant regardless of whether these cases were included or not, they were retained in the analysis. 37 Kruskall-Wallis tests resulted in comparable significance levels for the AAQ, WBSI, and substance use scale (p < 001) but i ifi a e le el fo the beha iou al di e a e e t ale de ea ed li htly to 001 De ial a coping? The WBSI and AAQ were used in the current study to measure participants’ propensities towards avoidant coping, along with four subscales from the Brief COPE (self-distraction, denial, substance use, and behavioural disengagement) that are thought to tap into avoidant coping (Chapman, Specht, & Cellucci, 2005). Histograms and Q-Q plots for each of the variables were inspected prior to conducting the planned multivariate analyses. All of the frequency distributions, with the exception of the AAQ for participants who had never self-injured, were significantly non-normal, D’s(190-217) ≥ .07, p’s ≤ .008). After converting the scores for the six coping variables into z-scores, a total of 10 scores (five each from the COPE denial and behavioural disengagement scales) were identified as possible 242 outliers because they were greater than 3.29. Since removing these cases did not alter the normality distributions, they were retained for subsequent analyses. Prior to conducting a MANOVA, a series of non-parametric bivariate correlations were carried out to check for multicollinearity. Although there were significant, positive relationships between all six coping variables, the strongest correlation—between the WBSI and the AAQ (rs = .58, p = 0.01)—was not high enough to warrant concern (Tabachnik & Fidell, 2007). Multivariate analyses of variance36 using Pillai’s trace showed that participants’ tendencies towards avoidant coping varied as a function of NSSI history, V = .11, F(6, 39) = 8.26, p < .001, partial eta squared = .11. The covariance matrices were significantly different (Box’s M = 111.50, F(21, 564687) = 5.22, p < .001). Follow-up ANOVAs37 with a Bonferroni corrected alpha of .008 indicated that participants who had self-injured reported significantly higher levels of thought suppression, experiential avoidance, substance use, and behavioural disengagement compared to participants who had never self-injured. To examine whether avoidant coping varies as a function of NSSI recency, participants who reported a lifetime history of NSSI were once again split into two groups; those who had self-injured in the past 12 months and those who had self- injured more than 12 months ago. This resulted in three levels of the group variable: No NSSI, Lifetime NSSI, and 12-month NSSI. All of the frequency distributions of variables at each level demonstrated some skewness and kurtosis. Normality tests confirmed that all but two of these distributions (the AAQ scores for No NSSI and 12-month NSSI) were significantly non-normal (D’s(56-217) ≥ .07, p’s ≤ .03). 38 Removing four multivariate outliers (p-values of Mahalanobis D2 < .001) increased the value of the F-statistic to 6.51. Since the test was significant regardless of whether these cases were included or not, they were retained in the analysis. y 39 Kruskal-Wallis tests comparing the three groups yielded comparable significance levels to the ANOVAs with one exception; denial decreased from p = .70 to p = .39. coping? 243 A MANOVA38 using Pillai’s trace showed a significant effect of recency of NSSI on avoidant coping by participants, V = 0.18, F(12, 750) = 6.10, p < .001, partial eta squared = .09. The covariance matrices were significantly different (Box’s M = 128.82, F(42,95396) = 2.97, p < .001). Follow-up ANOVAs39 using a Bonferroni corrected alpha of .008 demonstrated the same pattern of results observed for the two-group comparison. The propensity towards thought suppression and experiential avoidance, as well as substance use and behavioural disengagement varied significantly as a function of NSSI recency (see Table 14). NSSI recency, however, did not have a significant effect on self-distraction or denial. Post-hoc Games-Howell comparisons indicated that participants who had self-injured in the past 12 months reported significantly higher levels of thought suppression than those who had self-injured more than 12 months ago or those who had never self-injured. Participants who had self-injured more than 12 months ago also reported significantly higher levels of thought suppression than those who had never self-injured. Self-reported experiential avoidance and using substances to cope was significantly higher for participants who had self-injured in the past 12 months, when compared to participants who had self-injured more than 12 months ago or those who had never self-injured. However, there were no significant differences in experiential avoidance or substance use for participants who had self-injured more than 12 months ago when compared to participants who had never self-injured. Finally, participants who had self-injured in the past 12 months reported significantly higher behavioural disengagement scores than those who had never self-injured, but not those who had self-injured more than 12 months ago. Participants who had self-injured more than 12 months ago did not report Participants who had self-injured more than 12 months ago did not report 244 significantly higher behavioural disengagement than participants who had never self-injured. 40 Participants were asked how many times they had self-injured in the previous two months, but given the extremely skewed frequency distribution (skewness = 11.35), I chose to create and subsequently utilise a categorical variable of self-injury versus no self-injury in the analyses. 3.4 Is NSSI predicted by negative emotions and thoughts? To determine whether negative emotions and thoughts predicted NSSI, the 224 participants who completed both the T1 and T2 surveys were divided into two groups: (1) participants (N = 201) who had not self-injured in the two months between T1 and T2 (No NSSI group), and (2) participants (N = 23) who had self- injured in the two months between T1 and T2 (NSSI group). Two binary logistic regressions40 using the forced entry method were conducted to test whether negative emotions and thoughts at T1 predicted group membership at T2. These models are presented in Table 15. Although both of the regression models were significant, none of the predictors added significant predictive value to the respective models. With only 23 participants reporting self-injury in the two months between survey completions, there was insufficient power to predict self-injury group membership. Consequently, both models correctly predicted group membership for the No NSSI group 100% of the time, but correctly predicted group membership for the NSSI group 0% of the time. Given that the sample size of participants who had self-injured between survey completions was too small to detect meaningful differences through logistic regression, cross-lagged panel correlations using maximum likelihood estimates were tested in AMOS to determine the causal direction of the relationships between negative intrapersonal experiences at T1 and new episodes of self-injury reported at T2. By excluding possible third variable effects, cross-lagged panel correlations permit causal inferences from correlational data (Kenny, 1975). 245 Table 15 Table 15 Two binary logistic regression models for NSSI versus No NSSI 95% CI for Odds Ratio B (SE) Lower Odds Ratio Upper Model 1: NSSI (N = 23) vs No NSSI (N = 194) Included Constant -4.03 (0.78)* Negative affect 0.48 (0.41) 0.72 1.62 3.63 Automatic Thoughts 0.03 (0.02) 0.99 1.03 1.07 Model 2: NSSI (N = 23) vs No NSSI (N = 201) Included Constant -4.37 (0.87)** Fear 0.15 (0.43) 0.51 1.16 2.68 Hostility 0.36 (0.45) 0.59 1.44 3.47 Guilt 0.52 (0.37) 0.81 1.68 3.48 Sadness -0.06 (0.38) 0.45 0.94 1.98 Note. Model 1: R2 = .20 (Hosmer & Lemeshow), .04 (Cox & Snell), .09 (Nagelkerke). Model Χ2(2) = 9.36, p < .01. * p < .05. Model 2: R2 = .46 (Hosmer & Lemeshow), .05 (Cox & Snell), .11 (Nagelkerke). Model Χ2(4) = 11.95, p < .05. ** p < .001. ote. Model 1: R2 = .20 (Hosmer & Lemeshow), .04 (Cox & Snell), .09 (Nagelkerke). Model Χ2(2) = 9.36, p < .01. * p < .05. odel 2: R2 = .46 (Hosmer & Lemeshow), .05 (Cox & Snell), .11 (Nagelkerke). Model Χ2(4) = 11.95, p < .05. ** p < .001. As is evident from Figures 10 to 15, all of the autoregressive paths were significant (p < .001) showing stability in negative emotions and thoughts over time (Martens & Haase, 2006). Note. p<.05, p<.001 Figure 10. Cross-lagged panel model of NSSI and global negative affect. .35* .77** .09 .17* -.05 .29** R2 = .56 R2 = .11 NSSI T2 Negative Affect T1 Negative Affect T2 NSSI T1 Negative Affect T1 Note. p<.05, p<.001 Figure 10. Cross-lagged panel model of NSSI and global negative affect. 246 Note. p < .05, p<.001 Figure 11. Cross-lagged panel model of NSSI and negative automatic thoughts. .27 .68** .13* .17* .10 .29** R2 = .50 R2 = .12 Automatic Thoughts T1 NSSI T1 Automatic Thoughts T2 NSSI T2 Automatic Thoughts T2 Automatic Thoughts T1 Note. p < .05, p<.001 Figure 11. Cross-lagged panel model of NSSI and negative automatic thoughts. Note. p<.01, *p<.001 Figure 12. Cross-lagged panel model of NSSI and hostility. Note. p < .05, p<.001 Figure 13. Cross-lagged panel model of NSSI and guilt. Table 15 .29 .72** .01 .21* .11 .29 R2 = .52 R2 = .12 Hostility T1 NSSI T1 Hostility T2 NSSI T2 .38 .82** .14* .01 -.04 .26** R2 = .65 R2 = .12 Guilt T1 NSSI T1 Guilt T2 NSSI T2 Note. p<.01, p<.001 Figure 12. Cross-lagged panel model of NSSI and hostility. .29* .72** .01 .21* .11 .29** R2 = .52 R2 = .12 Hostility T1 NSSI T1 Hostility T2 NSSI T2 Figure 12. Cross-lagged panel model of NSSI and hostility. Note. p < .05, p<.001 Figure 13. Cross-lagged panel model of NSSI and guilt. .38* .82** .14* .01 -.04 .26** R2 = .65 R2 = .12 Guilt T1 NSSI T1 Guilt T2 NSSI T2 Note. p < .05, p<.001 Figure 13. Cross-lagged panel model of NSSI and guilt. 247 Note. p < .05, *p<.001 Figure 14. Cross-lagged panel model of NSSI and fear. Note. p < .05, p<.001 Figure 15. Cross-lagged panel model of NSSI and sadness. Two of the cross-lag paths were significant at an alpha level of .05. The path from .25 .68** .11 .15* .00 .30 R2 = .47 R2 = .12 Fear T1 NSSI T1 Fear T2 NSSI T2 .31 .72** .09 .16* .05 .30** R2 = .54 R2 = .11 Sadness T1 NSSI T1 Sadness T2 NSSI T2 Note. p < .05, p<.001 Figure 14. Cross-lagged panel model of NSSI and fear. .25* .68** .11 .15* .00 .30** R2 = .47 R2 = .12 Fear T1 NSSI T1 Fear T2 NSSI T2 .11 Note. p < .05, p<.001 Figure 14. Cross-lagged panel model of NSSI and fear. Note. p < .05, p<.001 Figure 15. Cross-lagged panel model of NSSI and sadness. .31 .72** .09 .16* .05 .30 R2 = .54 R2 = .11 Sadness T1 NSSI T1 Sadness T2 NSSI T2 .31 Note. p < .05, p<.001 Figure 15. Cross-lagged panel model of NSSI and sadness. Two of the cross-lag paths were significant at an alpha level of .05. The path from negative automatic thoughts at T1 to self-injury at T2 was significant (β = .13, p = .048), but the path from self-injury at T1 to negative automatic thoughts at T2 was non-significant (β = .13, p = .06). 41 Herr’s syntax is based on equations presented by Mackinnon and Dwyer (1993). Table 15 The path from guilt at T1 to self-injury at T2 was significant (β = .14, p = .03), but the path from self-injury at T1 to guilt at T2 was non- significant (β = -.04, p = .31). These results suggest that dispositional automatic negative thoughts and guilt may be causal factors for later self-injury, but that self- injury is not a causal factor for automatic negative thoughts and guilt. 248 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? 41 Herr’s syntax is based on equations presented by Mackinnon and Dwyer (1993). 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? The sample size of people who had self-injured between survey completions was too small to test mediation over time; instead, I used the T1 data to determine whether a dispositional tendency towards avoidance (i.e., experiential avoidance and thought suppression) mediated relationships between negative intrapersonal experiences (i.e., emotions and thoughts) and NSSI. Given that lifetime prevalence of NSSI was a dichotomous variable and standard mediation analyses are conducted with continuous variables, I used SPSS syntax41 written by Nathaniel Herr (www.nrhpsych.com/mediation/logmed.html) specifically for mediation analyses with dichotomous variables. Mediation was determined using Baron and Kenny’s (1986) criteria: mediation is observed if the predictor (X) correlates with both the outcome (Y) and the mediator (M), and M then predicts Y while controlling for X. Full mediation is observed when the relationship between X and Y while controlling for M is reduced to zero (Baron & Kenny, 1986). Partial mediation is observed when there is a reduction in the significance level of the relationship between X and Y when including M. Each mediation analysis was followed up with a Sobel test. Experiential avoidance did not mediate the relationships between NSSI and negative affect (see Figure 16), fear (see Figure 18), hostility (see Figure 20), guilt (see Figure 22), sadness (see Figure 24), or negative automatic thoughts (see Figure 26). Thought suppression, however, was identified as a partial mediator in five models. As is evident from the figures, thought suppression partially mediated the relationships between NSSI and negative affect (see Figure 17), fear (see Figure 19), hostility (see Figure 21), sadness (see Figure 23), and automatic thoughts (see Figure 27). This suggests that specific forms of experiential avoidance (i.e., thought suppression) may underlie engagement in NSSI. 249 ** p < .001 * p < .05, p < .001 Figure 16. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.11, SE = 0.07, p = .91. Figure 17. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.98, SE = 0.07, p = .048. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? Experiential Avoidance 6.87* 0.00 0.88* (0.87) Thought Suppression 10.02 0.88* (0.69**) 0.02 Negative Affect Negative Affect NSSI NSSI * p < .001 Figure 16. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.11, SE = 0.07, p = .91. Experiential Avoidance 6.87* 0.00 0.88* (0.87) Negative Affect NSSI p < .05, p < .001 Figure 17. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.98, SE = 0.07, p = .048. Thought Suppression 10.02* 0.88* (0.69) 0.02 Negative Affect NSSI * < 001 Experiential Avoidance 6.87* 0.00 0.88* (0.87) Negative Affect NSSI p < .05, p < .001 Thought Suppression 10.02* 0.88* (0.69) 0.02 Negative Affect NSSI Experiential Avoidance Thought Suppression Negative Affect * p < .05, p < .001 * p < .001 Figure 17. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.98, SE = 0.07, p = .048. Figure 17. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.98, SE = 0.07, p = .048. Figure 17. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is partially mediated by thought suppression. The Figure 16. Unstandardised regression coefficients for the relationship between negative affect and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.11, SE = 0.07, p = .91. unstandardised regression coefficient for the relationship between negative affect and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.98, SE = 0.07, p = .048. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? * p < .05, *** p < .001 * p < .05, p < .01, * p < .001 Figure 18. Unstandardised regression coefficients for the relationship between fear and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between fear and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 1.64, SE = 0.06, p = .10. Figure 19. Unstandardised regression coefficients for the relationship between fear and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between fear and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 3.17, SE = 0.06, p = .002. Experiential Avoidance 5.88* 0.03 0.58* (0.42) Thought Suppression NSSI 8.14 0.58* (0.35) 0.03 * Fear Fear NSSI * p < .05, * p < .001 Figure 18. Unstandardised regression coefficients for the relationship between fear and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between fear and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 1.64, SE = 0.06, p = .10. Experiential Avoidance 5.88* 0.03 0.58*** (0.42) Fear NSSI p < .05, p < .01, * p < .001 Thought Suppression NSSI 8.14* 0.58* (0.35) 0.03 * Fear * < 05 * < 001 Experiential Avoidance 5.88* 0.03 0.58*** (0.42) Fear NSSI Experiential Avoidance Thought Suppression 8.14** Fear * p < .05, p < .01, * p < .001 * p < .05, p < .01, * p < .001 * p < .05, * p < .001 Figure 18. Unstandardised regression coefficients for the relationship between fear and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between fear and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 1.64, SE = 0.06, p = .10. Figure 19. Unstandardised regression coefficients for the relationship between fear and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between fear and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 3.17, SE = 0.06, p = .002. p < .01, * p < .001 Figure 21. Unstandardised regression coefficients for the relationship between hostility and NSSI which is partially mediated by thought suppression. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? Unstandardised regression coefficients for the l ti hi b t t ti th ht d NSSI hi h i Experiential Avoidance 4.90* 0.01 0.63* (0.58) Thought Suppression NSSI 7.42 0.63* (0.45*) 0.03 Sadness Sadness Experiential Avoidance 0.35* 0.02 0.05* (0.04*) Thought Suppression NSSI 0.53* 0.05** (0.03) 0.03* Automatic Thoughts Automatic Thoughts NSSI NSSI *** p < .001 * p < .05, p < .01, * p < .001 Figure 24. Unstandardised regression coefficients for the relationship between sadness and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.64, SE = 0.06, p = .52. Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.33, SE = 0.07, p = .02. Experiential Avoidance 4.90* 0.01 0.63* (0.58*) Thought Suppression NSSI 7.42* 0.63* (0.45) 0.03* Sadness Sadness NSSI * p < .05, p < .01, * p < .001 Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.33, SE = 0.07, p = .02. Thought Suppression NSSI 7.42* 0.63* (0.45*) 0.03 Sadness * p < .001 Figure 24. Unstandardised regression coefficients for the relationship between sadness and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.64, SE = 0.06, p = .52. Experiential Avoidance 4.90* 0.01 0.63* (0.58) Sadness NSSI p < .05, p < .01, * p < .001 Thought Suppression NSSI 7.42* 0.63* (0.45*) 0.03 Sadness * 001 Experiential Avoidance 4.90* 0.01 0.63* (0.58) Sadness NSSI Experiential Avoidance Thought Suppression Sadness p < .05, p < .01, * p < .001 * p < .05, p < .01, * p < .001 * p < .001 Figure 24. Unstandardised regression coefficients for the relationship between sadness and NSSI which is not mediated by experiential avoidance. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.64, SE = 0.06, p = .52. Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.33, SE = 0.07, p = .02. Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.33, SE = 0.07, p = .02. Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.33, SE = 0.07, p = .02. * p < .001 * p < .05, p < .01, * p < .001 Figure 26. Unstandardised regression coefficients for the relationship between automatic thoughts and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between automatic thoughts and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.88, SE = 0.06, p = .38. Figure 27. Unstandardised regression coefficients for the relationship between automatic thoughts and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between automatic thoughts and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.91, SE = 0.06, p = .004. Experiential Avoidance 0.35* 0.02 0.05* (0.04*) Thought Suppression NSSI 0.53* 0.05** (0.03) 0.03* Automatic Thoughts Automatic Thoughts NSSI * p < .05, p < .01, * p < .001 Thought Suppression NSSI 0.53* 0.05 (0.03) 0.03* Automatic Thoughts * p < 001 Experiential Avoidance 0.35* 0.02 0.05* (0.04) Automatic Thoughts NSSI Experiential Avoidance Thought Suppression 0.03* Automatic Thoughts * p < .05, p < .01, * p < .001 * p < .05, p < .01, * p < .001 * p < .001 Figure 26. Unstandardised regression coefficients for the Figure 27. Unstandardised regression coefficients for the relationship between automatic thoughts and NSSI which is partially mediated by thought suppression. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? The unstandardised regression coefficient for the relationship between hostility and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 2.69, SE = 0.06, p = .01. Thought Suppression 7.74* 0.85* (0.64) 0.03* Hostility NSSI * p < .001 p < .01, * p < .001 Figure 20. Unstandardised regression coefficients for the relationship between hostility and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between hostility and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 1.36, SE = 0.06, p = .17. Figure 21. Unstandardised regression coefficients for the relationship between hostility and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between hostility and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 2.69, SE = 0.06, p = .01. Experiential Avoidance 4.77* 0.02 0.85* (0.75) Thought Suppression 7.74 0.85* (0.64*) 0.03 Hostility Hostility NSSI NSSI * p < .001 Experiential Avoidance 4.77* 0.02 0.85* (0.75) Hostility NSSI * p < .01, * p < .001 Thought Suppression 7.74* 0.85* (0.64) 0.03* Hostility NSSI Experiential Avoidance Thought Suppression 7.74* 0.02 Hostility p < .01, * p < .001 p < .01, * p < .001 * p < .001 Figure 21. Unstandardised regression coefficients for the relationship between hostility and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between hostility and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 2.69, SE = 0.06, p = .01. Figure 20. Unstandardised regression coefficients for the relationship between hostility and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between hostility and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 1.36, SE = 0.06, p = .17. 250 * p < .001 * p < .001 Figure 22. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between guilt and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0, SE = 0.06, p = 1. Figure 23. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by thought suppression. The unstandardised regression coefficient for the relationship between guilt and NSSI controlling for thought suppression is in parentheses. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? Sobel’s test = 1.80, SE = 0.07, p = .07. Experiential Avoidance 5.05* 0.00 0.75* (0.75*) Thought Suppression 7.75* 0.75* (0.61) 0.02 Guilt Guilt NSSI NSSI p < .001 Figure 23. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by thought suppression. The unstandardised regression coefficient for the relationship between guilt and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.80, SE = 0.07, p = .07. Thought Suppression 7.75* 0.75* (0.61) 0.02 Guilt NSSI p < 001 Experiential Avoidance 5.05* 0.00 0.75* (0.75) Guilt NSSI p < .001 Thought Suppression 7.75* 0.75* (0.61) 0.02 Guilt NSSI Experiential Avoidance Thought Suppression p < .001 Figure 23. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by thought suppression. The unstandardised regression coefficient for the relationship between guilt and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 1.80, SE = 0.07, p = .07. Figure 22. Unstandardised regression coefficients for the relationship between guilt and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between guilt and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0, SE = 0.06, p = 1. * p < .001 * p < .05, p < .01, * p < .001 Figure 24. Unstandardised regression coefficients for the relationship between sadness and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.64, SE = 0.06, p = .52. Figure 25. Unstandardised regression coefficients for the relationship between sadness and NSSI which is partially mediated by thought suppression. The unstandardised regression coefficient for the relationship between sadness and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.33, SE = 0.07, p = .02. *** p < .001 * p < .05, p < .01, * p < .001 Figure 26. Unstandardised regression coefficients for the l ti hi b t t ti th ht d NSSI hi h i Figure 27. 3.5 Does avoidance mediate relationships between negative intrapersonal experiences and NSSI? The unstandardised regression coefficient for the relationship between automatic thoughts and NSSI controlling for thought suppression is in parentheses. Sobel’s test = 2.91, SE = 0.06, p = .004. Figure 26. Unstandardised regression coefficients for the relationship between automatic thoughts and NSSI which is not mediated by experiential avoidance. The unstandardised regression coefficient for the relationship between automatic thoughts and NSSI controlling for experiential avoidance is in parentheses. Sobel’s test = 0.88, SE = 0.06, p = .38. 251 4. DISCUSSION 4. DISCUSSION Non-suicidal self-injury was highly prevalent in this self-selected sample of first year Psychology students, with almost half of the participants reporting a lifetime history of NSSI. These rates are generally higher than those identified in studies of American university students using the same self-injury measure (25%- 38%; Brown, 2009; Gratz et al., 2002), but of course cannot be used to infer true prevalence rates among this population. For instance, a random sample of university students found that only 17% reported self-injuring in their lifetimes (Whitlock et al., 2006). The participants with a history of NSSI in the current study had typically engaged in more than one type of self-injury; the most common types (i.e., cutting, severe scratching, and sticking sharp objects into one’s skin) were generally consistent with what has been reported in other research (Gratz et al., 2002; Hoff & Muehlenkamp, 2009). Comparisons between the participants who had, and had not, self-injured revealed several significant differences. To briefly review, it was proposed that people who had self-injured would report experiencing more negative intrapersonal experiences in general than people who had never self-injured and would use more avoidant coping methods. Furthermore, I anticipated that negative emotions and thoughts at T1 would predict engagement in self-injury at T2, and that these relationships may be mediated by dispositional tendencies towards avoidant coping. 4.1 Experience of negative emotions and thoughts 4.1 Experience of negative emotions and thoughts The intensity and frequency of negative intrapersonal experiences reported by participants not only differed according to whether or not they had self-injured, but also according to how recently they had self-injured. Participants who had self- injured in the last 12 months consistently reported the highest levels of general negative affect, emotions (i.e., fear, hostility, guilt, and sadness), and automatic thoughts compared to those who had self-injured more than 12 months ago or never self-injured. 252 The same pattern of results was observed for negative emotional experiences that had occurred in the past week. Symptoms of depression, stress, and anxiety were significantly more common among participants who had self-injured within the past 12 months than those who had a lifetime or no history of NSSI. In contrast, participants who had self-injured more than 12 months ago did not differ on symptoms of depression, anxiety, or stress from participants who had never self- injured. Moreover, participants with a lifetime history of NSSI did not report experiencing more negative automatic thoughts than participants who had never self-injured. However, these participants did endorse higher negative affect, hostility, guilt, and sadness than those without a history of self-injury, providing evidence that there are some significant differences in the general emotional experiences of people who have self-injured more than a year ago compared to people who have never self-injured. Taken together, these results show that people who have self- injured report significantly more intense, aversive emotions and a higher frequency of negative automatic thoughts than people who have not self-injured, and that these negative emotional and cognitive experiences are heightened for those with a more recent history of NSSI. These results are consistent with studies that show NSSI is associated with depression and anxiety disorders (Hintikka et al., 2009; Hoff & Muehlenkamp, 2009), and with the wealth of evidence that self-injury is primarily used as a strategy to deal with intense, negative affect (Klonsky, 2007). Given that negative cognitions feature prominently in both depression and anxiety (Dozois, Seeds, & Collins, 2009), it is unsurprising that the people who self-injured most recently also reported experiencing the highest frequency of negative automatic thoughts. 4.2 Tendencies towards avoidant coping As hypothesised, participants who had self-injured were significantly more likely to show a propensity towards avoidant coping strategies than participants who had never self-injured. However, this difference was only apparent with four 253 out of the six coping styles examined. Experiential avoidance, thought suppression, substance use, and behavioural disengagement were more commonly endorsed by participants who had self-injured and varied as a function of self-injury recency. This lends further support to the contention outlined within the EAM that self-injury is best understood as one of several avoidant behaviours in a functional response class (Chapman et al., 2006). In contrast, the use of self-distraction and denial did not differ between the groups. The absence of a significant difference for coping through self-distraction was particularly surprising in light of the evidence that suggests NSSI is an effective form of distraction (Brown et al., 2002; Polk & Liss, 2009). It may be that self- distraction is a commonly used coping strategy for people with and without a history of self-injury, but that the difference lies in the behaviours that people engage in to successfully distract themselves. For example, people without a history of NSSI may routinely distract themselves through watching TV or exercising, whereas people who self-injure may routinely distract themselves through cutting or burning. 4.3 Do negative emotions and thoughts cause self-injury? It is clear from the results that participants who had self-injured within the past 12 months were the most likely to endorse experiencing negative emotions and unwanted thoughts, and to report a tendency towards avoidant coping, but the causal direction between these variables cannot be established with cross-sectional data. To allow for causal inferences, two time-points were included in the current study. However, only a few people reported self-injuring between surveys which necessitated revising the data analysis plan. Although experimental methods are traditionally employed to test causal relationships, these methods are not ethically appropriate for self-injury research. Instead, cross-lagged panel correlations (Kenny, 1975) were used to test causality. Simple cross-lagged panel models of over-time data revealed that both guilt and negative automatic thoughts predicted subsequent self-injury. The self-referential 254 characteristics that define guilt and negative automatic thoughts also underpin the self-punishment function of NSSI, which has been consistently identified as one of the most commonly endorsed motivations for self-injury (Briere & Gil, 1998; Brown et al., 2002; Swannell et al., 2008). Therefore it is possible that guilt and automatic thoughts predict NSSI because these negative intrapersonal experiences are alleviated through self-punishing acts such as self-injury. 4.5 Strengths Given the paucity of longitudinal studies on NSSI, conducting this study across two time points was a methodological strength. As predictors of new incidents of NSSI, negative automatic thoughts and guilt likely functioned as EOs or SD’s for self-injurious episodes. Future studies should further examine the role of self-referential thoughts and emotions in predicting NSSI. Certainly, the inclusion of the short version of the Automatic Thoughts Questionnaire (Netemeyer et al., 2002) in the survey was a strength of the current study as few researchers have examined the role of cognitions in precipitating NSSI and none, to my knowledge, have investigated the predictive value of negative automatic thoughts. This is clearly an important area for future research. 4.4 Specific types of avoidance may underlie self-injury Experiential avoidance is conceptualised as a process that involves avoiding emotions, thoughts, or physical sensations (Hayes et al., 1996), and yet it did not act as a mediator in any of the analyses. In contrast, thought suppression, which has been identified as a specific form of experiential avoidance (Chapman et al., 2006; Hayes et al., 1996), partially mediated the relationships between lifetime NSSI and all of the negative intrapersonal experiences assessed, with the exception of guilt. It is intuitive that thought suppression, rather than general experiential avoidance, would mediate the relationship between negative automatic thoughts and NSSI, but it is less intuitive that thought suppression rather than general experiential avoidance would mediate the relationships between negative emotions and NSSI. It may be that the AAQ as a measure of experiential avoidance fails to fully capture the defining characteristics of this behavioural process. Indeed, Hayes and colleagues (1996) comment on the limitations associated with reducing generalised avoidant tendencies, which have the potential to manifest in a myriad of specific actions, to a nine item measure. Alternatively, unsuccessful attempts at cognitive regulation through thought suppression (Najmi et al., 2007), rather than general avoidance, may be one of the key mechanisms through which negative emotions lead to self-injurious behaviour. Negative emotions may trigger, or be triggered by, negative cognitions, which in turn could lead to thought suppression. However, given that thought suppression is more likely to increase than decrease unwanted thoughts, self-injury may be subsequently utilised as a more effective avoidant coping strategy (Najmi et al., 2007). 255 4.6 Limitations Given that I have discussed the analytic limitations imposed by the small sample of people who reported new episodes of NSSI at T2 several times in this chapter, I will not dwell on these here. Suffice it to say that future research studies designed to test similar hypotheses should take into account the need for larger samples or longer time periods between survey completions. Indeed, two months was an arbitrary time limit, chosen for pragmatic rather than theoretical reasons. Another pragmatic choice—conducting this study with only university students—is a limitation in that these results may not generalise to other populations. However, this is not necessarily a flaw given the atypically high rates of self-injurious behaviours and psychopathology among university students compared with other community groups (Briere & Gil, 1998; Stallman, 2010; Whitlock et al., 2006). This suggests that researchers studying NSSI should pay particular attention to university students. General discussion ‚All of us tend to use the behavior that was effective in the past at getting the outcome we desire.‛ (Cipani & Schock, 2007, p. 72) ‚All of us tend to use the behavior that was effective in the past at getting the outcome we desire.‛ (Cipani & Schock, 2007, p. 72) 4.7 Conclusions Negative intrapersonal experiences and avoidant coping styles were shown to vary not only as a function of NSSI history, but also as a function of NSSI recency. Particular negative intrapersonal experiences (i.e., automatic thoughts and guilt) also 256 predicted new episodes of self-injury. Finally, thought suppression was identified as a partial mediator of relationships between negative intrapersonal experiences and NSSI, although these results need to be replicated using over-time data. These results have a number of clinical implications. Students with a history of NSSI appear to be contending with significantly higher levels of general negative affect, aversive emotions, and cognitions than students who have never self-injured. Although these levels are not necessarily clinically significant in all cases, these students may be at a heightened risk of future self-injury episodes given their higher baseline rate of negative intrapersonal experiences (Brown et al., 2007). Clinicians should be mindful that even those students who have not self-injured in a year or more may still be struggling to regulate their emotions and cognitions. Other avoidant coping strategies, such as substance use, should also be assessed in students who have self-injured as these may be substituting for NSSI (Chapman et al., 2006). 257 1. INTRODUCTION Does NSSI function primarily as an experientially avoidant behaviour within Aotearoa New Zealand? Based on the three empirical studies I conducted for this thesis, the answer is yes; for many people, on a majority of occasions, self-injury provided an escape from unwanted, negative emotions and thoughts. Of course, as is to be expected, one functional model does not fit all episodes of self-injury. Intrapersonal escape/avoidance did predominate across studies, but some participants’ self-injurious behaviours also functioned as an adrenalin rush reminiscent of a drug high (i.e., intrapersonal access), a visceral form of communicating to others when support was needed (i.e., interpersonal access), and an escape from taxing demands or personal responsibilities (i.e., interpersonal escape/avoidance). Interviewing people for my first study about their experiences of NSSI provided a rich, detailed, and highly nuanced foundation for the subsequent studies in this thesis. Indeed, it was impossible to do justice to the complexity inherent in each person’s most recent episode of self-injury, let alone the learning histories that preceded those episodes. Nonetheless, functionally assessing specific episodes of self-injurious behaviour did give me an understanding of why participants were motivated to injure themselves, and allowed me to hypothesise about the ways in which their behaviours were being reinforced and maintained over time. It was apparent from my analysis that complex and, in many cases, concurrent reinforcement schedules were operating for participants. This reinforcement, for the most part, was intrapersonally mediated; interpersonal functions were atypical. A minority of participants did report accessing attention or 258 support subsequent to their self-injury, which may have reinforced their behaviour. However, this support or attention was often temporally delayed and seldom identified as a motivating factor behind their self-injury, making it less likely that these behaviours served an interpersonal function. Intrapersonal functions once again predominated over interpersonal functions in my second study, a survey of people from across Aotearoa New Zealand, who had all self-injured within the past 12 months. Participants endorsed intrapersonal functions, for both their most recent and global self-injury episodes, significantly more often than interpersonal functions. More specifically, they most often reported self-injuring to regulate affect, punish themselves, and mark their distress; motivations for NSSI that were similarly reported in my interview study. 1. INTRODUCTION Participants also retrospectively reported decreased negative affect and cognitions following their most recent episode of self-injury, which supports Chapman and colleagues’ (2006) proposal that NSSI is primarily maintained through negative reinforcement and thus functions as a form of experiential avoidance. Consistent with prior conceptualisations of self-injury as a self-help strategy (Favazza & Conterio, 1988), one of the two themes identified from a thematic analysis of the open-ended, survey responses detailing the consequences of NSSI was self becomes helper. These positive consequences of NSSI appeared to be both intrapersonally and interpersonally mediated. Participants utilised self-injury to regulate emotions, as well as access support and/or treatment. Furthermore, a few participants identified the presence of a physical wound as a positive consequence of self-injury. However, participants also identified a number of aversive consequences of their most recent episode of NSSI, which were captured in the second theme, self becomes transgressor. Self-injury was perceived by many participants as a transgression of socio-cultural norms and following their NSSI episode, people reported needing to conceal evidence of their self-injury from others, being judged by others for self-injuring, and feeling concerned that their behaviour had caused others to suffer. 259 The paradox of self-injury as a behaviour that diminishes and provokes distress was apparent in both my interview and survey studies. Although the aversive consequences engendered by self-injury could have functioned to suppress the behaviour, this seemed unlikely in the majority of cases since they typically followed reinforcing consequences. Instead, it is probable that many of the participants would have self-injured again, especially in light of the powerful intrapersonal functions served by their self-injury. My first two studies provided compelling evidence of the utility of NSSI as a coping strategy for negative emotions and cognitions, leading me to question whether people who self-injure could be differentiated from those who do not self- injure on the basis of their general intrapersonal experiences and coping styles. For my final study, I compared the emotional, cognitive, and coping experiences of people with a history of NSSI to those who have never self-injured to determine whether there were any significant differences between these two groups. Building on the results of my interview and survey analyses, my third study focused on investigating the broader emotional and cognitive experiences of people who self- injure, and whether these experiences would predict engagement in NSSI over time. 1. INTRODUCTION People with a history of self-injury did report experiencing significantly higher levels of negative emotions and thoughts on a general basis than people who did not have a history of NSSI, and they were also significantly more likely to use avoidant coping strategies, such as substance use and thought suppression. The strength of these differences varied according to NSSI recency. Furthermore, negative automatic thoughts and guilt predicted new episodes of self-injury, lending weight to the key premise of the EAM (Chapman et al., 2006) that people self-injure to decrease or eliminate negative affect, but also emphasising the importance of negative cognitions in precipitating self-injury. Finally, cross-sectional analyses demonstrated that thought suppression acted as a partial mediator of the relationship between negative intrapersonal experiences and NSSI. 260 Taken together, the findings from my three studies help to support and further clarify the extant literature on the functions of self-injury (Klonsky, 2009; Klonsky & Glenn, 2009; Nock & Prinstein, 2005), as well as provide insight into the cognitive antecedents and consequences of self-injurious behaviours, which have been largely neglected in the NSSI literature. Although this preliminary research into the functions of NSSI in Aotearoa New Zealand is a useful starting point, several questions remain open to debate. In particular, I will discuss what the self-injury mindset may tell us about the interrelationship between emotions and thoughts, where NSSI ‘fits’ in relation to other coping strategies, whether certain coping strategies (such as NSSI) qualify as disorders, and the clinical implications of conceptualising self-injury as an experientially avoidant behaviour. Finally, the strengths and limitations of my studies will be considered within the context of directions for future research. 2. THE SELF-INJURY MINDSET The difficulty of understanding how the complex interrelationship between emotions and cognitions impacts on the initiation, reinforcement, and maintenance of self-injurious behaviours is an issue which has surfaced repeatedly throughout this thesis. As identified by Nock (2008), few studies examine the cognitive precipitants of self-injury, with many researchers focusing almost exclusively on how self-injury functions as an emotion regulation strategy. However, my research demonstrates that ruling out, or downplaying, the role of cognitions in self-injury is premature. Certainly, the current evidence base about why people self-injure is too limited to conclude, as proposed by Chapman et al. (2006) in the EAM, that self- injury functions primarily as a form of emotional avoidance. Instead, it was apparent across my three studies that specific types of cognitions play a significant role in motivating people to engage in self-injury and also allow people to discriminate that reinforcement will be available following self- injury. Negative automatic thoughts featured prominently in people’s narrative descriptions of the antecedents of self-injury and were identified as being 261 significantly higher before, than after, self-injury in the cross-sectional, survey study. Furthermore, negative automatic thoughts predicted new episodes of NSSI over time. These studies suggest that negative automatic thoughts function as EOs for self-injurious behaviours; in other words, they motivate people to injure themselves on purpose. As I discussed in Chapter 4 (see p. 116), EOs have a value-altering effect through determining how potent a specific reinforcer will be in a particular situation and a behaviour-altering effect by increasing the likelihood of a person engaging in behaviours that are typically followed by that reinforcer (Laraway et al., 2003; Michael, 1993; Miltenberger, 2004). As EOs, negative automatic thoughts therefore not only influence the strength of reinforcement following self-injury, but also how frequently people engage in self- injurious behaviours. Of course, aversive emotions also appeared to function as EOs in all three of my studies, which highlights the importance of learning more about how emotional and cognitive EOs interact to reinforce NSSI. In the interview study, many people acknowledged that they made a decision to self-injure once they had surpassed a particular threshold of distress. Although they most easily identified the emotional components of this distress, it was clear that negative cognitions were also present. 2. THE SELF-INJURY MINDSET Part of people’s struggle to identify cognitive antecedents seemed to stem from their use of emotion language (e.g., ‚I felt<‛) to describe cognitive processes, which is customary for English speakers (Westbrook, Kennerley, & Kirk, 2007). Two examples from the interview study were provided by Owen and Natalie respectively: ‚I felt like I was yeah useless‛ and ‚I kind of felt like everyone had abandoned me‛. When couched as feelings, these thoughts appear to be more authoritative and immutable than they actually are which may have exacerbated participants’ negative emotional experience prior to self-injuring. This is one way in which negative emotions and thoughts may interact to motivate people to self- injure. 262 Another way in which negative cognitions precipitated self-injury was in their role as SD’s. The evidence for this contention is drawn from the interview study where several participants described how once they had surpassed a particular threshold of distress, their perspective became constricted to the point where they were unable to problem-solve alternative solutions to NSSI. Once in this mindset, they became convinced that self-injuring was the only way they could effectively alleviate their suffering. The presence of constricted cognition as a precipitant of self-injury supports Chapman et al.’s (2006) hypothesis that people who self-injure may tend towards experiential avoidance because they are unable to generate more helpful coping strategies when emotionally aroused. In these situations, particular thresholds or combinations of emotional and cognitive EOs appeared to activate, or at least occur alongside, verbal rules about the utility of self-injury. Such rules were evidence of the cognitive constriction experienced by some participants prior to self-injury and allowed them to discriminate that relief would be available following NSSI. Chapman et al. (2006) do briefly suggest that the interaction between verbal rules and emotional distress may play a role in maintaining self-injurious behaviour, but they do not address the complexities of this interaction within the EAM, preferring instead to focus on emotional precipitants and the process of emotional avoidance. However, the interrelationships between emotions and cognitions (both negative, automatic thoughts and verbal rules) evident in my research suggests that it is prudent at this point in time to adopt the more inclusive term of experiential avoidance, which implicates both emotions and thoughts in the reinforcement and maintenance of NSSI, until further research on the role of cognitive processes in self- injury has been conducted. 2. THE SELF-INJURY MINDSET Given that NSSI does appear to function primarily as a form of experiential avoidance, it may be both clinically and theoretically useful to refer to NSSI as an experientially avoidant coping strategy. 263 3. IS IT USEFUL TO CONCEPTUALISE NSSI AS A COPING STRATEGY? 3. IS IT USEFUL TO CONCEPTUALISE NSSI AS A COPING STRATEGY? Before defining self-injury as a form of avoidant coping, it is first necessary to determine what constitutes a coping strategy and how, if conceptualised as such, NSSI fits with other ways of coping. Unfortunately, the extant coping literature, which is beset with contradictions, provides little clarification about the parameters of different coping mechanisms (see Skinner, Edge, Altman, & Sherwood, 2003). In an effort to organise this literature to better inform future research on coping, Skinner et al. (2003) comprehensively reviewed the convergences and divergences among 100 measures of coping. Within their conception of coping as ‚an organizational construct used to encompass the myriad actions individuals use to deal with stressful experiences‛ (p. 217), Skinner et al. (2003) contend that coping is best viewed as a four-level hierarchy. Adaptive processes are at the top level of the hierarchy; these encompass families of coping, which in turn incorporate ways of coping, which are made up of coping instances. Classifying NSSI within this system (see Figure 28) may prove to be a useful way of organising the different functions of NSSI in relation to other coping behaviours. At the lowest level, the instances of coping reflect the infinite variations of self-injurious behaviours that people could use to hurt themselves on purpose. These instances of coping are then grouped into ways of coping (e.g., cognitive and emotional avoidance) at the second level of the hierarchy. Other ways of coping identified by Skinner et al. (2003) under the rubric of escape include behavioural avoidance, denial, and wishful thinking. Escape is the third level in the hierarchy and is classified as a family of coping. Families of coping serve particular functions for people, which in turn are linked to adaptive processes (i.e., level four of the hierarchy). Problem-solving, helplessness, and support-seeking are examples of other families of coping, while another adaptive process is gathering and ensuring the availability of social resources (Skinner et al., 2003). For example, the interpersonal access function of NSSI could be classified as gathering social support. 264 Figure 28. NSSI within an adaptive coping hierarchy. Adapted from ‚Searching for the structure of coping: A review and critique of category systems for classifying ways of coping,‛ by E.A. Skinner, K. Edge, J. Altman and H. Sherwood, 2003, Psychological Bulletin, 129, p. 218. 3. IS IT USEFUL TO CONCEPTUALISE NSSI AS A COPING STRATEGY? Coordinate actions and contingencies in the environment Cognitive avoidance Hitting with a belt Scratching with nails Punching a wall Cutting with a razor Burning with lighter Adaptive Process Coping Family Ways of Coping Instances of Coping Escape Emotional avoidance Coordinate actions and contingencies in the environment Emotional avoidance Cognitive avoidance Burning with lighter Figure 28. NSSI within an adaptive coping hierarchy. Adapted from ‚Searching for the structure of coping: A review and critique of category systems for classifying ways of coping,‛ by E.A. Skinner, K. Edge, J. Altman and H. Sherwood, 2003, Psychological Bulletin, 129, p. 218. Applying Skinner et al.’s (2003) hierarchy to NSSI raises several questions about the ways in which self-injury has been conceptualised to date. For example, Skinner and colleagues categorise emotion regulation within the coping family of self-reliance. They propose that self-reliance is adaptive because it allows people to protect their social resources. Within this taxonomy, it is not clear how NSSI could be described as form of emotion regulation and avoidance at the same time as they are distinct ways of coping. It is possible that instances of NSSI could function to regulate emotions, but those episodes would be separate from instances of NSSI that function as avoidance. However, in the NSSI literature, affect regulation reasons for self-injury are typically subsumed under the function of intrapersonal escape/avoidance (Klonsky & Glenn, 2009; Nock & Prinstein, 2004), which implies that self-injury is an emotion regulation strategy because it allows people to successfully escape or avoid their negative emotions. 265 Although such a fastidious approach to understanding why people self-injure may seem excessive, it is possible that isolating the mechanisms that underlie NSSI will necessitate clarifying the relationships between specific ways of coping (i.e., cognitive avoidance versus emotional regulation). Undoubtedly, clarification would further develop our understanding of how each of the single-function models (see pp. 68-76) relate to one another. Conceptualising particular self-injury behaviours as instances of coping that serve an adaptive function is also consistent with behavioural models of self-injury. 4. SHOULD COPING STRATEGIES BE CLASSIFIED AS DISORDERS? 5. CLINICAL IMPLICATIONS 5. CLINICAL IMPLICATIONS The conspicuous lack of empirically supported treatments for NSSI has been commented on by a number of authors (Klonsky & Muehlenkamp, 2007; Nock, 2010); indeed, the need for research into effective treatments for self-injury has been cited as a reason to classify NSSI as a disorder (Muehlenkamp, 2005). If, as my findings suggest, NSSI does function primarily as an experientially avoidant coping behaviour within Aotearoa New Zealand, it may be advisable for clinicians working here to employ interventions that aim to decrease intrapersonal avoidance. Two particularly promising interventions for people who utilise self-injury to avoid, or escape from, negative intrapersonal experiences are DBT (Linehan, 1993a, 1993b) and ACT (Hayes et al., 2006). As third-wave cognitive behavioural therapies, DBT and ACT both target experiential avoidance, but the way in which specific skills are conceptualised and taught to clients can differ (Lynch et al., 2006). The philosophy of dialectics lies at the heart of DBT and is concerned with interrelatedness, opposing forces, and the inevitability of change (Linehan, 1993a). Dialectical Behaviour Therapy has been identified as an effective treatment for self- injury among people diagnosed with BPD (Bohus et al., 2004; Kliem, Kröger, & Kosfelder, 2010; Stanley, Brodsky, Nelson, & Dulit, 2007), but no research has been conducted to determine whether it is a similarly efficacious treatment for people, without BPD, who self-injure. Two particularly promising interventions for people who utilise self-injury to avoid, or escape from, negative intrapersonal experiences are DBT (Linehan, 1993a, 1993b) and ACT (Hayes et al., 2006). As third-wave cognitive behavioural therapies, DBT and ACT both target experiential avoidance, but the way in which specific skills are conceptualised and taught to clients can differ (Lynch et al., 2006). The philosophy of dialectics lies at the heart of DBT and is concerned with i e e a e ess, opposi g o ces, a e i e i a i i y o c a ge ( i e a , 993a) Dialectical Behaviour Therapy has been identified as an effective treatment for self- injury among people diagnosed with BPD (Bohus et al., 2004; Kliem, Kröger, & Kosfelder, 2010; Stanley, Brodsky, Nelson, & Dulit, 2007), but no research has been conducted to determine whether it is a similarly efficacious treatment for people, without BPD, who self-injure. 4. SHOULD COPING STRATEGIES BE CLASSIFIED AS DISORDERS? At the start of this thesis, I discussed the current proposal for an NSSI Disorder to be included in the DSM-5 (American Psychiatric Association, 2010). My understanding of NSSI as a coping behaviour, as stated in Chapter 1 (see p. 26), conflicts with the argument that self-injury should be categorised as a disorder. Certainly, as instances of coping, specific episodes of self-injury do not count as disordered in and of themselves; rather, what could be considered to be maladaptive is the sustained use of self-injury as a form of avoidance. As such, continuously avoiding or escaping distress by self-injuring may qualify as a disorder. However, if it is the use of avoidance that is deemed unhelpful, then it seems more practical to adopt a functional perspective and view avoidance as a functional diagnostic dimension (cf. Hayes et al., 1996). This is consistent with the argument that NSSI belongs in a functional response class with other avoidant behaviours (Chapman et al., 2006). The alternative structural approach could potentially result in a long list of comorbid disorders, which on the surface may appear to have little in common, but ultimately serve the same function (Dougher & Hayes, 2000). Furthermore, a diagnosis of NSSI may encourage clinicians to assess and treat self- injury as a prescribed list of symptoms, rather than as a complex, dynamic way of coping that shifts in response to particular environmental contexts. However, if it is the use of avoidance that is deemed unhelpful, then it seems more practical to adopt a functional perspective and view avoidance as a functional diagnostic dimension (cf. Hayes et al., 1996). This is consistent with the argument that NSSI belongs in a functional response class with other avoidant behaviours (Chapman et al., 2006). The alternative structural approach could potentially result in a long list of comorbid disorders, which on the surface may appear to have little in common, but ultimately serve the same function (Dougher & Hayes, 2000). Furthermore, a diagnosis of NSSI may encourage clinicians to assess and treat self- injury as a prescribed list of symptoms, rather than as a complex, dynamic way of coping that shifts in response to particular environmental contexts. 266 5. CLINICAL IMPLICATIONS Turning to ACT, a protocol has been developed for treating adolescents who self-injure (Rowland, 2011) but, to my knowledge, the use of ACT to treat NSSI has never been empirically tested. Despite this lack of research, ACT appears to be a highly appropriate treatment for NSSI because it aims to decrease experiential avoidance through promoting psychological flexibility (Hayes et al., 2006). Turning to ACT, a protocol has been developed for treating adolescents who self-injure (Rowland, 2011) but, to my knowledge, the use of ACT to treat NSSI has never been empirically tested. Despite this lack of research, ACT appears to be a highly appropriate treatment for NSSI because it aims to decrease experiential avoidance through promoting psychological flexibility (Hayes et al., 2006). Experiential avoidance, within an ACT framework, is conceptualised as a barrier to acceptance; for someone who routinely escapes negative intrapersonal experiences through self-injury, acceptance would entail remaining in contact with their unwanted feelings or thoughts (Hayes et al., 2006). Psychologically flexible people Experiential avoidance, within an ACT framework, is conceptualised as a barrier to acceptance; for someone who routinely escapes negative intrapersonal experiences through self-injury, acceptance would entail remaining in contact with their unwanted feelings or thoughts (Hayes et al., 2006). Psychologically flexible people 267 are able to consciously connect with the present moment, and choose to engage in behaviours that enable them to live according their values (Hayes et al., 2006). Since it is beyond the purpose of this thesis to engage in an in-depth comparison of the potential for these two therapies to reduce self-injurious behaviours, I have instead chosen to discuss how particular strategies, which are routinely used in DBT and ACT protocols, could be used to prevent people from becoming overwhelmed by their negative emotions and thoughts to the point where their cognition constricts and they become trapped in the self-injury mindset. More specifically, I will focus on how teaching people mindfulness, cognitive defusion, and distress tolerance skills may help them to successfully regulate their emotions without resorting to NSSI. Mindfulness-based approaches to psychopathology, which involve teaching clients to focus their awareness on the present moment, appear to be growing in number and popularity (Chiesa & Malinowski, 2011). 5. CLINICAL IMPLICATIONS A fundamental element of mindfulness as defined by Kabat-Zinn is the absence of judgement (Elliston, 2002); that is, people are encouraged to develop the ability to simply notice what they are experiencing without judging those experiences as inherently good or bad (Hayes et al., 2006). Practising mindfulness may reduce the distress associated with particular emotions and thoughts for people who self-injure, which, in turn, could alleviate the sense, as described by participants in my interview study, of mounting pressure. Certainly, if thoughts and emotions are simply noticed, rather than judged as aversive and overwhelming, it is less likely that people will experience the same sense of urgency to escape from these intrapersonal events. Within a DBT framework, distress tolerance skills evolve out of, and build on, mindfulness training (Linehan, 1993a). Distress tolerance is defined as: the ability to perceive one’s environment without putting demands on it to be different, to experience your current emotional state without attempting to change it, and to observe your own thoughts and action patterns without attempting to stop or control them. (Linehan, 1993a, p. 96) the ability to perceive one’s environment without putting demands on it to be different, to experience your current emotional state without attempting to change it, and to observe your own thoughts and action patterns without attempting to stop or control them. (Linehan, 1993a, p. 96) 268 It is not difficult to see how distress tolerance may prove to be an essential component of self-injury prevention and intervention efforts in light of my findings that the interaction between intense, negative emotions and self-referential cognitions dominated as NSSI precipitants in studies one and two, and guilt and negative, automatic thoughts actually predicted new episodes of NSSI in study three. Distress tolerance would require people to perceive, observe, and experience their aversive emotions and thoughts instead of engaging in self-injurious behaviours, and is closely related to the notion of acceptance within ACT (Hofman & Asmundson, 2008). Cultivating a willingness to remain in contact with distressing thoughts and emotions may not only help people tolerate such experiences, but over time may reduce the likelihood that they reach the threshold of distress where self-injury, in the words of Lucy, becomes inevitable (see p. 130). Certainly, ACT contends that struggling to control unwanted, intrapersonal experiences is futile and counter- productive; such struggles may exacerbate these experiences and, in all likelihood, will heighten distress (Harris, 2006). 6. WHERE TO FROM HERE? Research on NSSI can be considered to be in its infancy; sophisticated study designs are needed to further develop our knowledge of why people start self- injuring and how these behaviours are maintained over time. One of the most critical areas for future research is how to effectively treat people who habitually self-injure, given the dearth of studies on NSSI interventions. In the following sections, I consider how the findings of my studies could be used to inform new research on self-injury and discuss limitations that need to be addressed in the future. Research on NSSI can be considered to be in its infancy; sophisticated study designs are needed to further develop our knowledge of why people start self- injuring and how these behaviours are maintained over time. One of the most critical areas for future research is how to effectively treat people who habitually self-injure, given the dearth of studies on NSSI interventions. In the following sections, I consider how the findings of my studies could be used to inform new research on self-injury and discuss limitations that need to be addressed in the future. Specifically, improved methods, a continued focus on the EAM and the functions of self-injury, an enhanced understanding of the interrelationship between emotions and cognitions, and more diverse samples are needed in order to develop a robust evidence base of NSSI research. Specifically, improved methods, a continued focus on the EAM and the functions of self-injury, an enhanced understanding of the interrelationship between emotions and cognitions, and more diverse samples are needed in order to develop a robust evidence base of NSSI research. 5. CLINICAL IMPLICATIONS For instance, as discussed earlier in this thesis (see p. 85), thought suppression usually increases the frequency of unwanted thoughts (Wegner & Zanakos, 1994). Cognitive defusion is another skill that may facilitate a reduction in distress over time through decreasing the believability of negative thoughts about oneself, others, and the world (Hayes et al., 2006). When people are fused with their cognitions, these thoughts are interpreted as literal truths or rules, and thus wield immense power over people’s behaviour (Harris, 2006). Therefore, the aim of cognitive defusion is to change the relationship that people have with their thoughts, rather than the content or frequency of those thoughts (Hayes et al., 2006). Teaching cognitive defusion to people who self-injure is likely to be helpful not only in reducing the believability of self-referential thoughts such as, ‚I am worthless‛, and thus the distress evoked by such thoughts, but also in challenging the literality and believability of verbal rules such as, ‚If I want to feel better, then I 269 need to hurt myself‛ (see pp. 131-133). For example, someone could defuse from this verbal rule by labelling it as a thought: ‚I am having the thought that if I want to feel better, then I need to hurt myself‛ (Hayes et al., 2006). In sum, ACT and DBT show considerable promise as treatment methods for a wide range of people who self- injure, but studies in support of this contention are urgently needed. 6.1 More sophisticated methods are necessary In Psychological research, as in literature, unreliable narrators (Booth, 1983) are to be expected. Memories of past events are fallible and the findings of my empirical studies, all of which relied exclusively on self-report methodologies, are undoubtedly influenced to some extent by retrospective bias. Such criticism, however, can be levelled at the majority of research conducted on NSSI to date and is typically an inescapable reality of studies designed to investigate what people think about and how they feel. Understanding the phenomenology of self-injury is essential to effectively prevent and treat these behaviours and, as such, self-report will always feature within NSSI research. However, the use of increasingly sophisticated methods, such as ecological momentary assessment (see Muehlenkamp et al., 2009; Nock et al., 270 2009) will not only help researchers reduce the error associated with retrospective bias, but will also facilitate a deeper understanding of the emotional, cognitive, and situational antecedents and consequences of NSSI. This understanding, in turn, will improve our knowledge of how self-injurious behaviours are reinforced and maintained over time. One of the limitations of the cross-sectional design of my first two studies is that I was unable to determine whether people’s self-injurious behaviours were actually reinforced as I do not know whether they self-injured again. Instead, I could only hypothesise that the majority of the episodes reported by participants were negatively reinforced and, as such, they were likely to engage in further NSSI. Including two time points in my third study was a strength because I was able to test whether specific intrapersonal experiences and coping styles predicted new incidents of self-injury. Certainly, over-time and longitudinal studies are desperately needed as the majority of work conducted on NSSI to date has been cross-sectional. As a result, we know very little about what factors are causally implicated in the initiation and maintenance of self-injurious behaviours. Preliminary evidence suggests well- established NSSI correlates, which have been hypothesised as risk factors for self- injury, do not actually predict new incidents of self-injury (Glenn & Klonsky, 2011), making longitudinal studies even more imperative. 6.2 How do emotions and cognitions interact to precipitate NSSI? Although my findings support the utility of conceptualising NSSI primarily as an experientially avoidant behaviour within the context of Aotearoa New Zealand, they also highlight the importance of developing a more sophisticated understanding of how cognitions and emotions interact to precipitate and reinforce self-injurious behaviours. Certainly, the EAM’s (Chapman et al., 2006) focus on emotional precipitants appears, in light of my findings about the self-injury mindset, to be overly simplistic. 271 Rather, it is clear from my research that the interaction between negative emotions and thoughts led participants to become overwhelmed, and, in some cases, once they surpassed a threshold of emotional intensity, they found it difficult to generate possible coping strategies other then self-injury. An important area of investigation for future research is to examine in greater depth how and why this process of cognitive constriction occurs, as well as the predictive power of verbal rules in this context. More specifically, it would be important to examine within- individual and between-group differences in how this threshold is reached and then surpassed. For instance, is the tipping point for self-injury completely idiosyncratic, based on individual learning histories and skill deficits, or do gendered, diagnostic, or culture-specific patterns in tipping points exist? Additionally, it should be noted that the relatively simplistic distinction made between emotions and cognitions when researching NSSI (including my studies) is becoming increasingly outdated when situated within the broader context of the emotion literature (cf. Barrett, 2009; Duncan & Barrett, 2007). For instance, Duncan and Barrett (2007) argue that at a neurobiological level, affect is a cognitive process and, as such, distinguishing between affect and cognition reflects an epistemological alignment with phenomenology, not ontology. They propose investigating why this distinction holds functional value for people is necessary to better understand the experience of emotions and cognitions. Such investigations within the emotion literature may usefully inform future research on how emotional and cognitive processes interact to precipitate, reinforce, and maintain self-injurious behaviour. 6 3 F th h th EAM i t d 6.3 Further research on the EAM is warranted Despite what is arguably an overly simplistic preoccupation with emotional avoidance, I contend nonetheless that the EAM (Chapman et al., 2006) is a useful framework for both research and clinical practice. However, it is the broader, more inclusive notion of experiential, rather than emotional, avoidance that should be utilised both empirically and clinically. Certainly, specific components of the model, such as why people who self-injure gravitate towards experientially avoidant coping 272 strategies in the first place, remain largely untested and would benefit from further research. Understanding why people utilise avoidance over and above other coping strategies provides information for clinicians about what skill deficits could be targeted in treatment. As I discussed in Chapter 3 (see pp. 82-83), Chapman and colleagues (2006) provide several suggestions as to why people who self-injure may evidence a heightened propensity towards experiential avoidance. It is possible that people who begin self-injuring experience emotions more intensely than people who do not ever self-injure (Chapman et al., 2006). A recent study showed that adolescents who had self-injured were more physiologically reactive when distressed than adolescents who had never self-injured (Nock & Mendes, 2008). Additionally, I identified in my over-time, survey study that students who had self-injured did report experiencing more intense, negative emotions in general than those who had never self-injured. However, neither of these studies was designed to determine whether these differences in emotional intensity existed prior to the onset of NSSI. Cohort studies with children and young adolescents are needed to examine whether emotional intensity predicts whether people begin engaging in NSSI. Linked to the notion of emotional intensity is the suggestion that people may be predisposed to NSSI because they are unable to tolerate emotional distress (Chapman et al., 2006). Low distress tolerance has been empirically associated with NSSI (Nock & Mendes, 2008) and is a target for therapeutic change among people diagnosed with BPD (Linehan, 1993a). Further research needs to be conducted to determine whether low distress tolerance is a risk factor for NSSI. experiential avoidance? Given that NSSI is conceptualised as one of many behaviours within the functional response class of experiential avoidance (Chapman et al., 2006), the similarities and differences between self-injury and functionally equivalent behaviours should be investigated in future research. In particular, understanding why people choose self-injury instead of, or in conjunction with, other experientially avoidant behaviours may have important implications for prevention and treatment efforts. Furthermore, such investigations are necessary to further test the specificity of the EAM (Chapman et al., 2006) to NSSI. For example, substance use is routinely conceptualised as an experientially avoidant behaviour (see pp. 80, 82, 84-86) and preliminary evidence suggests that people may replace self-injury with substance use (Brown et al., 2007). It is possible that this occurs within the context of developmental shifts; certainly, substance use is a more socially acceptable, avoidant behaviour to engage in as one grows older. However, it is likely that there are other variables, aside from social acceptability, that distinguish self-injury from substance use. Future studies could focus on identifying the factors (i.e., intrapersonal, interpersonal, and situational) that place people at risk of engaging in different forms of experiential avoidance. 6.4 What are the similarities and differences between NSSI and other forms of 6.4 What are the similarities and differences between NSSI and other forms of experiential avoidance? 6.3 Further research on the EAM is warranted Finally, it is possible that people who self-injure have a propensity for experientially avoidant coping strategies because they lack access to alternative coping mechanisms or they may possess such skills but find it too difficult to employ them when emotionally overwhelmed (Chapman et al., 2006).The latter hypothesis is more consistent with the findings in my research where people found it difficult to think of alternative coping strategies when they reached specific tipping points. 273 Examining these questions within a research context will undoubtedly develop our understanding of why people begin self-injuring and what types of interventions are most appropriate when attempting to reduce self-injurious behaviours. 6.5 How do functions of self-injury change over time? Applying behavioural theory to the study of NSSI among typically developing populations (Chapman et al., 2006; Klonsky & Glenn, 2009; Linehan, 1993a, 1993b; Nock & Prinstein, 2004, 2005) has significantly advanced our understanding of why people self-injure. Longitudinal studies are needed to further develop this evidence base, particularly with regards to how functions of self-injury change within people over time (Nock & Prinstein, 2004). Given the functions of a 274 behaviour are context dependent (Anderson, 2007), it seems probable that the within-person functions of NSSI will be influenced by factors that govern the parameters of people’s environments, such as developmental stage. Ecological momentary assessment techniques will also undoubtedly refine our current understanding of the functions of NSSI as retrospective functional assessments are likely to be subject to memory biases. For example, it is possible that retrospective self-reports may be tapping into the verbal rules people hold about NSSI rather than the behavioural functions. Preliminary results from one of the few ecological momentary assessment studies of NSSI show that the functions of self- injury are largely consistent with those endorsed in retrospective studies (Nock et al., 2009), but further research is needed to replicate these results. 6.6 What does NSSI communicate to others? Although NSSI does appear to function primarily as an experientially avoidant behaviour both within Aotearoa New Zealand and in international samples (Klonsky, 2007, 2011; Nock & Prinstein, 2004), interpersonal functions can play an important role in the initiation and maintenance of self-injury. In both my interview and cross-sectional survey studies, there were a number of participants who described utilising self-injury as a means of avoiding interpersonal responsibilities or seeking support. To better understand the complexity of how interpersonal functions of NSSI are reinforced, maintained, or punished, it would be useful to conduct further research on how and why NSSI functions as a form of communication to others. Additionally, it would be valuable to compare the semiotics of self-injury (i.e., what the behaviours signify to others) across cultures, sub-cultures, genders, and age groups, and how often messages conveyed through NSSI are received as intended. Certainly, several participants in my interview study provided examples of how the reasons behind their self-injurious behaviour had been misinterpreted by 275 others. Angela described being labelled as emo42 by people who saw evidence of her self-injury, a judgement she found ‚very offensive‛. She described self-injury, when associated with this subculture, as a public, group activity that was discussed ‚almost boastfully‛. For Angela, emo self-injury appeared to be distasteful because it primarily fulfilled an attention-seeking function. This directly contrasted with her use of NSSI as a self-help strategy. The belief that some groups use NSSI as a status symbol was echoed in Emily’s observation that the popularity of self-injury at her all-girls’ secondary school was evidence of a ‚glamorisation of dysfunction‛. While self-injury contagion has been studied among clinical populations (Matthews, 1968; Rosen & Walsh, 1989), the normalisation of self-injury within particular peer groups in community-based samples remains largely unexamined. Certainly, if such normalisation is prevalent, identifying what factors place young people at risk of succumbing to peer pressure to self-injure would be an important area for future research. 42 The word emo signifies a disparaging stereotype of someone who is overly sensitive or emotional (Emo, n.d.). 6.7 Diverse samples are needed As commented on earlier in this thesis, much of the research on NSSI has been conducted with female Caucasians (Shaw, 2002); my studies are no exception. The majority of the participants in all three of the studies I conducted were female and the samples lacked ethnic diversity. Furthermore, in my third study, participants who did not complete the Time 2 survey were significantly more likely to be male, perhaps suggesting that males are less willing to participate in research on NSSI. Unwillingness to participate could stem from perceptions that self-injury is a predominantly female problem or a reluctance to disclose self-injury. Although Pākehā are in the majority in Aotearoa New Zealand, they are overrepresented in my research compared to general population statistics (Bascand, 2007). As such, the lack of ethnic diversity observed across my three studies does not 42 The word emo signifies a disparaging stereotype of someone who is overly sensitive or emotional (Emo, n.d.). 276 reflect the current social demographics of Aotearoa New Zealand. This limitation should be addressed in future research especially given that definitions of NSSI are socio-culturally bound, as discussed in Chapter 1. Within Aotearoa New Zealand, researchers have an obligation to conduct themselves in accordance with the principles of the Treaty of Waitangi and particular attention thus needs to be paid to understanding Māori conceptualisations of NSSI and how these behaviours may manifest among Māori. Certainly, researching and treating self-injury as an experientially avoidant behaviour may not be appropriate for Māori or other ethnic groups, particularly if avoidant behaviours are considered socially acceptable or culturally adaptive in such communities. 7. CONCLUSION Non-suicidal self-injury is an important topic for future research as it appears to be a highly prevalent behaviour among certain groups and our understanding of why people begin, and continue, to self-injure is limited. However, I believe that it is equally important not to sensationalise the prevalence of NSSI in our communities; although many researchers unproblematically assert that the incidence of NSSI is rising, there is little evidence to confirm this suspicion. Alarmist statements such as that contained in one journal article title which referred to adolescent NSSI as ‚the latest epidemic [emphasis added] to assess and treat‛ (Miller & Smith, 2008, p. 178) misrepresent the current state of knowledge in this area. Certainly, I am cognisant that the explosion of research interest in NSSI may have helped to establish self-injury as one of the behaviours in the symptom pool of Western cultures, thus legitimising it as a signifier of distress (Shorter, 1987) and possibly fuelling an increase in the prevalence of these behaviours. Researchers have a responsibility to reflect on how they present NSSI in scientific discourse as these forms of knowledge have the potential to exert a significant influence over the way in which people behave (Watters, 2010). 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Ethnicity and deliberate self-injury: A review of the literature. In N. Robertson (Ed.), Māori and psychology: Research and practice - The proceedings of a symposium sponsored by the Māori and Psychology Research Unit. Hamilton: Māori & Psychology Research Unit. 308 Wykes, T., & Callard, F. (2010). Diagnosis, diagnosis, diagnosis: Towards DSM-5. Journal of Mental Health, 19, 301-304. doi:10.3109/09638237.2010.494189 Yates, T.M. (2004). The developmental psychopathology of self-injurious behavior: Compensatory regulation in posttraumatic adaptation. Clinical Psychology Review, 24, 35-74. doi:10.1016/j.cpr.2003.10.001 Yates, T.M., Carlson, E.A, & Egeland, B. (2008). A prospective study of child maltreatment and self-injurious behavior in a community sample. Development and Psychopathology, 20, 651-671. doi:10.1017/S0954579408000321 Yip, K. (2005). A multi-dimensional perspective of adolescents’ self-cutting. Child and Adolescent Mental Health, 10, 80-86. doi:10.1111/j.1475-3588.2005.00122.x Zahl, D.L., & Hawton, K. (2004). Repetition of deliberate self-harm and subsequent suicide risk: Long-term follow-up study of 11,583 patients. The British Journal of Psychiatry, 185, 70-75. Zanarini, M.C., Laudate, C.S., Frankenburg, F.R., Reich, D.B., & Fitzmaurice, G. (2011). Predictors of self-mutilation in patients with borderline personality disorder: A 10-year follow-up study. Journal of Psychiatric Research, 45, 823-828. doi:10.1016/j.jpsychires.2010.10.015 Zinck, A. Supervisor: Dr Marc Wilson School of Psychology PO Box 600 Victoria University of Wellington Ph: (04) 463 5225 Email: marc.wilson@vuw.ac.nz 05.010 (2008). Self-referential emotions. Consciousness and Cognition, 17, 496-505. doi:10.1016/j.concog.2008.03.014 Zlotnick, C., Shea, M.T., Pearlstein, T., Simpson, E., Costello, E., & Begin, A. (1996). The relationship between dissociative symptoms, alexithymia, impulsivity, sexual abuse, and self-mutilation. Comprehensive Psychiatry, 37, 12-16. Robyn Langlands, PhD Student School of Psychology PO Box 600 Victoria University of Wellington Ph: (04) 463 5233 extension 8605 Email: robyn.langlands@vuw.ac.nz e experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 2.1, June 2008 Introduction You are invited to take part in the following study. The study will involve the followin 1) Completing a questionnaire about the different types of non-suicidal deliberate self-harm behaviours you may have engaged in, as well as how often and severe these behaviours have been. 1) Completing a questionnaire about the different types of non-suicidal deliberate self-harm behaviours you may have engaged in, as well as how often and severe these behaviours have been. 2) Potentially participating in one or two interviews to discuss deliberate self-harm behaviours, depending on the results of your questionnaire. The interview(s) will last approximately one to two hours. 3) Completing a questionnaire (if you have taken part in the interview(s)) about participating in this research study. This questionnaire will include the opportunity to provide suggestions for future research on the topic of deliberate self-harm. Your participation is entirely voluntary (your choice). You do not have to take part in this study, and if you do choose not to take part, this will not affect any future care or treatment. If you do agree to take part, you are free to withdraw from the study at any time, without having to give a reason and this will in no way affect your continuing health care. If you do withdraw from the study before completing the questionnaire(s) or interview(s), all the data that has been collected in relation to you (e.g. completed questionnaires or interview transcripts) will be immediately destroyed. You do not have to answer all the questions, and you may stop the interview at any time. Participation in this study will be stopped should the Principal Investigator feel it is not in your best interest to continue. You may have support from a friend, family or whanau member to help you understand the risks and/or benefits of this study and any other explanation you may require. This study has received ethical approval from the Multi- region Ethics Committee which reviews National and Multi-regional studies. Page 1 of 4 The Principal Investigator intends to interview at least 12-15 participants. The Principal Investigator intends to interview at least 12-15 participants. Where will the interviews be conducted? The interviews will take place either at the mental health clinic or counselling service that the participant attends (if appropriate), or at the School of Psychology at Victoria University’s Kelburn Campus. What are the aims of the study? Deliberate self-harm is a seldom investigated topic in New Zealand and as a result, very little is known about New Zealanders’ experiences of self-harm. The purpose of this study is to explore what kinds of thoughts, feelings and events may lead adolescents and young adults to engage in deliberate self-harm, and the purpose(s) that self-harm fulfils for them. This study is being conducted as part of a PhD thesis. The Principal Investigator is interested in learning about your experiences of deliberate self-harm from your perspective. The study will not involve you participating in any form of therapy. The ultimate goal of the thesis is to identify factors that trigger and maintain non-suicidal self-harm behaviours in order to improve mental health treatments for people who self-harm. Who is being asked to participate in this study? Individuals will be asked to participate in the study if they are between the ages of 16 and 34, can speak English fluently, and have engaged in deliberate self-harm within the past 12 months. Individuals will be excluded if they have an intellectual disability, have only engaged in deliberate self-harm during episodes of mania or psychosis, or if they are currently experiencing a manic or psychotic episode. How were participants selected for this study, and who selected them? There are two groups of participants involved in this study—individuals who are engaged in public mental health and counselling services, and individuals recruited from the community. In regards to individuals from mental health and counselling services, the Principal Investigator approached mental health clinicians to inform them about the study. These clinicians were asked to pass on information about the study to any of their clients, between the ages of 16 to 34 years old, who have engaged in deliberate self-harm behaviour(s) within the past 12 months and who may be interested in participating in the study. To recruit participants from the community, the Principal Investigator placed advertisements about the study in community newspapers, newsletters, and venues inviting individuals who have engaged in deliberate self-harm behaviour(s) to contact her if they would like to participate in the study. Are there any risks? Deliberate self-harm can be a very difficult topic to talk about and there is a risk that some of the questions asked may bring up past memories or feelings that are unpleasant or distressing. If you do become distressed during the interview, you can stop the interview at any time. If you find that you feel upset after the interview, you are welcome to contact Robyn Langlands. Robyn will also explore options for further support with you at the end of the interview. What will happen during the study? 6. Robyn will contact you a day or two after the interview(s) to ask whether the study has raised any questions for you that you would like answered. 7. Within 2 weeks of completing the interview(s), you will receive a questionnaire to fill in about your experiences of participating in a research study on deliberate self-harm. This questionnaire will give you the opportunity to provide suggestions about how this type of research can be improved. What about privacy? No material which could personally identify you will be used in any reports on this study. The information will be used as part of the researcher’s PhD thesis and may be published in an academic journal or presented at conferences. In any publication or presentation, information will be provided in such a way that you cannot be identified. Some publications require that the data described is made available to competent professionals. If the data is requested by other professionals, it will be provided in such a way that no-one will be able to identify individual participants. What will happen during the study? If you agree to participate in this study, the following will take place: If you agree to participate in this study, the following will take place: 1. Robyn Langlands, the Principal Investigator, will send you a questionnaire to complete about deliberate self-harm behaviour(s). Page 2 of 4 Page 2 of 4 2. Depending on the results of your questionnaire, you may be asked to participate in an interview to discuss deliberate self-harm behaviours. You will be given a list of questions that will be asked during the interview to enable you to decide whether you will be comfortable discussing such topics. p 3. If you decide you would like to participate in the interview, Robyn will arrange a suitable time and place to meet you for the interview. You are welcome to bring a support person with you to the interview. 3. If you decide you would like to participate in the interview, Robyn will arrange a suitable time and place to meet you for the interview. You are welcome to bring a support person with you to the interview. 4. You will be asked to sign a consent form to show that you understand what the study is about and that you agree to participate. If you have a mental health clinician, by giving your consent you will also be giving permission for Robyn to contact your mental health clinician to inform them that you are taking part in the study and to confirm what particular mental health diagnoses you have been given. The information that you share with Robyn during the interview will not be discussed with your clinician unless she believes that you are danger of harming yourself or someone else, in which case she is legally required to break confidentiality. 5. During the interview, Robyn will ask you a number of questions about your self-harm behaviour(s). This interview will take approximately 1-2 hours and will be audio-taped for later transcription. If you or Robyn does not believe that you have covered all the necessary information within the interview, you will be given the opportunity to participate in a second interview. The tapes and transcripts from the interview(s) will be stored in a locked filing cabinet following the study. 6. Robyn will contact you a day or two after the interview(s) to ask whether the study has raised any questions for you that you would like answered. What are the benefits? There is no guarantee that you will receive any benefits from this project. However, this project does have the potential to lead to better mental health care and support for people who engage in deliberate self-harm. Page 3 of 4 The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 2.1, June 20 Will I be paid to participate in this project? You will be given a movie voucher to the value of one movie ticket for completing the initial questionnaire. If you participate in the interview, you will be given the choice of a petrol voucher, Farmer’s voucher or The Warehouse voucher to the value of $30 to reimburse you for your time and travel expenses. Finally, if you complete the questionnaire on research participation, you will be given a movie voucher to the value of one movie ticket. Where can I get more information about the study? If you would like further information about this project, please contact Robyn Langlands, email: robyn.langlands@vuw.ac.nz, phone: (04) 463 5233 extension 8605. If you do phone Robyn and you are directed through to an answer phone, please leave your name and number, and she will call you back as soon as possible. The answer phone is private and Robyn will be the only person who has access to the messages left on the phone. What will happen at the end of the study? You will be asked whether you would like to be sent a transcript of your interview(s) once the data has been transcribed. You will also be given the option of receiving a brief summary of the common themes that arose from the interviews once the data has been transcribed and analysed. What if I have concerns about the project? If you have any questions or concerns about your rights as a participant in this research study, you can contact an independent health and disability advocate. This is a free service provided under the Health and Disability Commissioner Act. 0800 555 050 0800 2787 7678 (0800 2 SUPPORT) advocacy@hdc.org.nz he experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 2.1, June 2008 Compensation In the unlikely event of a physical injury as a result of your participation in this study, you may be covered by ACC under the Injury Prevention, Rehabilitation and Compensation Act. ACC cover is not automatic and your case will need to be assessed by ACC according to the provisions of the 2002 Injury Prevention Rehabilitation and Compensation Act. If your claim is accepted by ACC, you still might not get any compensation. This depends on a number of factors such as whether you are an earner or non- earner. ACC usually provides only partial reimbursement of costs and expenses and there may be no lump sum compensation payable. There is no cover for mental injury unless it is a result of physical injury. If you have ACC cover, generally this will affect your right to sue the investigators. If you have any questions about ACC, contact your nearest ACC office or the Principal Investigator. Page 4 of 4 Page 4 of 4 General Information and Deliberate Self-Harm Inventory (Gratz, 2001) Name: _____________________________________________________________________________ Date of birth: _______________________________________________________________________ Gender (circle one): Female Male Nationality: ________________________________________________________________________ Ethnicity: __________________________________________________________________________ Have you ever received a mental health diagnosis or diagnoses? (circle one): Yes No If yes, what was the diagnosis or diagnoses? ___________________________________________ ____________________________________________________________________________________ How old were you when received the diagnosis or diagnoses? ___________________________ ____________________________________________________________________________________ Phone number: _____________________________________________________________________ Postal Address: _____________________________________________________________________ Email Address: _____________________________________________________________________ General Information and Deliberate Self-Harm Inventory (Gratz, 2001) Name: _____________________________________________________________________________ Date of birth: _______________________________________________________________________ Gender (circle one): Female Male Nationality: ________________________________________________________________________ Ethnicity: __________________________________________________________________________ Have you ever received a mental health diagnosis or diagnoses? (circle one): Yes No If yes, what was the diagnosis or diagnoses? ___________________________________________ ____________________________________________________________________________________ How old were you when received the diagnosis or diagnoses? ___________________________ ____________________________________________________________________________________ Phone number: _____________________________________________________________________ Postal Address: _____________________________________________________________________ Email Address: _____________________________________________________________________ neral Information and Deliberate Self-Harm Inventory (Gratz, 2001 This questionnaire asks about a number of different things that people sometimes do to hurt themselves. Please be sure to read each question carefully and respond honestly. Often, people who do these kinds of things to themselves keep it a secret, for a variety of reasons. However, honest responses to these questions will provide us with greater understanding and knowledge about these behaviours and the best way to help people. Please answer yes to a question only if you did the behaviour intentionally, or on purpose, to hurt yourself. Compensation Do not respond yes if you did something accidentally (e.g., you tripped and banged your head on accident). Also, please be assured that your responses are completely confidential. This questionnaire should take you approximately 15 minutes to complete. 1 1 1 1. Have you ever intentionally (i.e., on purpose) cut your wrist, arms, or other area(s) of your body (without intending to kill yourself)? (circle one): Yes No If yes:  How old were you when you first did this? _________________  How many times have you done this? _________________  When was the last time you did this? _________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) ________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? _________________ 2. Have you ever intentionally (i.e., on purpose) burned yourself with a cigarette (circle one): Yes No If yes:  How old were you when you first did this? _________________  How many times have you done this? _________________  When was the last time you did this? _________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) _________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? _________________ 3. Have you ever intentionally (i.e., on purpose) burned yourself with a lighter or a match? (circle one): Yes No If yes:  How old were you when you first did this? _________________  How many times have you done this? _________________  When was the last time you did this? _________________ 3. Have you ever intentionally (i.e., on purpose) burned yourself with a lighter or a match? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) _________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? Compensation __________________ 2 2 Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 5. Have you ever intentionally (i.e., on purpose) carved pictures, designs, or other marks into your skin? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________ How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 6. Have you ever intentionally (i.e., on purpose) severely scratched yourself, to the extent that scarring or bleeding occurred? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 6. Have you ever intentionally (i.e., on purpose) severely scratched yourself, to the extent that scarring or bleeding occurred? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 3 3 7. Have you ever intentionally (i.e., on purpose) bitten yourself, to the extent that you broke the skin? (circle one): __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 11. Have you ever intentionally (i.e., on purpose) stuck sharp objects such as needles, pins, staples, etc. into your skin, not including tattoos, ear piercing, needles used for drug use, or body piercing? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 12. Have you ever intentionally (i.e., on purpose) rubbed glass into your skin? (circle one): Yes No 10. Have you ever intentionally (i.e., on purpose) used bleach or oven cleaner to scrub your skin? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 11. Have you ever intentionally (i.e., on purpose) stuck sharp objects such as needles, pins, staples, etc. into your skin, not including tattoos, ear piercing, needles used for drug use, or body piercing? 7. Have you ever intentionally (i.e., on purpose) bitten yourself, to the extent that you broke the skin? (circle one): (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 12. Have you ever intentionally (i.e., on purpose) rubbed glass into your skin? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 11. Have you ever intentionally (i.e., on purpose) stuck sharp objects such as needles, pins, staples, etc. into your skin, not including tattoos, ear piercing, needles used for drug use, or body piercing? (circle one): Yes No If 11. Have you ever intentionally (i.e., on purpose) stuck sharp objects such as needles, pins, staples, etc. into your skin, not including tattoos, ear piercing, needles used for drug use, or body piercing? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 11. Have you ever intentionally (i.e., on purpose) stuck sharp objects such as needles, pins, staples, etc. 7. Have you ever intentionally (i.e., on purpose) bitten yourself, to the extent that you broke the skin? (circle one): Ye o If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 8. Have you ever intentionally (i.e., on purpose) rubbed sandpaper on your body? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 9. Have you ever intentionally (i.e., on purpose) dropped acid onto your skin? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 9. Have you ever intentionally (i.e., on purpose) dropped acid onto your skin? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 4 4 10. Have you ever intentionally (i.e., on purpose) used bleach or oven cleaner to scrub your skin? (circle one): Yes No If yes:  How old were you when you first did this? 7. Have you ever intentionally (i.e., on purpose) bitten yourself, to the extent that you broke the skin? (circle one): into your skin, not including tattoos, ear piercing, needles used for drug use, or body piercing? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 5 13. Have you ever intentionally (i.e., on purpose) broken your own bones? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 14. Have you ever intentionally (i.e., on purpose) banged your head against something, to the extent that you caused a bruise to appear? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 15. Have you ever intentionally (i.e., on purpose) punched yourself, to the extent that you caused a bruise to appear? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________ Wh th l t ti did thi ? 15. Have you ever intentionally (i.e., on purpose) punched yourself, to the extent that you caused a bruise to appear? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? 7. Have you ever intentionally (i.e., on purpose) bitten yourself, to the extent that you broke the skin? (circle one): __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 6 6 16. Have you ever intentionally (i.e., on purpose) prevented wounds from healing? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 17. Have you ever intentionally (i.e., on purpose) done anything else to hurt yourself that was not asked about in this questionnaire? If yes, what did you do to hurt yourself? _________________________________________________________________________________  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 16. Have you ever intentionally (i.e., on purpose) prevented wounds from healing? (circle one): Yes No If yes:  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 17. Have you ever intentionally (i.e., on purpose) done anything else to hurt yourself that was not asked about in this questionnaire? If yes, what did you do to hurt yourself? _________________________________________________________________________________  How old were you when you first did this? __________________  How many times have you done this? 7. Have you ever intentionally (i.e., on purpose) bitten yourself, to the extent that you broke the skin? (circle one): __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ 17. Have you ever intentionally (i.e., on purpose) done anything else to hurt yourself that was not asked about in this questionnaire? If yes, what did you do to hurt yourself? _________________________________________________________________________________  How old were you when you first did this? __________________  How many times have you done this? __________________  When was the last time you did this? __________________  How many years have you been doing this? (If you are no longer doing this, how many years did you do this before you stopped?) __________________  Has this behaviour ever resulted in hospitalisation or injury severe enough to require medical treatment? __________________ Mensline Free telephone Counselling Available 5:30pm-11pm, 7 nights a week Ph: 0800 MENSLINE (636 754) Website: http://www.mensline.org.nz/ Available 10am-10pm daily Ph: 0800 787 797 Website: http://www.adanz.org.nz/ADANZ/Home Available 10am-10pm daily Ph: 0800 787 797 Website: http://www.adanz.org.nz/ADAN Your time and contribution to this research is much appreciated. 7 7 Support Organisations Deliberate self-harm can be a very difficult topic to talk about and as a result, some of the questions you have been asked about today may have brought up past memories or feelings that are unpleasant or distressing. If this is the case, please feel free to contact me on (04) 463 5233 extension 8605 or at robyn.langlands@vuw.ac.nz. Alternatively, you may wish to call, email or check out the website of one of the following support organisations: Deliberate self-harm can be a very difficult topic to talk about and as a result, some of the questions you have been asked about today may have brought up past memories or feelings that are unpleasant or distressing. If this is the case, please feel free to contact me on (04) 463 5233 extension 8605 or at robyn.langlands@vuw.ac.nz. Alternatively, you may wish to call, email or check out the website of one of the following support organisations: LifeLine Free telephone counselling Available 24 hours a day, 7days a week Ph: 0800 111 777 Website: http://www.lifeline.org.nz/ Free email counselling Email: chris@lifeline.co.nz Website: http://www.elifeline.co.nz/ Youthline Free telephone counselling Available 24 hours a day, 7days a week Ph: 0800 376 633 Website: http://www.youthline.co.nz/ Email or Text Support Email: talk@youthline.co.nz TXT: 027 4 YOUTHS (027 4968 847) Mensline Free telephone Counselling Available 5:30pm-11pm, 7 nights a week Ph: 0800 MENSLINE (636 754) Website: http://www.mensline.org.nz/ Alcohol and Drug Helpline Available 10am-10pm daily Ph: 0800 787 797 Website: http://www.adanz.org.nz/ADANZ/Home Warmline Free phone support service staffed by volunteers specifically for people who use mental health services. Volunteers are people who have used mental health services themselves. Available 7.00pm-1.00am, Tuesday-Sunday Ph: 0800 200 207 Buddies Buddies offers peer support to people who self identify as experiencing a mental illness or who have used mental health services. Ph: (04) 385 2104 or (021) 960 060 Email: buddies.wn@paradise.net.nz Visit: Level 6 NZEI House 178 Willis Street, Wellington LifeLine Free telephone counselling Available 24 hours a day, 7days a week Ph: 0800 111 777 Website: http://www.lifeline.org.nz/ Free telephone counselling Available 24 hours a day, 7days a week Ph: 0800 376 633 Website: http://www.youthline.co.nz/ Interview Questions Please read the following questions carefully. The purpose of this study is to understand how you experience self-harm from your perspective, while at the same time addressing the research questions listed below. As a result, Robyn cannot guarantee that she will ask you all of these questions or that these will be the only questions she will ask you during the interview(s). The questions do, however, provide you with an idea about what kinds of topics will be discussed during the interview. Please consider carefully if you would be comfortable answering these questions before you decide whether you would like to participate in an interview.  Can you tell me about the last time that you deliberately harmed yourself without intending to kill yourself?  Can you tell me about the last time that you deliberately harmed yourself without intending to kill yourself?  Can you tell me as much as you can remember about what led up to this episode of self-harm?  Can you describe for me how you were feeling prior to harming yourself?  How did you feel during this particular episode of self-harm?  How did you feel afterwards?  How did you feel afterwards?  Can you tell me what you were thinking about prior to harming yourself?  What were you thinking about during this particular episode of self-harm?  Did those thoughts change in any way after the episode of self-harm? [If yes…] What were you thinking about after you had harmed yourself?  Did you have to deal with any particular consequences from that episode of self-harm? [If yes…] Can you tell me about them?  Thinking about what we’ve talked about in relation to this particular episode of self-harm, would you consider this a typical episode of self-harm for you? [If no…] What was different about this episode compared to other times in the past when you’ve harmed yourself?  Is there anything that you’d like to tell me about in relation to your self-harm that we haven’t discussed?  Can you tell me what you were thinking about prior to harming yourself?  Did those thoughts change in any way after the episode of self-harm? [If yes…] What were you thinking about after you had harmed yourself?  Did you have to deal with any particular consequences from that episode of self-harm? Warmline Free phone support service staffed by volunteers specifically for people who use mental health services. Volunteers are people who have used mental health services themselves. Buddies offers peer support to people who self identify as experiencing a mental illness or who have used mental health services. Ph: (04) 385 2104 or (021) 960 060 Email: buddies.wn@paradise.net.nz Visit: Level 6 NZEI House 178 Willis Street, Wellington Available 7.00pm-1.00am, Tuesday-Sunday Ph: 0800 200 207 Available 7.00pm-1.00am, Tuesday-Sunday Ph: 0800 200 207 1 [If yes…] Can you tell me about them?  Thinking about what we’ve talked about in relation to this particular episode of self-harm, would you consider this a typical episode of self-harm for you? [If no…] What was different about this episode compared to other times in the past when you’ve harmed yourself?  Is there anything that you’d like to tell me about in relation to your self-harm that we haven’t discussed? Consent to Contact Clinician The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Consent to Contact Clinician, Version 1, July 2008 Consent to Contact Clinician I give permission for the Principal Investigator to inform my mental health clinician that I am participating in this study. I give permission for the Principal Investigator to confirm with my clinician any mental health diagnoses that I have if this is necessary. Full name: The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Consent to Contact Clinician, Version 1, July 2008 1 f 1 Page 1 of 1 Exploring the experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours your family’s home phone number) I_________________________________________ (full name) hereby consent to take part in this study. Date: Please complete the following: I would like to receive a copy of my interview transcript.  Yes  No I would like to receive a summary of the research results.  Yes  No I would like to be contacted by Robyn Langlands in the future with regards to  Yes  No other studies she is conducting on deliberate self-harm behaviours for her PhD. Please write your contact details below. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ What is the best time of day to contact you? (please circle one): Morning Afternoon Evening Is the form of contact you’ve chosen (please circle one): Private Shared (e.g. personal email address or cell number) (e.g. your family’s home phone number) I_________________________________________ (full name) hereby consent to take part in this study. Date: Signature: Please complete the following: Please complete the following: I would like to receive a copy of my interview transcript.  Yes  No I would like to receive a summary of the research results.  Yes  No I would like to be contacted by Robyn Langlands in the future with regards to  Yes  No other studies she is conducting on deliberate self-harm behaviours for her PhD. Please write your contact details below. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ What is the best time of day to contact you? (please circle one): Morning Afternoon Evening Is the form of contact you’ve chosen (please circle one): Private Shared (e.g. personal email address or cell number) (e.g. your family’s home phone number) I_________________________________________ (full name) hereby consent to take part in this study. Date: I would like to receive a copy of my interview transcript.  Yes  No I would like to receive a summary of the research results.  Yes  No I would like to be contacted by Robyn Langlands in the future with regards to  Yes  No other studies she is conducting on deliberate self-harm behaviours for her PhD. I would like to receive a copy of my interview transcript.  Yes  No I would like to receive a summary of the research results.  Yes  No I would like to be contacted by Robyn Langlands in the future with regards to  Yes  No other studies she is conducting on deliberate self-harm behaviours for her PhD.  Yes  No  Yes  No  Yes  No I would like to receive a copy of my interview transcript. _ (full name) hereby consent to take part in this study. _ (full name) hereby consent to take part in this study. Exploring the experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours Consent Form  I have read and I understand the information sheet dated June 2008 for volunteers taking part in the study designed to explore the deliberate self-harm experiences of adolescents and young adults.  I have read and I understand the information sheet dated June 2008 for volunteers taking part in the study designed to explore the deliberate self-harm experiences of adolescents and young adults.  I have read the list of interview questions for the study.  I have had the opportunity to discuss this study. I am satisfied with the answers I have been given.  I have had the opportunity to use whanau support or a friend to help me ask questions and understand the study.  I understand that taking part in this study is voluntary (my choice) and that I may withdraw from the study at any time and this will in no way affect my continuing health care.  I understand that my participation in this study is confidential and that no material which could identify me will be used in any reports on this study.  I understand that my interview will be audio-taped.  I understand the compensation provisions for this study.  I have had time to consider whether to take part.  I know who to contact if the study distresses me in any way.  I know who to contact if I have any questions about the study. The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Consent Form, Version 3, July 2008 The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Consent Form, Vers Page 1 of 2 Page 1 of 2 Please complete the following: I would like to receive a copy of my interview transcript.  Yes  No I would like to receive a summary of the research results.  Yes  No I would like to be contacted by Robyn Langlands in the future with regards to  Yes  No other studies she is conducting on deliberate self-harm behaviours for her PhD. Please write your contact details below. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ What is the best time of day to contact you? (please circle one): Morning Afternoon Evening Is the form of contact you’ve chosen (please circle one): Private Shared (e.g. personal email address or cell number) (e.g. Date: Signature: Full names of Principal Investigator: Contact Phone Number for Principal Investigator: Evaluation of Research Participation This questionnaire should take approximately 15 minutes to complete. Please note that when a question refers to ‘the study’, this includes both the first questionnaire you completed and the interview(s) you participated in. Please circle the response that best describes your experiences of taking part in this study: describes your experiences of taking part in this study: 1) The study was explained thoroughly to me before I took part. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 2) It was my choice to take part in the study (i.e., I could have said no even if other people wanted me to say yes). 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 3) I knew I could skip questions or parts of the study if I wanted to. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 4) I knew I could stop being in the study at any time. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 5) During the interview, I knew that I could ask to take a break whenever I wanted. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 1.1, July 2008 Page 1 of 4 1) The study was explained thoroughly to me before I took part. 1) The study was explained thoroughly to me before I took part. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 2) It was my choice to take part in the study (i.e., I could have said no even if other people wanted me to say yes). 2) It was my choice to take part in the study (i.e., I could have said no even if other people wanted me to say yes). 2) It was my choice to take part in the study (i.e., I could have said no even if other people wanted me to say yes). Not sure Agree 3) I knew I could skip questions or parts of the study if I wanted to. 3) I knew I could skip questions or parts of the study if I wanted to. 3) I knew I could skip questions or parts of the study if I wanted to. Contact Phone Number for Principal Investigator: The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Consent Form, Version 3, July 2008 ts and young adults who engage in non-suicidal deliberate self-harm behaviours, Consent Form, Version 3, July 2008 Page 2 of 2 Page 2 of 2 Page 2 of 2 Evaluation of Research Participation 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 4) I knew I could stop being in the study at any time. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 5) During the interview, I knew that I could ask to take a break whenever I wanted. Not sure 5 Strongly agree Agree ing the interview, I knew that I could ask to take a break whenever I wanted. The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 1.1, July 2008 Page 1 of 4 6) The interviewer made me feel comfortable during the interview. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 7) The interviewer showed respect for my feelings and experiences during the interview. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 8) I am glad that I took part in this study. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 9) Being in this study made me feel distressed. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 10) Being in this study made me feel upset. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 11) Being in this study made me feel good about myself. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 12) I regret participating in this study. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 13) Being in this study made me feel sad. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree 14) I believe that by being in the study I am helping other people who self-harm. 1 2 3 4 5 Strongly disagree Disagree Not sure Agree Strongly agree Th i f d l d d l h i i id l d lib lf h b h i V i 1 1 J l 2008 P 2 f 13) Being in this study made me feel sad. Not sure Agree 14) I believe that by being in the study I am helping other people who self-harm. 15) Knowing what I know now about participating in the study, I would still choose to take part in this research. Evaluation of Research Participation 15) Knowing what I know now about participating in the study, I would still choose to take part in this research. 16) Do you have any suggestions as to how to improve research studies about deliberate self-harm? If yes, please write them below. ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 17) Do you have any other comments about your experiences of participating in this study? If yes, please write them below. ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ The experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 1.1, July 2008 Page 3 of 4 16) Do you have any suggestions as to how to improve research studies about deliberate self-harm? If yes, please write them below. Page 4 of 4 Page 4 of 4 he experiences of adolescents and young adults who engage in non-suicidal deliberate self-harm behaviours, Version 1.1, July 2008 Coding scheme1 “Unpleasant events” are defined as members of a set of events (e.g., death of a close relative, losing one’s job), stimuli (e.g., strong electric shock), and behaviours (e.g., cleaning a mess) which are experienced by the individual to whom they occur as unpleasant, painful, noxious, or distressing. 1) First, code each response according to the following categories (responses may be given more than one code): Code Category Definition 01 Health and well-being Events that involve injury, physical pain, and danger to the self or to important others. These can be specific psychopathological events (e.g., a binge) but do not include ongoing mental health problems (e.g., bulimia). 02 Achievement-Academic-Job Events that involve work, school, and other competitive situations; achievement related failures, disappointments, and difficulties. 03 Domestic, day-to-day inconveniences Events that involve noise, crowding, minor physical discomforts, mishaps, delays, and accidents. 04 Interpersonal Relationships (e.g., sexual, marital, friendships, family, collegial) Events concerning rejection, separation, loss, and other disappointing and painful interpersonal experiences. These events can include memories of negative interpersonal experiences. 05 Legal Events involving encounters with the police, courts of law, lawsuits, incarceration, and other legal problems. 06 Material-Financial Events involving financial losses and problems. 07 Death Related The actual or remembered death of an important other or events which remind the individual of the reality of death (e.g., seeing a corpse). 08 Other Events that could not be coded in any of the above categories. 88 No specific event General response that did not refer to a specific event (e.g., I was feeling overwhelmed). 99 Insufficient Information Response did not contain enough information about the event to be coded. “Unpleasant events” are defined as members of a set of events (e.g., death of a close relative, losing one’s job), stimuli (e.g., strong electric shock), and behaviours (e.g., cleaning a mess) which are experienced by the individual to whom they occur as unpleasant, painful, noxious, or distressing. “Unpleasant events” are defined as members of a set of events (e.g., death of a close relative, losing one’s job), stimuli (e.g., strong electric shock), and behaviours (e.g., cleaning a mess) which are experienced by the individual to whom they occur as unpleasant, painful, noxious, or distressing. 2) Second, code each response again according to the following categories: Code Category Definition 13 Self Self-related events that involve only the individual. These can include memories of past events that involved others. 1 Adapted from the Unpleasant Events Schedule (Lewinsohn, Mermelstein, Alexander, & MacPhillamy, 1983). Debriefing information Thank you for completing this survey. In this study, we were interested in the thoughts, feelings, and events that lead people to engage in non-suicidal self-injury. Researchers have identified that one of the main reasons why people injure themselves on purpose is to manage overwhelming, negative feelings such as sadness or anger. Other reasons people give for self-injury include: to punish themselves, to feel more in control, and to stop feeling numb. Understanding why people self-injure is essential in order to effectively treat this behaviour. Very little research has been conducted in Aotearoa/New Zealand to explore non-suicidal self-injury. This research will help us to learn more about why people in Aotearoa/New Zealand injure themselves on purpose and, ultimately, may help us to identify the most effective treatments for non-suicidal self- injury. Thank you once again for taking the time to participate in this study. If you know of anyone else who has injured themselves on purpose in the past 12 months, is at least 16 years of age, lives in Aotearoa/New Zealand, and you think they may be interested in completing this survey, please pass on the study details to them. 14 Other Events which involve interacting with other people at the time (i.e., not memories of past interactions). Free telephone counselling available 24 hours a day, 7 days a week Ph: (09) 5222 999 (within Auckland) 0800 543 354 (outside Auckland) Website: http://www.lifeline.org.nz Free email counselling Email: chris@elifeline.co.nz Website: http://www.elifeline.co.nz Youthline Free telephone counselling available 24 hours a day, 7 days a week Ph: 0800 376 633 Website: http://www.youthline.co.nz Email: talk@youthline.co.nz Free TXT using your mobile phone to 234 Free telephone counselling available 24 hours a day, 7 days a week Ph: (09) 5222 999 (within Auckland) 0800 543 354 (outside Auckland) Website: http://www.lifeline.org.nz Free email counselling Email: chris@elifeline.co.nz Website: http://www.elifeline.co.nz Need to talk to someone? If you have found completing this survey distressing in any way, it may be helpful for you to talk to a friend, relative, or other support person (e.g., teacher, minister, counsellor) about how you are feeling. You could also call, email, or check out the websites of one of the following support organisations: Warmline Website: http://www.wellink.org.nz/services/warmline.htm Rainbow Youth Rainbow Youth is an Auckland-based organisation providing support, information, advocacy and education for lesbian, gay, bisexual, transgender, intersex, fa'afafine, and takataapui young people (aged between 13 and 28) and their friends, family and whanau. Phone: (09) 376 4155 Email: info@rainbowyouth.org.nz Website: http://www.rainbowyouth.org.nz Youthline Free telephone counselling available 24 hours a day, 7 days a week Ph: 0800 376 633 Website: http://www.youthline.co.nz Email: talk@youthline.co.nz Free TXT using your mobile phone to 234 Free telephone counselling available 24 hours a day, 7 days a week Ph: 0800 376 633 Website: http://www.youthline.co.nz Email: talk@youthline.co.nz Free TXT using your mobile phone to 234 Warmline Warmline is a peer-run service, staffed by people who have all experienced mental illness in a real way. Warmline’s service is free, and open to anyone who feels affected in some way by mental illness. Available 7.00pm-1.00am, Tuesday-Sunday Ph: 0800 200 207 Website: http://www.wellink.org.nz/services/warmline.htm Warmline Warmline is a peer-run service, staffed by people who have all experienced mental illness in a real way. Warmline’s service is free, and open to anyone who feels affected in some way by mental illness. Available 7.00pm-1.00am, Tuesday-Sunday Ph: 0800 200 207 Website: http://www.wellink.org.nz/services/warmline.htm Rainbow Youth 2 2 Need to talk to someone? If you need to talk to someone, please contact one of the following:  Lifeline 0800 543 354  Depression Helpline 0800 111 757  Youthline 0800 376 633  Warmline 0800 200 207 Do you need support? If you are feeling distressed or upset, it may be helpful for you to talk to a friend, relative, or other support person (e.g., teacher, minister, counsellor) about how you are feeling. Alternatively, you could contact one of the following. In crisis? If you need urgent mental health support, please phone 111 or one of the following Crisis and Assessment Treatment Teams (CATT): Crisis and Assessment Treatment Teams (CATT):  If you live in the greater Wellington region (i.e., Wellington, Porirua, or Kapiti) phone 494 9169.  If you live in the Hutt Valley, phone 566 6999 and ask for the CAT team. Need a referral or counselling? Angelique O'Connell is a registered Clinical Psychologist who works in the Victoria Psychology Clinic, located on the 5th floor of the Easterfield Building. Phone 463 6400 or email: psychclinic@vuw.ac.nz to book an appointment. Victoria University has a free, confidential Counselling Service for students. Phone 463 5310 or email: counselling-service@vuw.ac.nz to book an appointment.
https://openalex.org/W3009473951	https://europepmc.org/articles/pmc7048112?pdf=render	English	null	Impact of protocolized diuresis for de-resuscitation in the intensive care unit	Critical care	2,020	cc-by	6,968	© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Objective: Administration of diuretics has been shown to assist fluid management and improve clinical outcomes in the critically ill post-shock resolution. Current guidelines have not yet included standardization or guidance for diuretic-based de-resuscitation in critically ill patients. This study aimed to evaluate the impact of a multi-disciplinary protocol for diuresis-guided de-resuscitation in the critically ill. Methods: This was a pre-post single-center pilot study within the medical intensive care unit (ICU) of a large academic medical center. Adult patients admitted to the Medical ICU receiving mechanical ventilation with either (1) clinical signs of volume overload via chest radiography or physical exam or (2) any cumulative fluid balance ≥0 mL since hospital admission were eligible for inclusion. Patients received diuresis per clinician discretion for a 2-year period (historical control) followed by a diuresis protocol for 1 year (intervention). Patients within the intervention group were matched in a 1:3 ratio with those from the historical cohort who met the study inclusion and exclusion criteria. Results: A total of 364 patients were included, 91 in the protocol group and 273 receiving standard care. Protocolized diuresis was associated with a significant decrease in 72-h post-shock cumulative fluid balance [median, IQR −2257 (− 5676–920) mL vs 265 (−2283–3025) mL; p < 0.0001]. In-hospital mortality in the intervention group was lower compared to the historical group (5.5% vs 16.1%; p = 0.008) and higher ICU-free days (p = 0.03). However, no statistically significant difference was found in ventilator-free days, and increased rates of hypernatremia and hypokalemia were demonstrated. Conclusions: This study showed that a protocol for diuresis for de-resuscitation can significantly improve 72-h post-shock fluid balance with potential benefit on clinical outcomes. Keywords: Critical illness, Diuretics, Resuscitation, Fluid therapy, Pharmacists, Mechanical ventilation harm has been demonstrated on pulmonary and renal function, as well as important clinical outcomes such as mortality and length of stay [1]. Despite the growing body of evidence supporting the adverse aspects of posi- tive fluid balance, fluid overload remains common in ICU patients [4]. Impact of protocolized diuresis for de- resuscitation in the intensive care unit Brittany D. Bissell1,2,3* , Melanie E. Laine1,2, Melissa L. Thompson Bastin1,2, Alexander H. Flannery1,2, Andrew Kelly4, Jeremy Riser4, Javier A. Neyra5, Jordan Potter6 and Peter E. Morris3 Bissell et al. Critical Care (2020) 24:70 https://doi.org/10.1186/s13054-020-2795-9 Bissell et al. Critical Care (2020) 24:70 https://doi.org/10.1186/s13054-020-2795-9 Introduction Indications for continuous infusion diur- esis included a lack of response to 200 mg or failure of sustained diuretic response resulting in failure to achieve goal fluid balance. After identification of appropriate patients for inclusion a net 24 h fluid balance (ranging from −1000 mL to − 3000 mL) was established during interdisciplinary rounds which was divided into three shift goal fluid balance tar- gets to assess at 8-h intervals. Upon establishment of goal, diuretic orders were entered, with dose selection based on previous diuretic exposure and baseline renal function. Orders included conditional diuretic orders if shift fluid balance targets were not met, basic metabolic panels, goal parameters, and hold parameters for adverse events (see Additional file 1). Combination diuresis was permitted once the maximum dose of furosemide was reached (200 mg IV) or potential hypernatremia. Avail- able options included metolazone 10 mg oral or chloro- thiazide 500 mg IV in instances when no enteral access was available. Indications for continuous infusion diur- esis included a lack of response to 200 mg or failure of sustained diuretic response resulting in failure to achieve goal fluid balance. removal are not clear, and clinical signs of fluid overload are delayed relative to onset of organ damage [5–7]. Standard of care diuretic treatment regimens may be in- adequate via sustained delays in initiation from shock resolution or inadequate dosing and follow-up. Addition- ally, apprehension for side effects can be seen, including serum creatinine rises and new onset acute kidney injury (AKI). However, the preponderance of adverse event data surrounding these medications is found in non-critical care populations, frequently non-translatable to patients in the ICU [8]. Previous protocols guiding volume removal in the crit- ically ill can be found in specific populations including acute decompensated heart failure, AKI, or RRT wean- ing, with protocolized approaches often improving clin- ical outcomes versus standard of care [9–11]. Further, while limited evidence is available steering diuretic de- resuscitation in the broad ICU population, protocols have relied upon dated monitoring parameters, including central venous or pulmonary artery occlusion pressures [12–14]. In this study, we aimed to evaluate the impact of a novel diuresis protocol utilizing common bedside monitoring parameters and simplified loop diuretic dos- ing on cumulative fluid balance over the first 72 h fol- lowing hemodynamic stability, as compared to standard of care. Introduction In order to ensure appropriate compliance during overnight hours with decreased staffing ratios, an order set was created requiring nursing evaluation of urine output at the designated intervals. Conditional medica- tion orders could be activated by the bedside nurse based on individual patient response and pharmacist- driven goal parameters. Diuresis hold parameters were established to minimize adverse events. The overall management of patients outside of diuresis protocol was left to physician discretion. Study outcomes h The primary outcome of this study was the net cumulative fluid balance 72 h following shock resolution. Secondary outcomes included ICU mortality, ICU length of stay, hos- pital length of stay, ventilator-free days, incidence of AKI (defined by KDIGO criteria), and the incidence of a severe metabolic disturbance including hypokalemia, hypernatre- mia, or de novo metabolic alkalosis, defined as a potassium Materials and methods Patient selection This was a pilot study to evaluate a service line level change in diuresis practice. Patients requiring mechanical ventilation with a net-positive or -even cumulative fluid or clinical signs of fluid overload determined via chest X-ray or physical exam between April 1, 2018, and April 1, 2019, received the diuresis protocol (see Additional file 1). Inclu- sion and exclusion criteria are summarized in Add- itional file 1. Patients were assessed for inclusion and exclusion daily while in the ICU. In order to approximate an experimental design using observational electronic health record (EHR) data, each patient visit within the intervention group was matched to three patient visits meeting the above inclusion and exclusion criteria from the historical time period of all Medical ICU admits be- tween January 2016 and December 2017 who received fur- osemide. Diuresis practices in the historical group were non-protocolized and left to physician discretion. Patients who met the inclusion criteria from the historical cohort who were not matched with a patient from the interven- tion group were excluded from the analysis to prevent sig- nificant heterogeneity between groups. Given the paucity of evidence surrounding diuresis in this population, investigators involved in this study per- formed an interim analysis to promote a quality improve- ment corollary to the protocol. A data monitoring committee (DMC) was formed for data analysis after 50% of chronologic study completion. The DMC consisted of the division chief, independent statistical committee (ISC), and non-committee physicians, pharmacists, and nursing. Approximately 6 months from protocol initiation, the ISC performed data extraction which was brought forward to the DMC, without statistical analysis. A protocol modi- fication occurred per the request of the DMC (see Additional file 1). This study protocol and modification were approved by the institutional review board. As this project was considered a quality improvement initiative, a waiver of informed consent was granted. Introduction Early intravenous (IV) fluid resuscitation is a necessary tool to improve hemodynamic stability and organ perfu- sion and possibly decrease mortality in critically ill pa- tients admitted to the intensive care unit (ICU) [1, 2]. However, the benefit of continued fluid administration after the first 24–48 h is unclear. Paradoxically, a positive fluid balance secondary to excess fluid accumulation has been associated with diverse and persistent detriment on a multitude of organ systems [3]. Perpetuating clinical One approach to correcting fluid balance is shifting focus onto the post- or de-resuscitation period with ap- propriate diuresis, or renal replacement therapy (RRT) in those non-responsive to diuresis, once hemodynamic sta- bility is achieved [5]. Effective diuresis may be challenged by many hindrances. An overall lack of standardization exists in identification of fluid-overloaded patients as opti- mal transition times between fluid resuscitation and fluid * Correspondence: brittany.bissell@uky.edu 1Department of Pharmacy Services, Neuro-Pulmonary Division, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA 2College of Pharmacy, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA Full list of author information is available at the end of the article * Correspondence: brittany.bissell@uky.edu 1Department of Pharmacy Services, Neuro-Pulmonary Division, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA 2College of Pharmacy, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA Full list of author information is available at the end of the article Bissell et al. Critical Care (2020) 24:70 Bissell et al. Critical Care (2020) 24:70 Page 2 of 10 Page 2 of 10 After identification of appropriate patients for inclusion a net 24 h fluid balance (ranging from −1000 mL to − 3000 mL) was established during interdisciplinary rounds which was divided into three shift goal fluid balance tar- gets to assess at 8-h intervals. Upon establishment of goal, diuretic orders were entered, with dose selection based on previous diuretic exposure and baseline renal function. Orders included conditional diuretic orders if shift fluid balance targets were not met, basic metabolic panels, goal parameters, and hold parameters for adverse events (see Additional file 1). Combination diuresis was permitted once the maximum dose of furosemide was reached (200 mg IV) or potential hypernatremia. Avail- able options included metolazone 10 mg oral or chloro- thiazide 500 mg IV in instances when no enteral access was available. Study intervention Patient identification occurred by the clinical pharmacist 7 days per week in collaboration with the medical team. Bissell et al. Critical Care (2020) 24:70 Page 3 of 10 Page 3 of 10 < 3 mmol/L, sodium > 150 mmol/L, or bicarbonate > 40 mmol/L with a pH of > 7.50, respectively. Ventilator-free days were defined as the number of days from day 1 to day 28 in which a patient was able to breathe without assistance with death as a competing risk with an assignment of zero free days. For time-dependent interventions, medication ad- ministration record medication scans were utilized for medication-related times, respiratory therapy documenta- tion was utilized for ventilator therapy, while admission, transfer, and discharge orders were collected for durations of stay. age, intervention versus standard therapy assignment, mechanical ventilation time to initiation of first dose of furosemide, net cumulative fluid balance prior to fur- osemide, and vasoactive therapy. If the intervention group was not to be identified as a significant covariate, it was predetermined that such would be manually en- tered into the final model to ascertain the point esti- mate. Collinearity was assessed with the use of variance inflation factors while goodness of fit was assessed with the Hosmer-Lemeshow test. Given the potential for pertinent changes in clinical practice that are unrelated to the protocol, an inter- rupted time series was performed. Further, given the subjective nature of the inclusion criterion clinical signs of fluid overload determined via chest X-ray or physical exam, a subgroup analysis was performed including only those included based on objective volume status (net positive cumulative fluid balance at furosemide start). A subgroup was also collected for pre- and post-protocol amendments to assure no significant impact on clinical outcome. Statistical analysis From our previous study of diluent change in the med- ical ICU, the average fluid balance in our patients at 72 h was positive 2.4 ± 5.1 l [15]. Based on these data, we calculated a sample size of 104 patients in each group to achieve a ≥2-l decrease in fluid balance at 72 h post- shock, maintaining an 80% power and an alpha of 0.05. g p p Continuous data were assessed for distribution and evaluated via t test or Mann-Whitney U, as appropriate. Chi-square or Fisher’s exact were utilized for categorical data. Data for analysis was pulled by a data analyst and validated with prospectively collected data, with discrep- ancies resolved by the analyst. The same inclusion and exclusion criteria used to enroll patients in the protocol were applied to selection of the control patients in the pre-protocol group. Mahalanobis distance matching was used to measure similarities of each patient in the con- trol and protocol group. Age, gender, insurance type, home county classification, admission source, diagnosis- related group (DRG) weight, sequential organ failure as- sessment (SOFA) score at time of diuresis initiation, baseline serum creatinine prior to first dose of furosem- ide, pre-diuretic fluid balance, time from ventilator to first diuretic administration, pre-diuretic vasopressor ad- ministration, chronic obstructive pulmonary disease (COPD) diagnosis, and acute respiratory distress syn- drome (ARDS) diagnosis were used as matching vari- ables in the distance calculation. Nearest neighbor matching was then used to select the three control visits “closest” to each protocol visit, based on the distance calculation. The utilization of DRG was chosen by data analysis experts to bolster the validity of the severity of illness scores between groups. Further, a test of inter- action was performed for patient enrollment pre- and post-protocol modification regarding the magnitude of difference on 72 h fluid balance. Results Over the study period, 832 patients met criteria for in- clusion upon screening, of which, 741 were excluded based on pre-defined exclusion criteria (Fig. 1). A total of 273 standard therapy patients who met study criteria were matched 3:1 to patients in the intervention group (n = 91), for a total of 364 study patients. The matching procedures resulted in balanced groups, based on the pre-defined variables used in the matching algorithm (Table 1). Further, no major difference in other baseline clinical criteria was found with the exception of a higher arterial pH in the intervention group, as well as a higher incidence of rhabdomyolysis on admission (see Add- itional file 1). No difference was demonstrated in the utilization of concomitant medications, other than a higher incidence of use of intravenous anti-viral medica- tions in the protocol group (Table 2). Regarding diuretic exposure, the diuresis protocol group received a higher dose of furosemide upon initiation, day 1–3, and cumu- latively; however, diuretic dosing and patient response was variable (Fig. 2). More patients in the protocol group received concomitant metolazone or acetazolamide ther- apy, while the standard therapy group had more adjunct- ive albumin use. The median (IQR) fluid balance within this study at 72-h post-shock resolution was 265 mL (−2283–3025) vs −2257 mL (−5676–920) in the historical and inter- ventional cohorts, respectively (p < 0.0001) (Table 3). There was also a significant difference in 24- and 48-h fluid balance in the intervention group when compared to the historical cohort. The test of interaction demon- strated no statistical significance regarding those A logistic regression model was defined a priori to be built for all-cause mortality. Forward selection was uti- lized with variables included in the model if p < 0.05 in the univariate analysis or if deemed biologically plausible and clinically relevant. These initial variables incorpo- rated into the model included SOFA score, DRG weight, Page 4 of 10 Bissell et al. Critical Care (2020) 24:70 Bissell et al. Critical Care Fig. 1 Selection of patients for study population Fig. 1 Selection of patients for study population Additional file 1). Given known limitations of serum cre- atinine as a marker of kidney function during acute ill- ness, a post hoc analysis was performed of RRT dependence at discharge. RRT dependence at discharge was found to be significantly higher in the standard ther- apy cohort compared to the protocol group. Results enrolled in the protocol before or after modification (see Additional file 1), and the subgroup analysis excluding those patients based on subjective clinical criteria (phys- ical exam findings, concern for pulmonary edema) showed similar findings (see Additional file 1). In the interrupted time series accounting for potential practice variation over time, no significance was demonstrated relative to time before or after intervention (see Add- itional file 1). However, a significant difference was dem- onstrated in 72-h post-shock fluid balance with protocol use (see Additional file 1). For the secondary outcomes, while patients had an additional ventilator-free day in the intervention group, this difference was not statisti- cally significant. Within the intervention cohort, there was a statistically significant increase in the rate of elec- trolyte disturbances, primarily driven by an increase in hypernatremia and hypokalemia, despite higher total po- tassium replacement in the intervention group. Regarding protocol compliance, a total of 204 patient days on protocol were available for evaluation. The most common indication for a furosemide hold was due to protocol discontinuation (see Additional file 1). A total of 27 deviations occurred within the 204 patient days, 8 for a decrease in dosing frequency prior to protocol modification, 2 for doses administered despite hold cri- teria, 2 missed nursing activations of conditional orders, and 12 inappropriate holds, 7 of which for unknown rea- sons, 1 for nursing concern regarding furosemide inter- val, and 4 for urine output. Eighteen patient days required a dose adjustment per protocol, 11 of which were driven by conditional orders. In-hospital mortality in the intervention group was lower compared to the historical group (5.5% vs 16.1%; p = 0.008). There was also a higher rate of ICU-free days, with these patients having 2 more days free of ICU care (p = 0.03). In multivariable analysis, protocolized therapy was associated with a 75% (32–91%) decreased odds of hospital mortality after adjustment for SOFA, fluid bal- ance upon furosemide initiation, time on mechanical ventilation prior to furosemide therapy, and age (see Discussion This study was the first to evaluate a volume de- resuscitation protocol utilizing pharmacologic diuresis in the medical intensive care unit. This study has several strengths, including the protocol with easily obtainable bedside monitoring parameters within the EHR, the multi-disciplinary approach to protocol development, Page 5 of 10 Bissell et al. Critical Care (2020) 24:70 Bissell et al. Critical Care (2 Table 1 Baseline characteristics Parameter Historical cohort (n = 273) Intervention cohort (n = 91) p value Matching parameter demographics Age (years)a 58 (48–68) 58 (46–70) 0.711 Male genderb 134 (49.1) 49 (53.8) 0.431 Medicare payerc 134 (49.1) 43 (47.3) 0.935 Medicaid payerc 97 (35.5) 36 (39.5) Commercial payerc 32 (11.7) 10 (10.9) Self-pay or government payerc 9 (3.3) 2 (2.2) Rural countyb 33 (12.1) 9 (9.8) 0.262 Urban areab 105 (38.5) 28 (30.8) Urban clusterb 135 (49.5) 54 (59.3) Non-matching parameter demographics Chronic kidney diseaseb 41 (15.0) 11 (12.1) 0.489 Cirrhosisb 40 (14.7) 8 (8.8) 0.152 Matching critical illness parameters and comorbidities Cumulative fluid balance at furosemide start (mL)a 2243 (0–5381) 1411 (−124–4438) 0.161 Vasopressor utilization prior to furosemideb,d 119 (43.6) 49 (53.8) 0.89 Time MV prior to furosemide (hours)a 45.5 (22–83) 52 (30.5–104) 0.155 Diagnostic-related group weighta 5.1 (2.3–5.9) 5.6 (2.4–6.3) 0.167 Prior SCr to furosemide (mg/dL)a 0.96 (0.74–1.29) 0.95 (0.75–1.44) 0.598 Sequential Organ Failure Assessment score a 6 (4–8) 6 (4–8) 0.875 Chronic obstructive pulmonary diseaseb 64 (23.4) 25 (27.5) 0.439 Acute respiratory distress syndromec 16 (5.9) 3 (3.3) 0.425 From emergency department (ED)c 65 (23.8) 14 (15.4) 0.301 From outside hospitalc 96 (35.2) 39 (42.9) From outside hospital via EDc 62 (22.7) 25 (27.5) From other intensive care unitc 5 (1.8) 2 (2.2) From floorb 45 (16.5) 11 (12.1) MV mechanical ventilation aWilcoxon rank sum, median (interquartile range) bChi-square test; number (percentage) cFisher’s exact, number (percentage) dVasopressors including norepinephrine, epinephrine, or vasopressin Historical cohort (n = 273) Intervention cohort (n = 91) p value utilization, and modification, frequency of monitoring, and selection of matching parameters. Several potential confounders on 72-h fluid balance were matched be- tween groups, systematically decreasing between-group difference. Further, results of the interrupted time series showed no significant difference in slopes of fluid bal- ance over time, while the association between improved 72-h post-shock fluid balance and intervention group remained significant (Fig. 3). utilization, and modification, frequency of monitoring, and selection of matching parameters. Discussion Several potential confounders on 72-h fluid balance were matched be- tween groups, systematically decreasing between-group difference. Further, results of the interrupted time series showed no significant difference in slopes of fluid bal- ance over time, while the association between improved 72-h post-shock fluid balance and intervention group remained significant (Fig. 3). our protocol prioritized intermittent dosing to decrease intravenous access concerns and protocolized electrolyte and safety monitoring [14]. With such, a significant in- crease in the rate of hypernatremia and hypokalemia was seen within the intervention group. No statistically signifi- cant difference in duration of mechanical ventilation wean was found. This does not correlate with previous evidence within the critically ill population, demonstrating in- creased ventilator-free days with conservative volume management [14]. Comparatively, while our study utilized more specific titration strategies and common bedside monitoring parameters, this was a single-center, non- randomized study and likely underpowered to detect a dif- ference in ventilator duration. We demonstrated a significant decrease in 72 h cumula- tive fluid volume with the addition of a diuresis protocol in the critically ill. This correlates with previous protocols within acute respiratory distress syndrome and heart fail- ure which demonstrated improved volume status with strategized diuresis without an increase in kidney failure [11, 14]. Unlike studies within the heart failure population, Key considerations to this study include a decrease in mortality and increased ICU-free days in the Bissell et al. Discussion Critical Care (2020) 24:70 Page 6 of 10 Table 2 Pharmacotherapy Parameter Historical cohort (n = 273) Intervention cohort (n = 91) p value Furosemide dosing Starting dose (mg) a 40 (20–40) 40 (40–40) 0.003 Day one total daily dose (mg) a 40 (40–60) 80 (40–120) < 0.0001 Day two total daily dose (mg)a 0 (0–40) 80 (20–120) < 0.0001 Day three total daily dose (mg) a 0 (0–20) 0 (0–80) 0.0007 Total cumulative dose (mg)a 80 (40–200) 240 (120–420) < 0.0001 Conversion to continuous infusion b 32 (11.7) 8 (8.8) 0.562 First to last dose furosemide (days) a 4.9 (1.4–12.4) 4.8 (3.1–9.8) 0.165 Diuresis adjuncts Metolazoneb 15 (5.5) 30 (32.9) < 0.0001 Chlorothiazidec 48 (17.6) 6 (6.6) 0.402 Acetazolamideb 14 (5.1) 14 (15.4) 0.001 Albuminc 29 (10.6) 2 (2.2) 0.009 Day one potassium supplementationa 40 (40–60) 60 (40–80) 0.007 Day two potassium supplementationa 40 (40–60) 60 (40–100) 0.002 Day three potassium supplementationa 50 (40–80) 70 (60–100) 0.002 Other medication exposure Total nephrotoxin exposurea 1 (1–2) 1 (1–2) 0.288 Aminoglycosideb 27 (9.9) 8 (8.8) 0.758 Beta-lactamb 227 (83.2) 75 (92.4) 0.872 Intravenous antiviralb 11 (4.0) 12 (13.2) 0.002 ACE inhibitor and/or ARBb 49 (17.9) 13 (14.3) 0.421 Amphotericin Bc 5 (1.8) 3 (3.3) 0.418 Intravenous sulfamethoxazole-trimethoprimc 19 (6.9) 4 (7.7) 0.465 Intravenous vancomycinb 153 (56.0) 51 (56.0) 1.000 Combination vancomycin and piperacillin-tazobactamb 88 (32.2) 30 (32.9) 0.897 ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker aWilcoxon rank sum, median (interquartile range) bChi-square test; number (percentage) cFisher’s exact, number (percentage) Regarding ventilator days, ventilation wean procedures are not standardized at this institution. Daily spontan- eous breathing trials are performed in all patients who meet criteria; however, extubation orders are left to pro- vider discretion. This lack of ventilator wean protocoli- zation may have affected ventilator-free days between groups. However, reintubation rates were in alignment with previous studies with ranges 13.8–22.6% and were not significantly different between groups which sup- ports relative uniformity on wean strategies [21]. intervention group. Known correlates of mortality within the sepsis population, including baseline weight and ad- mission source, were included as parameters within the regression model [16–18]. The variables previously cor- related with mortality were accounted for in the match- ing criteria of this cohort. Studies demonstrate that almost ubiquitous organ dysfunction has been associated with positive volume status in the ICU. Discussion It is possible that the implication of volume de-resuscitation seen in the current study could be casually linked with mortality, in line with a vast number of previous studies demonstrat- ing the impact of fluid status on survival rates aside of its effect on ventilator days; however, this study can only show correlation given the nature of its design. Particu- larly, patients in the intervention group also had a de- crease in RRT dependence at discharge. RRT receipt prior to hospital discharge has been associated with pro- gression to end stage renal disease, cardiovascular dis- ease, and increased mortality [19, 20]. Further of note, changes to the institutional nursing- driven electrolyte replacement protocol occurred mid- implementation (see Additional file 1). The protocol modification sought more aggressive potassium replace- ment; however, nursing adherence was not evaluated. As follow-up potassium evaluations were mandated with protocol implementation, it is possible that incidences of hypokalemia were increased secondary to more frequent monitoring relative to the historical cohort; however, Bissell et al. Critical Care (2020) 24:70 Page 7 of 10 Bissell et al. Critical Care Fig. 2 Furosemide dose and 72-h cumulative fluid balance per group Fig. Fig. 2 Furosemide dose and 72-h cumulative fluid balance per group A key limitation to this study is the lack of randomization and blinding. Given the nature of the protocol, blinding to the medical staff was not possible. A pre- and post-intervention study was chosen given the lack of blinding. It was anticipated that an overall change in practice may occur over the study timeframe given in- creased awareness of the detrimental effects of fluid overload and approach to diuretic dosing in critically ill patients, a phenomenon recently found in management of septic shock [22, 23]. However, given the limited time lapse between the historical group and protocol imple- mentation and lack of emergence of guidelines regarding volume de-resuscitation, changes in overall approaches to care based on external factors were unlikely. To limit potential bias further, patients were matched on a large number of relevant variables and objective outcome measures were utilized, with the exception of the DRG weight. However, the authors opted for inclusion of this variable versus International Classification of Disease coding given its consideration for up to eight diagnoses, including the primary diagnosis, and up to six proce- dures performed during the stay, likely increasing its frequency of serum potassium collections were not re- corded. Discussion In regard to rates of hypernatremia, providers were permitted to request continuation of furosemide despite elevated sodium levels, likely resulting in the subsequent increased rate of metolazone use in the intervention group. There was a significant difference in cumulative fluid balance that was likely due to higher furosemide exposure in the intervention group, as dem- onstrated in previous protocols of furosemide in acute kidney injury [10]. The significant increase in episodes of hypernatremia and hypokalemia are predictable and re- versible within this strategy. If replicated in future ran- domized trials, improvements in ICU length of stay and mortality may take precedence over concern for electro- lyte abnormalities. Future protocol designs should account for these episodes of hypernatremia and hypokalemia with creation of more explicit electrolyte replacement rules. Further, electrolyte derangements may be of greater con- sideration in an alternative ICU population, including car- diothoracic/cardiology critical care. Patient-specific factors should be taken into consideration with implementation of this protocol. frequency of serum potassium collections were not re- corded. In regard to rates of hypernatremia, providers were permitted to request continuation of furosemide despite elevated sodium levels, likely resulting in the subsequent increased rate of metolazone use in the intervention group. There was a significant difference in cumulative fluid balance that was likely due to higher furosemide exposure in the intervention group, as dem- onstrated in previous protocols of furosemide in acute kidney injury [10]. The significant increase in episodes of hypernatremia and hypokalemia are predictable and re- versible within this strategy. If replicated in future ran- domized trials, improvements in ICU length of stay and mortality may take precedence over concern for electro- lyte abnormalities. Future protocol designs should account for these episodes of hypernatremia and hypokalemia with creation of more explicit electrolyte replacement rules. Further, electrolyte derangements may be of greater con- sideration in an alternative ICU population, including car- diothoracic/cardiology critical care. Patient-specific factors should be taken into consideration with implementation of this protocol. Bissell et al. Discussion Critical Care (2020) 24:70 Page 8 of 10 Table 3 Clinical outcomes Parameter Historical cohort (n = 273) Intervention cohort (n = 91) p value Clinical outcomes 72 h fluid balance (mL)d 265 (−2283–3025) −2257 (−5676–920) < 0.0001 48-h fluid balance (mL) d 309 (−1267–2434) −1799(−3884–1092) < 0.0001 24-h fluid balance (mL)a 101 (−963–1622) −692 (−1833–697) 0.0002 Ventilator-free days (days) a 19 (10–22) 20 (15–23) 0.098 Overall adverse eventb,e 74 (27.1) 37 (40.6) 0.015 Ventilator days (days) a 8 (5–13) 5 (5–12) 0.441 Furosemide to extubation (hours) a 70 (24–147) 58 (23–122) 0.282 Re-intubation rateb 57 (20.8) 17 (18.6) 0.652 ICU-free days (days) a 17 (7–21) 19 (13–22) 0.030 ICU days (days) a 8.6 (6.2–13.5) 8.1 (5.9–12.8) 0.513 In-hospital mortalityc 44 (16.1) 5 (5.5) 0.008 Safety outcomes Bolus administration after furosemidec 4 (1.5) 0 (0) 0.576 Vasopressor administration after furosemideb 65 (23.8) 19 (20.9) 0.566 Tachyarrhythmiab 50 (18.3) 15 (16.4) 0.693 In-hospital mortalityc 44 (16.1) 5 (5.5) 0.008 RRT receipt in ICUc 17 (6.2) 0 (0) < 0.0001 RRT dependence at dischargec 14 (5.1) 0 (0) 0.025 Acute kidney injuryf 62 (22.7) 22 (24.2) 0.775 Hypokalemiac 0 3 (3.3) 0.015 Hypernatremiab 19 (6.9) 19 (20.9) 0.001 Metabolic alkalosisc 3 (1.1) 1 (1.1) 1.000 aWilcoxon rank sum, median (interquartile range) bChi-square test; number (percentage) cFisher’s exact, number (percentage) dStudent’s t test, average (standard deviation) eOverall adverse event; serum creatinine rise, hypokalemia, hypernatremia, or metabolic alkalosis fAcute kidney injury; serum creatinine 1.5 times baseline serum creatinine, serum creatinine increase of at least 0.3 mg/dL Table 3 Clinical outcomes dStudent’s t test, average (standard deviation) eOverall adverse event; serum creatinine rise, hypokalemia, hypernatremia, or metabolic alkalosis fAcute kidney injury; serum creatinine 1.5 times baseline serum creatinine, serum creatinine increase of at least 0.3 mg/dL our patients were excluded for active vasoactive therapy or AKI. Clinical inertia is a consideration, particularly given this protocol’s pilot nature. Further, consideration must be made for a lag in adaptation, particularly in times of low staffing. objectiveness versus retrospective chart review. Regard- less, it is still possible for potential residual confounders on illness severity to have been missed. Given that vol- ume overload and positive fluid balance may be markers of severity of illness rather than a parameter for early di- uresis intervention, the differences in mortality and length of stay must be replicated in a larger, randomized controlled trial for confirmation. Discussion Worth nothing, true blinding in a randomized controlled trial would likely be unfeasible by nature of the protocol design and a parallel design could subject the trial to potential for a signifi- cant Hawthorne effect. g Lastly, the selection of outcome parameters is worth mentioning. We evaluated 72-h net cumulative fluid balance in accordance with previous literature; how- ever, evidence suggests that fluid balance documenta- tion is not always accurate. The utilization of EHR flowsheets decreases potential for error in ICU docu- mentation. The frequency in documentation required via the protocol aligns with standard of care within the ICU. Recent studies have challenged the validity of net cumulative fluid balance in the ICU and its re- lationship to body weight or clinical signs of fluid overload [26, 27]. Because this practice is not tightly protocolized, we did not utilize body weight as a monitoring parameter. However, it is possible that Protocol modifications in the study may also be seen as a potential limiting factor. However, in the subgroup analysis performed, protocol inclusion did not appear to significantly impact the primary result. Additionally, the inclusion rate appeared relatively low at 11%. Recent studies have demonstrated small recruitment rates within the critically ill [24, 25]. A significant portion of Bissell et al. Critical Care (2020) 24:70 Page 9 of 10 Fig. 3 Interrupted time series analysis of 72-h post-shock fluid balance over time. Pre-intervention slope R2 = 0.0092, p = 0.099; post-intervention slope R2 = 0.018, p = 0.185 daily weight monitoring would assist in clinical deci- sion making and outcome measures. controlled trials are needed to confirm or refute the potential benefits of de-resuscitation, through protocol-driven diuresis, on important patient cen- tered outcomes, such as ICU length of stay, ventilator-free days, and in-hospital mortality, as sug- gested by observed associations in the present study. This study demonstrated that a pharmacist-driven di- uresis protocol of volume de-resuscitation was signifi- cantly associated with a lower cumulative fluid balance at 72 h post-shock. The addition of the diuresis protocol was likely effective for a multitude of reasons, including the overall increased awareness of avoidance of volume overload and tailored diuresis utilization, the standardization of doses and follow-up monitoring, as well as an increase in furosemide dosing as demon- strated in this study. However, with increased dosing of furosemide, increased rates of adverse events were found, namely hypernatremia and hypokalemia. Funding Financial support for this study was provided by the American Society of Health-Systems Pharmacists Foundation New Investigator Grant. Authors’ contributions BB and ML designed the protocol. AF, BB, ML, and MT assisted in the patient enrollment and data collection. AF, BB, ML, MT, and JP collected the data. AK and JR assisted in the statistical analysis and retrospective data collection. JN and PM assisted with the protocol implementation and paper design. All authors read and approved the final manuscript. Acknowledgements We would like to thank the physician and nursing staff and leadership who played an integral part in the implementation and improvement of the project. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13054-020-2795-9. y Supplementary informatio 1186/s13054-020-2795-9. Additional file 1. Supplementary Digital Content This file includes relevant study protocols, definitions, as well as subgroup analyses and additional informational tables beyond manuscript content. Conclusion Using a diuresis protocol for volume de-resuscitation, we demonstrated a significant decrease in net cumu- lative fluid balance at 72 h following shock resolution, with potential benefit on clinical outcomes including renal recovery, mortality, and ICU length of stay. Al- though this study supports the implementation of a diuresis protocol in the ICU, larger randomized Discussion Risk ver- sus benefit of active volume de-resuscitation and electro- lyte fluctuations must be considered. The increased mortality and decreased number of ICU-free days in the standard therapy group are hypothesis-generating, par- ticularly given the lack of difference between-groups in ventilator-free days. Supplementary information References 1. Sakr Y, Rubatto Birri PN, Kotfis K, et al. Higher fluid balance increases the risk of death from sepsis: results from a large international audit. Crit Care Med. 2017;45(3):386–94. 1. Sakr Y, Rubatto Birri PN, Kotfis K, et al. Higher fluid balance increases the risk of death from sepsis: results from a large international audit. Crit Care Med. 2017;45(3):386–94. 24. Walters SJ, Bonacho Dos Anjos Henriques-Cadby I, Bortolami O, et al. Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ open. 2017;7(3):e015276. 2. Tigabu BM, Davari M, Kebriaeezadeh A, Mojtahedzadeh M. Fluid volume, fluid balance and patient outcome in severe sepsis and septic shock: a systematic review. J Crit Care. 2018;48:153–9. 25. Keh D, Trips E, Marx G, et al. Effect of hydrocortisone on development of shock among patients with severe sepsis: the HYPRESS randomized clinical trial. JAMA. 2016;316(17):1775–85. 3. Malbrain ML, Marik PE, Witters I, et al. Fluid overload, de-resuscitation, and outcomes in critically ill or injured patients: a systematic review with suggestions for clinical practice. Anaesthesiol Intensive Ther. 2014;46(5): 361–80. 26. Perren A, Markmann M, Merlani G, Marone C, Merlani P. Fluid balance in critically ill patients. Should we really rely on it? Minerva Anestesiol. 2011; 77(8):802–11. 4. 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Author details 1 1Department of Pharmacy Services, Neuro-Pulmonary Division, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA. 2College of Pharmacy, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA. 3College of Medicine, Department of Internal Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, University of Kentucky, 740 S. Limestone, Lexington, KY 40536, USA. 4Performance Analytics Center of Excellence, University of Kentucky, 800 Rose Street, H110, Lexington, KY 40536, USA. 5College of Medicine, Department of Internal Medicine, Bone and Mineral Metabolism, University of Kentucky, 800 Rose Street, MN668, Lexington, KY 40536, USA. 6Department of Pharmacy Services, Beaumont Hospital, 3601 W 13 Mile Road, Royal Oak, MI 48073, USA. 19. Brahmbhatt SA, Armanyous S, Lioudis M, Heyka RJ, Wong LP, Demirjian S. High Incidence of transition to ESRD in patients discharged with dialysis dependent AKI: The Cleveland Clinic experience. Presented in poster format at Kidney Week 2017 in New Orleans (Oct. 31-Nov. 5). Abstract FR-PO129. 20. Sueyoshi K, Watanabe Y, Inoue T, Ohno Y, Nakajima H, Okada H. Predictors of long-term prognosis in acute kidney injury survivors who require continuous renal replacement therapy after cardiovascular surgery. PLoS One. 2019;14(1):e0211429. 21. Esteban A, Frutos F, Tobin MJ, et al. A comparison of four methods of weaning patients from mechanical ventilation. Spanish Lung Failure Collaborative Group. N Engl J Med. 1995;332(6):345–50. 22. Rivers E, Nguyen B, Havstad S, et al. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001;345(19): 1368–77. Received: 12 November 2019 Accepted: 17 February 2020 Received: 12 November 2019 Accepted: 17 February 2020 23. Rowan KM, Angus DC, Bailey M, et al. Early, goal-directed therapy for septic shock - a patient-level meta-analysis. N Engl J Med. 2017;376(23):2223–34. Competing interests The authors declare that they have no competing interests. 18. de Grooth HJ, Postema J, Loer SA, Parienti JJ, Oudemans-van Straaten HM, Girbes AR. Unexplained mortality differences between septic shock trials: a systematic analysis of population characteristics and control-group mortality rates. Intensive Care Med. 2018;44(3):311–22. The authors declare that they have no competing interests. Availability of data and materials The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. Page 10 of 10 Page 10 of 10 Page 10 of 10 Bissell et al. Critical Care (2020) 24:70 Bissell et al. Critical Care Ethics approval and consent to participate prospective, open-label, sequential period pilot study. Crit Care Med. 2018; 46(8):1217–23. prospective, open-label, sequential period pilot study. Crit Care Med. 2018; 46(8):1217–23. This work was performed at the University of Kentucky HealthCare. Institutional review board approval was received (MEDXP Protocol 42820). 16. Motzkus CA, Chrysanthopoulou SA, Luckmann R, Rincon TA, Lapane KL, Lilly CM. ICU admission source as a predictor of mortality for patients with sepsis. J Intensive Care Med. 2018;33(9):510–6. sepsis. J Intensive Care Med. 2018;33(9):510–6. Consent for publication Not applicable. Consent for publication Not applicable. 17. Pepper DJ, Demirkale CY, Sun J, et al. Does obesity protect against death in sepsis? A retrospective cohort study of 55,038 adult patients. Crit Care Med. 2019;47(5):643–50. Publisher’s Note 6. Jacobs R, Jonckheer J, Malbrain M. Fluid overload FADEs away! Time for fluid stewardship. J Crit Care. 2018;48:458–61. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 7. Rivers EP. Fluid-management strategies in acute lung injury--liberal, conser ati e or both? N Engl J Med 2006 354(24) 2598 600 7. Rivers EP. Fluid management strategies in acute lung injury liberal, conservative, or both? N Engl J Med. 2006;354(24):2598–600. conservative, or both? N Engl J Med. 2006;354(24):2598–600. 8. Brisco MA, Zile MR, Hanberg JS, et al. Relevance of changes in serum creatinine during a heart failure trial of decongestive strategies: insights from the DOSE trial. J Card Fail. 2016;22(10):753–60. from the DOSE trial. J Card Fail. 2016;22(10):753–60. 9. Raurich JM, Llompart-Pou JA, Novo MA, Talavera C, Ferreruela M, Ayestarán I. Successful weaning from continuous renal replacement therapy. Associated risk factors. J Crit Care. 2018;45:144–8. 10. Bagshaw SM, Gibney RTN, Kruger P, Hassan I, McAlister FA, Bellomo R. The effect of low-dose furosemide in critically ill patients with early acute kidney injury: a pilot randomized blinded controlled trial (the SPARK study). J Crit Care. 2017;42:138–46. 11. Barsuk JH, Gordon RA, Cohen ER, et al. A diuretic protocol increases volume removal and reduces readmissions among hospitalized patients with acute decompensated heart failure. Congest Heart Fail. 2013;19(2):53–60. 12. Schuller D, Lynch JP, Fine D. Protocol-guided diuretic management: comparison of furosemide by continuous infusion and intermittent bolus. Crit Care Med. 1997;25(12):1969–75. 13. Ostermann M, Alvarez G, Sharpe MD, Martin CM. Frusemide administration in critically ill patients by continuous compared to bolus therapy. Nephron. 2007;107(2):c70–6. 14. Wiedemann HP, Wheeler AP, Bernard GR, et al. Comparison of two fluid- management strategies in acute lung injury. N Engl J Med. 2006;354(24): 2564–75. 15. Magee CA, Bastin MLT, Laine ME, et al. Insidious harm of medication diluents as a contributor to cumulative volume and hyperchloremia: a
https://openalex.org/W2883423100	http://www.scielo.br/pdf/rmat/v23n2/1517-7076-rmat-23-02-e12032.pdf	English	null	Influence of defects on the irreversible phase transition in the Fe-Pd doped with Co and Mn	Matéria	2,018	cc-by	5,287	ISSN 1517-7076 artigo e-12032, 2018 ISSN 1517-7076 artigo e-12032, 2018 ABSTRACT The appearance of BCT martensite in Fe-Pd-based ferromagnetic shape memory alloys, which develops at lower temperatures than the thermoelastic martensitic transition, deteriorates the shape memory properties. In a previous work performed in Fe70Pd30, it was shown that a reduction in defects density reduces the non thermoelastic FCT-BCT transformation temperature. In the present work, the influence of quenched-in- defects upon the intensity and temperature of the thermoelastic martensitic (FCC-FCT) and the non thermoelastic (FCT-BCT) transitions in Fe-Pd doped with Co and Mn is studied. Differential scanning calorimetric and mechanical spectroscopy studies demonstrate that a reduction in the dislocation density the stability range of the FCC-FCT reversible transformation in Fe67Pd30Co3 and Fe66.8Pd30.7Mn2.5 ferromagnetic shape memory alloys. Keywords: Fe-Pd-Co; Fe-Pd-Mn; ferromagnetic shape memory alloys; martensitic transformation; dislocation dynamics. Influence of defects on the irreversible phase transition in the Fe-Pd doped with Co and Mn Federico Guillermo Bonifacich1, Osvaldo Agustín Lambri1, Damián Gargicevich1, Griselda Irene Zelada1, José Ignacio Pérez-Landazábal2,3, Vicente Recarte2,3, Vicente Sánchez-Alarcos2,3 Federico Guillermo Bonifacich1, Osvaldo Agustín Lambri1, Damián Gargicevich1, Griselda Irene Zelada1, José Ignacio Pérez-Landazábal2,3, Vicente Recarte2,3, Vicente Sánchez-Alarcos2,3 1 CONICET-UNR-Laboratorio de Materiales, Escuela de Ingeniería Eléctrica, Centro de Tecnología e Investigación Eléctrica, Facultad de Ciencias Exactas, Ingeniería y Agrimensura, Avda. Pellegrini 250, CP: 2000, Rosario, Santa Fe, A ti g e-mail: bonifaci@fceia.unr.edu.ar; olambri@fceia.unr.edu.ar; gargi@fceia.unr.edu.ar; gizelada@fceia.unr.edu.ar 2 Departamento de Física, Universidad Pública de Navarra, Campus de Arrosadía 31006, Pamplona, Navarra, Spain. 3 Institute for Advanced Materials (INAMAT), Universidad Pública de Navarra, Campus de Arrosadía 31006, Pamplona, Navarra, Spain. p p p e-mail: ipzlanda@unavarra.es; recarte@unavarra.es; vicente.sanchez@unavarra.es Corresponding Author : Federico Guillermo Bonifacich 1. INTRODUCTION In this sense, mechanical spectroscopy which is a powerful and sensitive technique to analyze the dynamics of structural defects like dislocations was used [18,19]. of these phase transformations and in their stabilities [15-17]. Indeed, in a recent work the influence of defects on the FCT-BCT transition temperature has been studied. The large misfit between the cell parameters of FCT and BCT martensites point out to a critical role of dislocations in the accommodation of these phases. In this sense, mechanical spectroscopy which is a powerful and sensitive technique to analyze the dynamics of structural defects like dislocations was used [18,19]. The aim of the present work is to study the role of quenched-in-defect on the FCC-FCT and FCT-BCT transformations temperatures in Fe-Pd ferromagnetic shape memory alloys doped with Co and Mn by means of mechanical spectroscopy and differential calorimetry studies. 2. MATERIALS AND METHODS Polycrystalline ingots of nominal composition (at.%) Fe67Pd30Co3 and Fe66.8Pd30.7Mn2.5 were prepared from high purity elements by arc melting under protective Ar atmosphere (called hereafter FePdCo and FePdMn). Ingots were homogenized in vacuum at 1273 K during 24 hours, and subsequently they were subjected to a 30 minutes annealing treatment at 1173 K in a vertical furnace; followed by quenching into iced water under vacuum. The compositions of the alloys were analyzed by energy dispersive X-ray spectroscopy (EDS) in a Jeol JSM-5610LV scanning electron microscope (SEM). Differential scanning calorimetry (DSC) measurements were carried out at a heating/cooling rate of 10K/min in a TA Q100 calorimeter under nitrogen protective atmosphere. Different thermal cycles, involving heating and cooling runs, with different both minimum and maximum temperatures, within the interval around 150 K and 723 K, were performed in order to induce or prevent the non-thermoelastic MT. For Fe67Pd30Co3 were performed eight thermal cycles and for Fe66.8Pd30.7Mn2.5 were performed nine thermal cycles. During the mechanical spectroscopy (MS) studies the simultaneous measurement of damping, Q-1 (or internal friction) and natural frequency (f, f2 being proportional to the shear elastic modulus) as a function of temperature were performed [18,20]. MS measurements were performed in a mechanical spectrometer based on an inverted torsion pendulum, assembled in the laboratory, under Ar at atmospheric pressure. The schematic representation of the experimental device is presented in Figure 1 taken from Ref. [21]. The maximum strain on the sample surface was 5x10-5. The measurement frequency was around 1 Hz. The heating and cooling rates employed in the tests were 1K/minute. Damping was calculated from the slope of the natural logarithm of the decaying amplitudes versus period number through equation (1), such that [18]     1 0 ln ln    Q n A An  (1) where An is the area of the nth decaying oscillation, A0 is the initial area of the starting decaying oscillation and n is the period number. For all these measurements the same initial and final values of the decaying amplitudes were used to avoid distortions linked to the appearance of amplitude dependent damping effects [20]. In order to study the presence of non-linear effects in the microstructure, amplitude dependent damping (ADD), i.e. 1. INTRODUCTION Ferromagnetic shape memory alloys (FSMA) have attracted much scientific and technological interest owing to a broad range of possible engineering applications. These functional materials have the unique properties they show as a result of the combination of a thermoelastic martensitic transformation (MT) and a magnetic transition. In fact, one of the most interesting properties is the high magnetoplasticity they show as a consequence of the magnetic field induced re-orientation of the magnetic martensitic variants. In addition, fundamental physics studies related to the coupling between structural, mechanical, magnetic and thermodynamic properties are field of intense work [1,2]. Concerning the Fe-Pd system, the face centered tetragonal (FCT) martensitic phase allows the magnetic shape memory effect by reorientation of martensite variants via twin boundary motion. The FCT martensite can be obtained when cooling face centered cubic (FCC) austentite through the MT in samples which were quenched in iced water after an annealing at 1173 K [3-5]. This structural transformation from FCC to FCT only takes place in a very narrow compositional range (29 < at% Pd < 32) and the transformation temperatures lie typically below room temperature (RT) [6,7]. At lower temperature an irreversible transformation (FCT-BCT) needs to be prevented in order to retain the shape memory properties [8-12]. These temperatures strongly depend on both the composition and the addition of a third element. In fact, in previous works, the partial substitution of Fe by Co and Mn on the structural and magnetic properties of Fe-Pd alloys has been investigated and MT temperatures dependence has been observed [13,14]. Moreover, it was a reported that with some specific concentration of Mn it is possible to prevent the FCT-BCT non-thermoelastic MT [14]. Nevertheless, there exist other different contributions like internal stresses, point defects, dislocations and other bi-, tri-dimensional microstructural defects that could play an important role in the characteristics Received on: 01/08/2017 Accepted on: 13/12/2017 Received on: 01/08/2017 Accepted on: 13/12/2017 10.1590/S1517-707620180002.0368 BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. of these phase transformations and in their stabilities [15-17]. Indeed, in a recent work the influence of defects on the FCT-BCT transition temperature has been studied. The large misfit between the cell parameters of FCT and BCT martensites point out to a critical role of dislocations in the accommodation of these phases. 3. RESULTS AND DISCUSSION The thermoelastic martensitic transformation (MT) has been studied firstly by DSC measurement. Figure 2 shows the DSC thermograms for FePdCo (a) and FePdMn (b) alloys, in the as-quenched state, from a temperature higher than the non-thermoelastic MT up to different maximum temperatures. In fact, the minimum temperature during the DSC measurements was 220 K and 273 K in order to prevent the non- thermoelastic MT in for FePdCo and FePdMn alloys, respectively. The exothermic (endothermic) peak observed on cooling (heating) corresponds to a direct (reverse) thermoelastic MT. Thermoelastic MT occurs at 260 K and 310 K for FePdCo and FePdMn, respectively. The MT temperature was obtained as the reaction peak temperature, i.e. MT temperature peak. The measured enthalpies are 0.8 J/g and 0.7 J/g for FePdCo and FePdMn, respectively [14,23,24]. The Curie temperatures can be observed thorough the change in the baseline at TC=603 K for FePdCo and TC=540 K for FePdMn [13,24]. After several thermal cycles up to different maximum temperatures, appreciable changes in the height and temperature of the MT from DSC thermograms were not detected [25]. A change in less than 3 K for the MT temperature was detected in the two alloys. On further cooling a non-thermoelastic (FCT-BCT) transformation takes place. In fact, thermograms shown in Figure 3 were performed at temperatures lower than the non-thermoelastic MT temperature. These thermograms show a series of peaks which correspond to the FCT-BCT transformation in FePdCo alloy. The approximate temperatures for the appearance of the non-thermoeleastic MT were 190 K and 240 K for FePdCo and FePdMn, respectively [13,14,25,26]. The FCT-BCT transformation is non-thermoelastic and the appearing BCT phase will remain (degrading the shape memory properties) unless the alloy is annealed at high temperature and subsequently quenched [6]. At temperatures lower than the non-thermoelastic transition both the reversible and the irreversible phases coexist. In this way, it is expected that the different cell parameters of the FCT phase and the BCT phase could be generate internal stresses in the alloy. In fact, it has been reported that in (at.%) Fe70Pd30 the large misfit between both phases, revealed by means neutron thermodiffraction measurements, must be adjusted by the creation of dislocations or twins boundaries to relief the stresses generated in the interphase area. Then the larger the quantity of dislocations in the alloy, the easier should be the irreversible transition to the BCT phase. 2. MATERIALS AND METHODS damping as a function of the maximum strain on the sample, ε0, was calculated from equation (2) [20,22]:      dn A d n ln 1 0     (2)      dn A d Q n ln 1 0 1      (2)      dn A d Q n ln 1 0 1      (2) Polynomials were fitted to the curve of the decaying areas of the torsional vibrations as a function of the period number by means of Chi-square fitting. Polynomials of degree higher than 1 indicate that Q-1 is a function of ε0, leading to the appearance of ADD effects, as it can be inferred easily. Subsequently the equation (2) was applied. This procedure allows to obtain damping as a function of the maximum strain (ε0) from free decaying oscillations [20,22]. The strength of the ADD, can be determined through the average slope of the Q-1(ε0) curve using the S coefficient in equation (3) [20,22]: 0 1      Q S (3) 0 1      Q S (3) where ΔQ-1 is the damping change corresponding to the full amplitude changes Δε0 measured in the whole oscillating strain range. where ΔQ-1 is the damping change corresponding to the full amplitude changes Δε0 measured in the whole oscillating strain range. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. Figure 1: Schematic representation of the experimental device used during mechanical spectroscopy measurement. S: sample. G: jaws B: excitation coils. E: masses for variable moment of inertia. Taken from Ref. [21]. Figure 1: Schematic representation of the experimental device used during mechanical spectroscopy measurement. S: sample. G: jaws B: excitation coils. E: masses for variable moment of inertia. Taken from Ref. [21]. 3. RESULTS AND DISCUSSION So it could be expected that a low density of dislocations must produce a reduction in the transition temperature [25]. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 200 300 400 500 600 700 HF (W/g) Temperature (K) 0.07 0.072 0.074 0.076 230 240 250 260 270 280 290 HF (W/g) Temperature (K) Reverse MT Tc (a) exo up -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 300 400 500 600 700 Temperature (K) HF (W/g) 0.082 0.086 0.09 0.094 0.098 280 290 300 310 320 330 Temperature (K) HF (W/g) Tc Reverse MT (b) exo up Figure 2: Heat flux (HF) as a function of temperature during the DSC measurement for (a) FePdCo and (b) FePdMn from a temperature higher than the non-thermoelastic MT temperature up to different maximum temperatures. Arrows indicate the MT and Tc temperatures. The inset shows a magnification of the MT temperature range. -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 200 300 400 500 600 700 HF (W/g) Temperature (K) 0.07 0.072 0.074 0.076 230 240 250 260 270 280 290 HF (W/g) Temperature (K) Reverse MT Tc (a) exo up -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 300 400 500 600 700 Temperature (K) HF (W/g) 0.082 0.086 0.09 0.094 0.098 280 290 300 310 320 330 Temperature (K) HF (W/g) Tc Reverse MT (b) exo up Figure 2: Heat flux (HF) as a function of temperature during the DSC measurement for (a) FePdCo and (b) FePdMn from a temperature higher than the non-thermoelastic MT temperature up to different maximum temperatures. Arrows indicate the MT and Tc temperatures. The inset shows a magnification of the MT temperature range. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. 0 0.004 0.008 0.012 0.016 0.02 0.024 0.028 160 200 240 280 320 HF (W/g) Temperature (K) FCC-FCT FCT-BCT exo up FePdCo FCC-FCT FCT-BCT FePdMn Figure 3: DSC thermograms performed down to temperatures lower than the non-thermoelastic transformation tempera- ture for FePdCo and FePdMn. Figure 3: DSC thermograms performed down to temperatures lower than the non-thermoelastic transformation tempera- ture for FePdCo and FePdMn. In order to study the influence of the non-thermoelastic MT on thermoelastic MT, mechanical spectroscopy measurement from temperatures below the FCT-BCT transformation temperature were carried out. 3. RESULTS AND DISCUSSION Figures 4 and 5 show the damping spectra and squared frequency (proportional to shear elastic modulus) curves, from 170 K up to different maximum temperatures, for FePdCo and FePdMn alloys, respectively. The maximum temperature reached in each consecutive thermal cycle was increased as follows: 603K, 603K, 673K, 773K and 773K. In Figures 4(a) and 5(a) a damping peak at around 450 K can be observed during the successive thermal cycles. During the second heating run up to the same temperature of the first, this peak decreases its height markedly in both alloys. In fact, this peak has been reported as P1 peak in Fe70Pd30 with and without BCT phase, and in Fe67Pd30Co3 and Fe66.8Pd30.7Mn2.5 without the appearance of BCT phase [24- 26]. The mechanism of the P1 peak was related with a dislocation dragging mechanisms controlled by the migration of vacancies without break-away and is related to the intrinsic properties of the austenitic phase [25]. Therefore, the net peak height (after background subtraction) is lower in a matrix with BCT phase than in a matrix without BCT phase, since the BCT phase is still present overlapped to the austenite matrix. In fact, in FePdCo and FePdMn alloys a 32% and 38% (mass) transform to the BCT martensite during the cooling down to 170 K, respectively. These amounts remain unless the alloy is annealed at high temperature and subsequently quenched [13]. After the first heating run up to 603 K, the P1 peak decreases its height markedly in both alloys, indicating that the density of quenched-in-defects was decreased [24]. On the other hand, damping spectra show higher values of damping in martensitic phase than in austenitic phase. In fact, the damping behaviour out of the range of MT peak is controlled by the background damping values of each phase. This behaviour is in agreement with the damping capacity of the twin boundary and dislocations in the martensitic phase, while in the austenitic phase only dislocation contribute to background damping values. Nevertheless, the damping background values in the martensitic phase decrease, as different annealing were performed. This indicates that, effectively, the density of point defects and dislocations decreases and consequently the interaction between twin boundaries and structural defects decreases too. This effect is presented more clearly after the first heating run up to 603 K and 773 K, so the maximum temperature was reached in consecutive cycles two times. 3. RESULTS AND DISCUSSION Then, damping capacity in the martensitic phase decreases and the contribution to the damping values is mainly controlled by twin boundaries motion [18,19]. Squared frequency curves, shown in Figures 4(b) and 5(b), exhibit the typical minimum at the MT temperature. At higher temperatures than the MT temperature, the modulus increases with the temperature accordingly with both the dragging mechanism (within the P1 peak temperatures) and the recovery of structure [25]. Moreover, a precipitation and decomposition processes of the γ phase are present in the FePdMn alloy. Indeed, the increases of the MT peak after the annealing at 773 K in FePdMn (Figure 5(b)) BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. was related with a mechanism that involves a precipitation and decomposition processes of the γ phase [27]. was related with a mechanism that involves a precipitation and decomposition processes of the γ phase [27]. The effect of the thermal cycles on the non-thermoelastic MT can be seen from the movement of the hillside towards lower temperatures in Figures 4(a) and 5(a). In fact, the FCT-BCT transition temperature moves towards lower temperatures mainly after the first thermal cycle at 603 K. This behavior indicates that the reduction of structural defects difficult the appearance of BCT. 0 1 2 3 4 5 6 7 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Damping, Q-1x103 Temperature (K) (a) P1 MT 0.8 1 1.2 1.4 1.6 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Temperature (K) Squared frequency (s -2) (b) MT Figure 4: Damping spectra (a) and squared frequency curves (b) as a function of temperature during successive thermal cycles for a FePdCo alloy from 170 K up to different final temperatures. Lines are a guide for the eyes. 3. RESULTS AND DISCUSSION 0 1 2 3 4 5 6 7 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Damping, Q-1x103 Temperature (K) (a) P1 MT 0 1 2 3 4 5 6 7 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Damping, Q-1x103 Temperature (K) (a) P1 MT 0.8 1 1.2 1.4 1.6 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Temperature (K) Squared frequency (s -2) (b) MT Figure 4: Damping spectra (a) and squared frequency curves (b) as a function of temperature during successive thermal cycles for a FePdCo alloy from 170 K up to different final temperatures. Lines are a guide for the eyes. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. 0 0.5 1 1.5 2 2.5 3 3.5 4 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Temperature (K) Damping, Q-1x103 (a) P1 MT 0.5 0.6 0.7 0.8 0.9 1 1.1 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Squared frequency (s -2) Temperature (K) (b) MT Figure 5: Damping spectra (a) and squared frequency curves (b) as a function of temperature during successive thermal cycles for a FePdMn alloy from 170 K up to different final temperatures. Lines are a guide for the eyes. The appearance of ADD has been checked through the behaviour of S parameter for the first and 0 0.5 1 1.5 2 2.5 3 3.5 4 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Temperature (K) Damping, Q-1x103 (a) P1 MT 0.5 0.6 0.7 0.8 0.9 1 1.1 200 300 400 500 600 700 800 603 K 603 K 673 K 773 K 773 K Squared frequency (s -2) Temperature (K) (b) MT Figure 5: Damping spectra (a) and squared frequency curves (b) as a function of temperature during successive thermal cycles for a FePdMn alloy from 170 K up to different final temperatures. Lines are a guide for the eyes. The appearance of ADD has been checked through the behaviour of S parameter for the first and second heating runs of the whole cycles shown in Figure 4 and 5. 3. RESULTS AND DISCUSSION S values as a function of temperature for as-quenched samples and after annealing to 603 K is shown in Figure 6. As it can be expected, the values differ from zero for temperatures lower than MT temperature since the mobility of twin boundaries. The higher values of S for the annealed samples and the decrease of the amount of quenched-in-defects, i.e. decrease of P1 peak, indicate that both dislocations and variants increase their mobility. On the other hand, the S values are zero for temperatures higher than MT temperature. The absence of ADD effects indicate that the dislocation movement is carried out without thermally activated break-away [18-20,22,28]. Therefore, the appearance of the BCT phase and the addition of substitutional atoms no modifies the mechanisms of the peak P1. Nevertheless, additional little peaks appear superimposed to P1 peak. These little peaks could be related with the interaction of substitutional atoms with the BCT phase retained in the γ phase. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. 0 10 20 30 40 50 60 70 80 150 200 250 300 350 400 450 500 550 S=Q-1/0 Temperature (K) MT MT FePdMn FePdCo Figure 6: S parameter as a function of temperature for the as-quenched (circles) and annealed at 603 K (open circles) state for a FePdCo and FePdMn alloys. Lines are a guide for the eyes. Figure 6: S parameter as a function of temperature for the as-quenched (circles) and annealed at 603 K (open circles) state for a FePdCo and FePdMn alloys. Lines are a guide for the eyes. Finally, in order to check that both the non-thermoelastic MT transition and the Mn and Co substitutional atoms have not influence on the relaxation mechanism controlling P1 peak, the activation energy and pre-exponential factor for the relaxation time corresponding to P1 peak were obtained from the Arrhenius plot (the peak temperature shifts measured at different oscillation frequencies) for both alloys [18]. In fact, Figure 7 shows the Arrhenius plot related to P1 peak for the two alloys. The values obtained were HaFePdCo= (1.0±0.1) eV and oFePdCo=810-(14±0.5) s, and HaFePdMn= (1.0±0.1) eV and oFePdMn=110-(13±0.5) s. These values are in agreement with those previous obtained in these alloys, FePdCo and FePdMn, without the appearance of the BCT phase [24]. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. BONIFACICH, F.G.; LAMBRI, O.A.; GARGICEVICH, D., et al., revista Matéria, v. 23, n. 2, 2018. Therefore, the behaviour of the P1 peak let to conclude that annealing of FePdCo and FePdMn alloys over 603 K leads to a recovery of the structure where the amount of point defects and dislocations decrease. So, it is expected that this recovery makes difficult the accommodation of the misfit strain between the FCT and the BCT phases, giving rise to a decrease in the temperature for the appearance of the irreversible BCT phase [25]. In fact, from Figures 4(a) and 5(a) it can be seen the movement of the hillside of the MT non- thermoelastic phase at lower temperatures, between 210 K and 250 K for FePdCo and between 200 K and 290 K for FePdMn. This movement of the hillside is directly related with the movement towards lower temperatures of the FCT-BCT transition. Indeed, Figure 8 shows a decrease in the temperature for the appearance of the BCT phase measured through DSC in Fe70Pd30 (full line) and MS in Fe70Pd30 (dashed line) [25], Fe67Pd30Co3 (dotted line) and Fe66.8Pd30.7Mn2.5 (alt-dashed line); from where a good correspondence between the curves can be observed. 170 180 190 200 210 220 230 240 250 0 1 2 3 4 Temperaure (K) Previous maximum temperature (K) DSC FePd MS FePd MS FePdMn MS FePdCo 603 603 673 773 773 170 180 190 200 210 220 230 240 250 0 1 2 3 4 Temperaure (K) Previous maximum temperature (K) DSC FePd MS FePd MS FePdMn MS FePdCo 603 603 673 773 773 Figure 8: FCT-BCT transformation temperature as a function of the maximum temperature of the thermal cycles. See explanation in the text. Figure 8: FCT-BCT transformation temperature as a function of the maximum temperature of the thermal cycles. See explanation in the text. 4. CONCLUSIONS The stability at low temperatures of the thermoelastic martensite in FePdCo and FePdMn alloys is restricted by the irreversible FCT-BCT phase transformation. The appearance of dislocations, as a result of the FCT- BCT strain misfit, and their dynamics has been analyzed by mechanical spectroscopy. The P1 damping peak, at around 450 K, related to the dislocation movement has been identified in these alloys with BCT non- thermoeleastic MT. The appearance of non-thermoeleastic MT and the addition of substitutional atoms do not modify the physical mechanisms controlling the P1 peak. Moreover, the behavior of the movement of the FCT-BCT transition temperature is not affected by the addition of substitutional atoms. Finally, a reduction of the dislocation density reduces the irreversible transformation temperature and so, increases the stability range of the FCC-FCT reversible transformation. 3. RESULTS AND DISCUSSION Moreover, the activation parameters are also in agreement with previous reported works in Fe70Pd30 with and without the appearance of the BCT phase [24,25]. Consequently, the driving force controlling the development of P1 is effectively the same as said before [25]. -3.5 -3 -2.5 -2 -1.5 -1 2.1 2.15 2.2 2.25 2.3 2.35 ln(1/ P) 1/TP x 103 (K-1) FePdCo - (1.0 ± 0.1) eV - 0 = 8x10-(14.0±0.5) s FePdMn - (1.0 ± 0.1) eV - 0 = 1x10-(13.0±0.5) s Figure 7: Arrhenius plot for FePdCo and FePdMn alloys related to P1 peak. -3.5 -3 -2.5 -2 -1.5 -1 2.1 2.15 2.2 2.25 2.3 2.35 ln(1/ P) 1/TP x 103 (K-1) FePdCo - (1.0 ± 0.1) eV - 0 = 8x10-(14.0±0.5) s FePdMn - (1.0 ± 0.1) eV - 0 = 1x10-(13.0±0.5) s Figure 7: Arrhenius plot for FePdCo and FePdMn alloys related to P1 peak. 6. BIBLIOGRAPHY [1] ULLAKKO, K., HUANG, J.K., KANTNER, R.C., et al., “Large magnetic-field-induced strains in Ni2MnGa single crystals”, Applied Physics Letters, v. 69, n. 13, pp. 1966-1968, 1996. 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https://openalex.org/W2811187570	https://digitalcommons.lsu.edu/cgi/viewcontent.cgi?article=2114&context=biosci_pubs	English	null	Structural modification of the tripeptide KPV by reductive “glycoalkylation” of the lysine residue	PloS one	2,018	cc-by	8,310	Structural modification of the tripeptide KPV by reductive Structural modification of the tripeptide KPV by reductive “glycoalkylation” of the lysine residue “glycoalkylation” of the lysine residue William T. Doerrler Louisiana State University Megan A. Macnaughtan Louisiana State University Carol M. Taylor Louisiana State University Follow this and additional works at: https://repository.lsu.edu/biosci_pubs Faculty Publications Department of Biological Sciences Faculty Publications This Article is brought to you for free and open access by the Department of Biological Sciences at LSU Scholarly Repository. It has been accepted for inclusion in Faculty Publications by an authorized administrator of LSU Scholarly Repository. For more information, please contact ir@lsu.edu. Louisiana State University Louisiana State University LSU Scholarly Repository LSU Scholarly Repository Faculty Publications Department of Biological Sc 6-1-2018 Structural modification of the tripeptide KPV by reductive Structural modification of the tripeptide KPV by reductive “glycoalkylation” of the lysine residue “glycoalkylation” of the lysine residue Abigael C. Songok Louisiana State University Pradip Panta Louisiana State University William T. Doerrler Louisiana State University Megan A. Macnaughtan Louisiana State University Carol M. Taylor Louisiana State University Louisiana State University Louisiana State University LSU Scholarly Repository LSU Scholarly Repository Editor: Israel Silman, Weizmann Institute of Science, ISRAEL Editor: Israel Silman, Weizmann Institute of Science, ISRAEL Received: December 7, 2017 Accepted: June 12, 2018 Published: June 28, 2018 Copyright: © 2018 Songok et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2018 Songok 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. Abigael C. Songok1, Pradip Panta2, William T. Doerrler2, Megan A. Macnaughtan1, Carol M. Taylor1* Abigael C. Songok1, Pradip Panta2, William T. Doerrler2, Megan A. Macnaughtan1, Carol M. Taylor1* 1 Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana, United States of America, 2 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * cmtaylor@lsu.edu * cmtaylor@lsu.edu Abstract Peptides that exhibit enzymatic or hormonal activities are regulatory factors and desirable therapeutic drugs because of their high target specificity and minimal side effects. Unfortu- nately, these drugs are susceptible to enzymatic degradation, leading to their rapid elimina- tion and thereby demanding frequent dosage. Structurally modified forms of some peptide drugs have shown enhanced pharmacokinetics, improving their oral bioavailability. Here, we discuss a novel glycomimetic approach to modify lysine residues in peptides. In a model sys- tem, the ε-amine of Ts-Lys-OMe was reductively alkylated with a glucose derivative to afford a dihydroxylated piperidine in place of the amine. A similar modification was applied to H- KPV-NH2, a tripeptide derived from the α-melanocyte stimulating hormone (α-MSH) reported to have antimicrobial and anti-inflammatory properties. Antimicrobial assays, under a variety of conditions, showed no activity for Ac-KPV-NH2 or the α- or ε-glycoalkylated analogs. Gly- coalkylated peptides did, however, show stability toward proteolytic enzymes. Recommended Citation Recommended Citation Songok, A., Panta, P., Doerrler, W., Macnaughtan, M., & Taylor, C. (2018). Structural modification of the tripeptide KPV by reductive “glycoalkylation” of the lysine residue. PLoS ONE, 13 (6) https://doi.org/ 10.1371/journal.pone.0199686 This Article is brought to you for free and open access by the Department of Biological Sciences at LSU Scholarly Repository. It has been accepted for inclusion in Faculty Publications by an authorized administrator of LSU Scholarly Repository. For more information, please contact ir@lsu.edu. RESEARCH ARTICLE OPEN ACCESS Citation: Songok AC, Panta P, Doerrler WT, Macnaughtan MA, Taylor CM (2018) Structural modification of the tripeptide KPV by reductive “glycoalkylation” of the lysine residue. PLoS ONE 13(6): e0199686. https://doi.org/10.1371/journal. pone.0199686 Structural modification of the tripeptide KPV by reductive “glycoalkylation” of the lysine residue Abigael C. Songok1, Pradip Panta2, William T. Doerrler2, Megan A. Macnaughtan1, Carol M. Taylor1* Introduction In the recent past, there has been a significant increase in the market for therapeutic peptides and proteins [1]. This interest is attributed to peptides’ high selectivity for their target, often with minimal side effects and toxicity [2]. Some problems that must be overcome for therapeu- tic peptides and proteins include proteolytic instability, immunogenicity, low oral bioavailabil- ity, and short half-life [3,4]. In order to enhance the pharmacokinetic properties of peptide drugs, various structural modifications have been effected. Examples of these modifications include N-methylation and the formation of cyclic peptides, which enhance membrane perme- ability and decrease susceptibility to enzymatic degradation [2,5]. Another strategy is to syn- thesize peptide analogs incorporating unnatural D-amino acids since they are less susceptible to proteolysis [6]. The half-life of a peptide can be increased using polymer conjugates, such as polyethyleneglycol (PEG) modified peptides. These PEGylated peptides have a larger hydrody- namic volume than their unmodified counterparts, which minimizes the elimination rate of Data Availability Statement: All relevant data are within the paper and its Supporting Information files, with the exception of the details for the X-ray crystallographic determination of compound 2 that is available at the CCDC with deposition number 1825648. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 1 / 14 Reductive "glycoalkylation" of KPV the drug through renal filtration [7]. Functional mimics utilizing non-peptidic foldamers (N,N ˈ-linked oligoureas coupled to amino acid sidechains) tested positive against S. aureus [8]. Other modifications include peptide lipidation [9], hydrophobic ion pairing [10], and com- plexation with cyclodextrin [1]. Regardless of the nature of the modification, the multi-faceted goal is to improve the target specificity, membrane permeation, stability, solubility, and oral bioavailability of the drug without altering the therapeutic activity. Herein we present a novel glycomimetic approach to modify the α- or ε-amino groups of lysine residues. As a model system, Nα-p-tosyl-L-lysine methyl ester (Ts-Lys-OMe) was modi- fied at the ε-position. Having established the best chemical reaction conditions for modifica- tion, the same approach was applied to the lysine residue in the C-amidated tripeptide, H-KPV-NH2. This sequence is the carboxy-terminal tripeptide of α-melanocyte stimulating hormone, (α–MSH, Ac-SYSMEHFRWGKPV-NH2) [11]. Both α-MSH and Ac-KPV-NH2 have anti-inflammatory [11] and antimicrobial activities [11,12]. Introduction Ac-KPV-NH2 is more attractive for drug development compared to full-length α-MSH because α-MSH has additional activity as a melanotropic peptide [11]. In addition, Ac-KPV-NH2 is chemically stable and is less costly to produce because of its small size. The mechanism of the Ac-KPV-NH2 tripeptide’s anti-inflam- matory action has received more attention than its antimicrobial activity. Elliott et al. reported a calcium signaling pathway for α-MSH and Ac-KPV-NH2, through the MC-R1 receptor [13]. They observed an elevation of intracellular calcium in human keratinocyte cells by adrenocorti- cotropic hormone (ACTH), α-MSH, Ac-KPV-NH2, and Ac-KPdV-NH2 (dV indicates D-valine in place of L-valine) in the presence of an adenosine agonist, which inhibits cAMP elevation [13]. Antimicrobial activity of Ac-KPV-NH2 has been reported for the multi-resistant human pathogens, S. aureus and Candida albicans [12], and antiviral activity, viz. HIV-1 [14]. The molecular basis for these activities remains unknown. Ac-KPV-NH2, along with its analogs and stereoisomers (Ac-dKPV-NH2, Ac-KPdV-NH2, Ac-KdPV-NH2, and Ac-dKPdV-NH2) [15], have similar anti-inflammatory activities to α-MSH. There are, however, conflicting studies as to whether the L-configuration of proline is essential for activity [16–18]. Antimicrobial peptides act via membrane disruption, initiated by electrostatic interactions and hydrogen bonding. Preferential affinity of such peptides for microbial membranes (typically negatively charged) rather than mammalian membranes (neutral) is attributed to their cationic nature. Giuliani et al. reviewed proposed disruption mechanisms in detail, including the barrel- stave, toroidal, aggregate channel, and carpet mechanisms [3]. Charnley et al. found that the cat- ionic lysine residue in Ac-KPdV-NH2 is not essential for antimicrobial activity [19]. Replacement of lysine with alanine in the sequence (Ac-APdV-NH2) did not affect the activity such that a gen- eral Ac-XPdV-NH2 or Ac-XPV-NH2 sequence was proposed. Modification of H-KPV-NH2 at the lysine residue is therefore not expected to interfere with the antimicrobial activity of the mol- ecule and can be used to enhance the peptide’s solubility and amphipathic properties. Herein we redefine the use of the term “glycoalkylation,” illustrated generically in Fig 1A as the modification of the ε-amino group of lysine. Schlimme et al. introduced the term for N- glycosylation of mono- and bicyclic dicarbonic acid imides using ribose [20]. The glycosylated imide [e.g., compound 1, Fig 1B] was in turn used as a glycoalkylating agent for lysine through a ring-opening reaction. Introduction In this paper, the α- or ε-amino group of a lysine residue is glycoalky- lated with a derivative of α-D-glucofuranose to incorporate the nitrogen into a piperidine diol ring, e.g., compound 2 in Fig 1C. Materials and methods Reagents were obtained from commercial sources and used without further purification except for triethylamine, piperidine, and collidine, which were distilled after overnight stirring in 2 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV Fig 1. Glycoalkylated lysine. (a) Lysine modification at the ε-amine with a generic sugar molecule; (b) Glycoalkylated lysine, as described by Schlimme et al. [20]; (c) ε-Glycoalkylated Ts-Lys-OMe, as described in this article. https://doi.org/10.1371/journal.pone.0199686.g001 Fig 1. Glycoalkylated lysine. (a) Lysine modification at the ε-amine with a generic sugar molecule; (b) Glycoalkylated lysine, as described by Schlimme et al. [20]; (c) ε-Glycoalkylated Ts-Lys-OMe, as described in this article. https://doi org/10 1371/journal pone 0199686 g001 https://doi.org/10.1371/journal.pone.0199686.g001 CaH2. Methanol was dried and distilled from magnesium turnings. Silica gel for flash column chromatography was obtained from Sigma (particle size 40–63 µm). Glass TLC plates were coated with silica gel 60G F254 manufactured by Merck Millipore. HPLC purification was per- formed on a Sorbent Purity C18 300A˚ 5 μm column (250 × 10.0 mm) with a flow rate of 1.0 mL/min and a gradient of 20–90% acetonitrile (+ 0.1% formic acid) over 20 min, monitoring UV-absorbance 218 and 254 nm. 1H and 13C NMR spectra were recorded on a Bruker AVIII- 400-Nanobay spectrometer, AV500-Prodigy or Bruker AVIII-400-3. Chemical shifts are expressed in ppm downfield of TMS, in deuterated solvents, as specified. Optical rotations were measured on a JASCO-P2000 polarimeter. High resolution mass spectrometry (HRMS) was carried out using an ESI TOF 6210 (electrospray ionization time-of-flight) mass spectrom- eter (Agilent Technologies). Streptomyces griseus pronase was purchased from VWR, and spec- ified to be 45,000 proteolytic units/g dry weight. A stock solution was prepared by dissolving 2 mg of the lyophilized powder in D2O (2 mL). Chemical synthesis Nα-Tosyl-Nε-1,2-O-isopropylidene-α-D-glucofuranose-L-lysine methyl ester (6). Flame-dried, 4 A˚ molecular sieves (143.0 mg) were added to a solution of 1,2-O-isopropyli- dene-α-D-glucofuranose-5-carbaldehyde 4 [21, 22] (132.9 mg, 0.77 mmol, 1.0 equiv) and triethylamine (300 μL, 216.6 mg, 2.15 mmol, 2.8 equiv) in dry methanol (20 mL). Nα-p-Tosyl- L-lysine methyl ester hydrochloride 5 (270.9 mg, 0.77 mmol, 1.0 equiv) was added as a solid in a single portion. The mixture was stirred at rt under N2 for 18 h. The molecular sieves were removed by filtration, washing well with methanol. The filtrate was concentrated to give imine. 1H NMR (400 MHz, CDCl3) δ 7.59 -N = CH-, d, J = 4.5 Hz. Sodium borohydride (40.0 mg, 1.06 mmol, 1.0 equiv) was added to a stirred solution of imine in dry methanol (15 mL) at 0˚C and stirred under N2 for 4 h. The reaction was quenched by dropwise addition of 2M HCl (600 µL), the mixture concentrated, and the residue parti- tioned between EtOAc (40 mL) and water (10 mL). The aqueous layer was further extracted with EtOAc (2 x 20 mL) and the combined organic extracts were concentrated. The residue was purified by flash chromatography on silica gel, eluting with 95:5 CH2Cl2-MeOH to afford 6 as a brownish solid (240.4 mg, 66%). Rf 0.56 (9:1 CH2Cl2-MeOH). [α]D 26 +9.53 (c 2.5, CHCl3). 1H NMR (400 MHz, CDCl3) δ 1.31 (s, 3H), 1.47–1.32 (m, 4H), 1.49 (s, 3H), 1.76–1.57 (m, 3H), 2.07 (dd, J = 13.2, 4.3 Hz, 1H), 2.40 (s, 3H), 2.62 (t, J = 6.6 Hz, 2H), 2.71 (dd, J = 12.4, 7.0 Hz, 1H), 2.87 (dd, J = 12.4, 3.3 Hz, 1H), 3.47 (s, 3H), 3.89 (dd, J = 7.3, 5.2 Hz, 1H), 4.37 3 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV (ddd, J = 14.2, 3.8, 3.5 Hz, 1H), 4.72 (t, J = 4.2 Hz, 1H), 5.80 (d, J = 3.7 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 21.5, 22.5, 26.1, 26.7, 28.3, 32.8, 36.5, 49.2, 52.2, 52.4, 55.6, 76.5, 80.4, 105.5, 111.0, 127.2(2C), 129.5(2C), 136.7, 143.6, 172.1. HRMS (ESI) calcd for C22H35N2O7S (M+H)+ 471.2159, obsd 471.2149. Nα-Tosyl-Nε-(2S,4R)-dihydroxypiperidine-L-lysine methyl ester (2). A solution of Nα- tosyl-Nε-1,2-O-isopropylidene-α-D-glucofuranose-L-lysine methyl ester (6) (105.0 mg, 0.24 mmol, 1.0 equiv) in TFA-water (2:1 v/v) solution was stirred for 3 h at rt. Chemical synthesis The TFA was co- evaporated with toluene, and the residue was diluted with water and lyophilized. The dried sample was dissolved in dry MeOH (3 mL) and cooled to 0˚C. Sodium borohydride (30.6 mg, 0.49 mmol, 2.0 equiv) was added and stirring continued for 4 h under N2. The reaction was quenched by the dropwise addition of 2M HCl (0.5 mL). The mixture was concentrated, and the residue purified by flash column chromatography on silica gel eluting with 9:1 CH2Cl2- MeOH. A solution of the purified product 2 in MeOH (1 mL) was kept at 4˚C, which led to crystallization (42.0 mg, 42%). Rf 0.37 (9:1 CH2Cl2-MeOH). [α]D 25 +7.3 (c 1.1, CHCl3). 1H NMR (400 MHz, CDCl3) δ 1.21 (dt, J = 11.0 Hz, 1H), 1.28–1.41 (m, 2H), 1.41–1.55 (m, 2H), 1.55–1.75 (m, 2H), 1.81 (t, J = 9.7 Hz, 2H), 2.19–2.30 (m, 1H), 2.38 (t, J = 7.3 Hz, 2H) 2.45 (s, 3H), 2.94 (dd, J = 10.5, 3.4 Hz, 2H), 3.44 (s, 3H), 3.66–3.71 (m, 2H), 3.86 (dd, J = 8.6, 5.5 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 20.1, 22.9, 25.0, 32.1, 41.3, 51.1, 55.6, 57.4, 59.4, 59.5, 64.9(2C), 126.8 (2C), 129.2(2C), 137.8, 143.4, 172.2. HRMS (ESI) calcd for C19H31N2O6S (M+H)+ 415.1903, obsd 415.1904. Fmoc-K(Boc)-PV-NH2 (10a). N-Hydroxysuccinimide (143.3 mg, 1.28 mmol, 1.0 equiv) and DCC (264.1 mg, 1.28 mmol, 1.0 equiv) were added to a solution of Fmoc-Lys(Boc)-OH (600.0 mg, 1.28 mmol, 1.0 equiv) in CH2Cl2 (20 mL) at 0˚C. The mixture was stirred for 20 min, warmed to rt, stirred for 4 h and filtered through a plug of cotton in a Pasteur pipette. The filtrate was concentrated, placed in the freezer for 2 h, filtered a second time and the fil- trate concentrated. The residue was dissolved in DMF (6 mL) and cooled in an ice bath. To the stirred mixture was added L-proline (147.4 mg, 1.28 mmol, 1.0 equiv) and diisopropylethyla- mine (268 µL, 199.0 mg, 1.54 mmol, 1.2 equiv). The mixture was stirred at 0˚C for 10 min, warmed to rt and stirred for 14 h. Dimethylformamide was removed by a stream of air. The residue was taken up in EtOAc (100 mL) and washed with 2M HCl (80 mL). The layers were separated, and the aqueous layer was further extracted with EtOAc (3 x 20 mL). PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV Boc-K(Fmoc)-PV-NH2 (10b). Boc-Lys(Fmoc)-OH (9b) (600.0 mg, 1.28 mmol) was treated, by analogy to the procedure described for the conversion of 9a to 10a, to afford 10b (360.0 mg, 42%) Rf 0.43 (9:1 CH2Cl2-MeOH). [α]D 25–55.7 (c 0.8, MeOH). 1H NMR (400 MHz, CD3OD) δ 0.98 (d, J = 2.1 Hz, 3H), 0.99 (d, J = 2.1 Hz, 3H), 1.43 (s, 9H), 1.43–1.64 (m, 5H), 1.72–1.81 (m, 1H), 1.95–2.17 (m, 5H), 3.13 (app. t, J = 6.2 Hz, 2H), 3.63 (dd, J = 16.0 Hz, 9.6 Hz, 1H), 3.79 (dd, J = 16.0 Hz, 6.8 Hz, 1H), 4.17–4.22 (m 1H), 4.21 (d, J = 6.6 Hz, 1H), 4.27– 4.34 (m, 1H), 4.35 (d, J = 6.9 Hz, 2H), 4.55 (dd, J = 8.0 Hz, 3.8 Hz, 1H), 7.31 (t, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.65 (d, J = 7.4 Hz, 2H), 7.79 (d, J = 7.4 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 17.2, 18.5, 22.5, 24.7, 27.4, 28.7, 29.1, 30.6, 30.9, 39.9, 47.1, 52.4, 58.3, 60.1, 66.2, 79.2, 119.6, 124.8, 126.8, 127.4, 141.2, 144.0, 157.4, 157.5, 172.6, 172.8, 174.7; HRMS (ESI) calcd for C36H50N5O7 (M+H)+ 664.3705, obsd 664.3710. αG’-K(Boc)PV-NH2 (11a). Piperidine (552 µL, 475.8 mg, 5.59 mmol, 5.6 equiv) was added to a solution of tripeptide 10a (797.8 mg, 1.20 mmol, 1.0 equiv) in dry DMF (20 mL). The reaction was stirred at rt for 30 min. The solvent was evaporated by a stream of air, the res- idue partitioned between CH2Cl2 (20 mL) and H2O (10 mL), and the layers separated. The aqueous layer was further washed with CH2Cl2 (3 x 10 mL) and lyophilized to afford the free amine that was used in the next reaction without further purification (Rf 0.59, 6:4:1 CHCl3- MeOH-H2O). Triethylamine (250 µL, 181.4 mg, 1.79 mmol, 3.0 equiv) and flame dried 4Å powdered molecular sieves (75.0 mg) were added to a solution of tripeptide amine (263.4 mg, 0.60 mmol, 1.0 equiv) in dry MeOH (3 mL). The mixture was stirred at rt and a solution of the aldehyde (328.3 mg, 1.91 mmol, 3.2 equiv) in dry MeOH (3 mL) was added. The mixture was left to stir at rt for 24 h. The reaction was filtered through a pad of Celite1 that was washed well with MeOH. The filtrate was cooled to 0˚C, NaBH4 (73.2 mg, 1.93 mmol, 3.2 equiv) was added, and the mixture was stirred for 4 h under N2. The reaction was quenched by dropwise addition of 2M HCl (250 µL). The mixture was concentrated, and the residue purified by flash column chromatography, eluting with 9:1 CH2Cl2-MeOH to afford the tripeptide 11a (194 mg, 27%) Rf 0.54 (9:1 CH2Cl2-MeOH). [α]D 25–68.7 (c 1.9, CHCl3). 1H NMR (500 MHz, CD3OD) δ 1.00 (d, J = 6.6 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 1.31 (s, 3H), 1.45 (s, 9H), 1.48 (s, 3H), 1.40–1.53 (m, 3H), 1.54–1.62 (m, 2H), 1.63–1.73 (m, 2H), 1.98–2.07 (m, 3H), 2.08–2.14 (m, 2H), 2.14– 2.20 (m, 1H), 2.58 (dd, J = 12.7, 6.6 Hz, 1H), 2.79 (dd, J = 12.7, 3.5 Hz, 1H) 2.89–3.12 (app. t, J = 5.1 Hz, 3H), 3.62–3.74 (m, 2H), 3.77–3.82 (m, 1H), 4.21 (d, J = 6.5 Hz, 1H), 4.25–4.30 (m, 1H), 4.59 (dd, J = 8.2, 4.3 Hz, 1H), 4.76 (app. t, J = 4.2 Hz, 1H), 5.78 (d, J = 3.7 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 17.1, 18.5, 22.4, 24.6, 25.0, 25.6, 27.4, 28.7, 29.4, 30.7, 32.4, 36.1, 39.7, 47.1, 50.0, 58.3, 58.8, 59.9, 77.9, 80.3, 105.7, 110.8, 157.1, 172.8 (2C), 174.2, 174,7. HRMS (ESI) calcd for C29H52N5O8 (M+H)+ 598.3810, obsd 598.3809. Reported for the major conformation only; two species were observed that were presumed to be rotamers about the prolyl amide bond. Boc-K(εG’)PV-NH2 (11b). Following the same series of reactions in the conversion of 10a to 11a above, compound 10b (125.0 mg, 0.19 mmol) was converted to 11b (41 mg, 60%). Rf 0.78 (6:4:1 CHCl3-MeOH-H2O). [α]D 25–59.3 (c 1.1, MeOH). PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Chemical synthesis The organic fractions were combined, filtered through anhydrous MgSO4 and concentrated to afford the dipeptide acid that was used directly without purification Rf 0.32 (9:1 CH2Cl2-MeOH). Valine amide hydrochloride (195.4 mg, 1.28 mmol, 1.0 equiv), HATU (535.5 mg, 1.41 mmol, 1.1 equiv), and 2,4,6-collidine (340 µL, 312.8 mg, 2.58 mmol, 2.0 equiv) were added to a stirred solution of Boc-Lys(Fmoc)-Pro-OH in CH2Cl2 (6 mL) at 0˚C. After 10 min, the reac- tion was warmed to rt and stirred for 18 h under N2. The mixture was concentrated and the tri- peptide 10a was isolated by flash column chromatography, eluting with 20:1 CH2Cl2-MeOH, as a colorless solid (134 mg, 44%) Rf 0.55 (9:1 CH2Cl2-MeOH). [α]D 25 +56.7 (c 1.4, DMSO). 1H NMR (400 MHz, CD3OD) δ 0.94 (d, J = 6.1 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 1.42 (s, 9H), 1.32–1.58 (m, 4H), 1.58–1.70 (m, 1H), 1.71–1.76 (m, 1H), 2.01–2.11 (m, 5H), 2.93–3.18 (m, 2H), 3.57–3.76 (m, 1H), 3.76–3.82 (m, 1H), 4.17–4.22 (m, 2H), 4.29–4.45 (m, 3H), 4.50 (dd, J = 7.6, 3.9 Hz, 1H), 6.70 (d, J = 7.5 Hz, NH), 7.07 (t, J = 5.5 Hz, NH) 7.30 (t, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.61 (d, J = 7.2 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ 18.2, 19.8, 23.0, 25.1, 28.7, 29.0, 29.7, 31.0 (2C), 39.9, 47.1 (2C), 52.9, 57.7, 59.8, 66.1, 77.8, 120.5, 125.8, 127.5, 128.0, 141.2, 144.3, 156.0, 156.6, 171.4, 171.5, 173.3; HRMS (ESI) calcd for C36H50N5O7 (M+H)+ 664.3705, found 664.3688. Does not integrate to a full proton due to proton exchange with CD3OD. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 4 / 14 By analogy to the procedure described for conversion of 11a to 12a, compound 11b (139.0 mg, 0.23 mmol) was converted to 12b. The crude product was purified by HPLC to afford ε-glycoalkylated 12b (24 mg, 23%). tR 15.6 min. Rf 0.20 (6:4:1 CHCl3-MeOH-H2O). [α]D 25–35.5 (c 0.4, MeOH). 1H NMR (500 MHz, CD3OD) δ 1.00 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.7 Hz, 3H), 1.19–1.35 (m, 1H), 1.41–1.54 (m, 2H), 1.42–1.64 (m, 2H), 1.66–1.76 (m, 1H), 1.78–1.92 (m, 3H), 1.96–2.16 (m, 4H), 2.20–2.29 (m, 2H), 2.50 (app. t, J = 7.7 Hz, 2H), 2.98 (dd, J = 10.6, 3.7 Hz, 2H), 3.63–3.66 (m, 1H), 3.68–3.75 (m, 3H), 3.91 (t, J = 6.2 Hz, 1H), 4.20 (d, J = 6.7 Hz, 1H), 4.58 (dd, J = 8.1, 4.6 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 17.1, 18.4, 22.5, 24.6, 25.8, 28.8, 30.7, 32.4, 41.7, 47.2, 51.9, 57.5, 58.4, 59.6 (2C), 60.1, 65.0 (2C), 171.4, 172.6, 174.7. HRMS (ESI) calcd for C21H40N5O5 (M+H)+ 442.3024, obsd 442.3032. Ac-KPV-NH2 (12c). A solution of tripeptide 10a (362.2 mg, 0.55 mmol, 1.0 equiv) in piperidine (544 µL, 470.0 mg, 5.50 mmol, 10.0 equiv) and DMF (5 mL) was stirred for 30 min. The solvent was evaporated by a stream of air, and the residue partitioned between CH2Cl2 (50 mL) and H2O (30 mL). The aqueous layer was further washed with CH2Cl2 (2 x 20 mL) and lyophilized to afford the free amine (232 mg, 96%). Rf 0.28 (9:1 CH2Cl2-MeOH). A portion of the free amine (94 mg, 0.213 mmol) was dissolved in a mixture of Ac2O-pyri- dine (1:1 v/v, 6 mL) and stirred for 15 h, concentrated and purified by flash column chroma- tography, eluting with 100:7 CH2Cl2-MeOH to give the acetylated tripeptide, Ac-K(Boc)- PV-NH2 (72 mg, 70%). Rf 0.50 (20:3 CH2Cl2-MeOH). [α]D 25–65.4 (c 1.5, CHCl3). 1H NMR (400 MHz, CD3OD) δ 0.99 (d, J = 3.4 Hz, 3H), 1.00 (d, J = 3.4 Hz, 3H), 1.43–1.56 (m, 4H), 1.45 (s, 9H), 1.59–1.72 (m, 1H), 1.77–1.87 (m, 1H), 1.96–2.19 (m, 5H), 1.98 (s, 3H), 3.06 (app. t, J = 6.0 Hz, 2H), 3.66–3.73 (m, 1H), 3.73–3.92 (m, 1H), 4.22 (app. 1H NMR (400 MHz, CD3OD) δ 1.00 (d, J = 2.1 Hz, 3H), 1.01 (d, J = 2.1 Hz, 3H), 1.33 (s, 3H), 1.45 (s, 9H), 1.49 (s, 3H), 1.40– 1.55 (m, 1H), 1.61–1.84 (m, 5H), 1.99–2.27 (m, 6H), 2.95–3.03 (m, 3H), 3.25 (dd, J = 12.8, 2.4 Hz, 1H), 3.66–3.27 (m, 1H), 3.83–3.88 (m, 1H), 4.18 (d, J = 6.6 Hz, 1H), 4.37 (t, J = 6.7 Hz, 1H), 4.42–4.48 (m, 1H), 4.55 (dd, J = 8.2, 4.2 Hz, 1H), 4.84 (t, J = 4.1 Hz, 1H), 5.89 (d, J = 3.5 Hz, 1H); 13C NMR (100 MHz, CD3OD,) δ 17.2, 18.5, 22.3, 24.7, 25.0, 25.7, 25.9, 27.3, 29.0, 30.7, 30.8, 36.2, 47.4, 48.0, 50.7, 52.0, 58.5, 60.3, 74.0, 79.2, 80.4, 105.9, 111.3, 156.5, 172.2, 172.9, 174.7. HRMS (ESI) calcd for C29H51N5O8 (M+H)+ 598.3810, obsd 598.3817. 5 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV αG-KPV-NH2 (12a). A solution of compound 11a (78.0 mg, 0.13 mmol, 1.0 equiv) in TFA-H2O (2:1 v/v, 4.5 mL) was stirred for 3.5 h. The mixture was diluted with toluene (20 mL) and concentrated. The residue was dissolved in MeOH and stirred at 0˚C. Solid NaHCO3 (35.3 mg) was added to neutralize the solution. NaBH3CN (16.3 mg, 0.26 mmol, 2.0 equiv) was added and the mixture stirred for 15 h. The reaction was quenched by the dropwise addition of 2M HCl (~600 µL), concentrated, and the residue subjected to HPLC to afford compound 12a (17.8 mg, 31%). tR 15.5 min. Rf 0.13 (6:4:1 CHCl3-MeOH-H2O). [α]D 25–28.9 (c 0.1, MeOH); 1H NMR (500 MHz, CD3OD) δ 1.01 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.7 Hz, 3H), 1.24 (app. q, J = 10.7 Hz, 1H), 1.29–1.52 (m, 2H), 1.62–1.78 (m, 3H), 1.79–1.92 (m, 1H), 1.94–2.13 (m, 5H), 2.15–2.28 (m, 2H), 2.31 (t, J = 10.0 Hz, 1H), 2.87–3.02 (m, 4H), 3.56 (dd, J = 10.1, 3.9 Hz, 1H), 3.60–3.78 (m, 3H), 3.88–3.92 (m, 1H), 4.18 (d, J = 6.8 Hz, 1H), 4.53 (dd, J = 8.4, 4.0 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 17.2, 18.4, 22.7, 24.4, 24.5, 27.0, 29.4, 30.7, 39.2, 41.6, 47.4, 55.3, 56.9, 58.5, 60.3, 65.2, 65.7 (2C), 171.0, 173.2, 174.7; HRMS (ESI) calcd for C21H40N5O5 (M+H)+ 442.3024, obsd 442.3029. H-K(εG)PV-NH2 (12b). t, J = 6.3 Hz, 1H), 4.54 (dd, J = 8.0, 3.9 Hz, 1H), 4.54–4.59 (m, 1H), 6.59 (br s, NH), 7.91 (d, J = 8.2 Hz, NH), 8.18 (d, J = 7.0 Hz, NH); 13C NMR (100 MHz, CD3OD) δ 17.1, 18.5, 20.9, 22.6, 24.7, 27.4(3C), 28.8, 29.3, 30.7 (2C), 39.6, 47.3, 51.3, 58.3, 60.1, 78.4, 157.1, 171.8, 171.9, 172.8, 174.7. HRMS (ESI) calcd for C23H42N5O6 (M+H)+ 484.3135, obsd 484.3130. Does not integrate for a full proton due to deuterium exchange. The acetylated tripeptide, Ac-K(Boc)PV-NH2 (72.0 mg, 0.19 mmol) was dissolved in a mix- ture CH2Cl2-TFA (1:1 v/v, 4 mL) and stirred at rt for 30 min. The mixture was concentrated, and the residue dissolved in toluene and concentrated again. The residue was purified by HPLC to afford the free amine 12c (40.8 mg, 71%). tR 16.2 min. Rf 0.36 (20:13:3:1 CHCl3- MeOH-H2O-NH3). [α]D 25 (c 0.6, MeOH). 1H NMR (400 MHz, CD3OD) δ 1.00 (d, J = 2.5 Hz, 3H), 1.01 (d, J = 2.5 Hz, 3H), 1.42–1.55 (m, 2 H), 1.62–1.73 (m, 3 H), 1.75–1.84 (m, 1 H), 1.96– 2.15 (m, 4H), 1.99 (s, 3H), 2.18–2.24 (m, 1H), 2.78 (t, J = 7.0 Hz, 2H), 3.66–3.71 (m, 1H), 3.86– 6 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV 3.92 (m, 1H), 4.18 (d, J = 6.9 Hz, 1H), 4.53 (dd, J = 8.4, 4.6 Hz, 1H), 4.60 (dd, J = 8.1, 5.9 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 17.1, 18.5, 20.9, 22.2, 24.7, 29.0 (2C), 30.6, 30.7, 39.7, 47.4, 51.1, 58.5, 60.1, 171.6, 171.8, 172.9, 174.7; HRMS (ESI) calcd for C18H34N5O4 (M+H)+ 384.2611, obsd 384.2606. Determination of stability of tripeptides to pronase. To a solution of each tripeptide in D2O (300 µL) was added 1M NH4HCO3 (20 µL) and 50 mM CaCl2 (40 µL). The pH of the resulting solution was adjusted to 7.0 by the addition of 3.7% HCl (10–12 µL). The volume was adjusted to 395 µL and the 1H-NMR spectrum recorded at 500 MHz. An aliquot (2 µL) of the 2 mg/mL pronase stock solution was added to the solution of tripeptide and the 1H-NMR spec- trum recorded at 15 min intervals for 1 h at RT. The solution was warmed to 37˚C using the NMR spectrometer’s variable temperature controller, and spectra recorded, at 15 min inter- vals, for 2 h. Chemical synthesis Aldehyde 4 was prepared from commercially available 1,2:5,6-di-O-isopropylidene-α-D-glu- cofuranose (3) according to literature procedures (Fig 2) [21–27]. Specifically, Barton- McCombie deoxygenation at C-3 [23,24], selective hydrolysis of the less substituted acetal and oxidative cleavage of the 5,6-diol afforded the requisite aldehyde 4 [21,22,27]. Aldehyde 4 has been subjected to reductive amination previously with benzylamine [28]. Hydroxylated piperi- dines have been prepared previously by condensation of carbohydrate-derived 1,5-dialdehydes with an amine [29–31]. Indeed, Steiner et al. performed such a “double reductive deamina- tion” with the ε-amino group of Boc-L-Lys-OMe en route to β-xylosidase inhibitors [32]. In the current context, we sought to perform two sequential glycoalkylations in a controlled fashion. Aldehyde 4 was condensed with the ε-amino group of lysine derivative 5. Evidence for imine formation was afforded by 1H NMR: there was no residual aldehyde signal (δ 9.68 ppm, RCH = O, d, J = 1.9 Hz) and the imine gave rise to a distinct new signal (δ 7.59 ppm, RCH = N, d, J = 4.5 Hz). Following verification of imine formation, reduction was performed under standard conditions to give the secondary amine 6. The next step in the synthesis of the 3,5-piperidinediol involved liberation of the masked aldehyde followed by an intramolecular reductive amination. Acid hydrolysis of the remaining acetal led to an equilibrium mixture of compounds: the two anomers of hemiacetal 7 and the open chain aldehyde 8. Reduction of the cyclic iminium ion led to formation of piperidine 2. From the crystal structure of compound 2, shown in Fig 3A, the piperidine-2,4-diol ring is symmetric along the ring plane passing through N and C4. Each hydroxyl group of the diol adopts an equatorial orientation. 1H NMR analysis of compound 2 confirmed the symmetry of the piperidine, showing three pairs of equivalent protons, Fig 3B: Hx (H2e and H6e); Hy (H2a and H6a); and Hz (H3 and H5). A doublet of doublet peak was observed at δ 2.94 correspond- ing to H2e, H6e with a large geminal coupling constant (J2e,2a and J6e,6a = 10.4 Hz) and a small vicinal coupling constant (J2e,3a and J6e,5a = 3.3 Hz). This small vicinal coupling constant places H3 and H5 in axial positions, consistent with the equatorial orientation of the hydroxyl groups in the crystal structure. The reaction was then incubated in an Imperial III incubator (LabLine) at 37˚C and transferred briefly to the NMR probe at room temperature periodically to monitor the reaction. 100 µg G-KPV-NH2 (12a); 200 µg K(G)PV-NH2 (12b) and 200 µg Ac-KPV-NH2 (12c) PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Chemical synthesis (a) ORTEP of the hydrate of compound 2 as determined by X-ray crystallography; (b) P pairs of equivalent hydrogens Hx, Hy, and Hz. https://doi.org/10.1371/journal.pone.0199686.g003 https://doi.org/10.1371/journal.pone.0199686.g003 https://doi.org/10.1371/journal.pone.0199686.g003 an aldehyde that underwent reductive aminocyclization to form the 3,5-dihydroxypiperidine ring at the α and ε-positions, respectively. Ac-KPV-NH2 (12c) was synthesized from com- pound 10a, in order to compare the activities of the two derivatives 12a and 12b with the activ- ity of 12c as previously reported in the literature. Fmoc deprotection of 12a, acetylation of the resulting amine with acetic anhydride, and Boc deprotection with TFA afforded 12c. an aldehyde that underwent reductive aminocyclization to form the 3,5-dihydroxypiperidine ring at the α and ε-positions, respectively. Ac-KPV-NH2 (12c) was synthesized from com- pound 10a, in order to compare the activities of the two derivatives 12a and 12b with the activ- ity of 12c as previously reported in the literature. Fmoc deprotection of 12a, acetylation of the resulting amine with acetic anhydride, and Boc deprotection with TFA afforded 12c. Chemical synthesis Having confirmed the structure and determined reaction conditions for “glycoalkylation,” similar conditions were utilized to modify the α- or ε-amino groups of the lysine residue in the tripeptide H-KPV-NH2. For site-specific modification, the lysine building block in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 7 / 14 Reductive "glycoalkylation" of KPV tripeptide synthesis had orthogonal protecting groups For α modification the protecting Fig 2. Reaction scheme 1. Synthesis of ε-glycoalkylated Ts-Lys-OMe (2). https://doi.org/10.1371/journal.pone.0199686.g002 Fig 2. Reaction scheme 1. Synthesis of ε-glycoalkylated Ts-Lys-OMe (2). Fig 2. Reaction scheme 1. Synthesis of ε-glycoalkylated Ts-Lys-OMe (2). https://doi.org/10.1371/journal.pone.0199686.g002 https://doi.org/10.1371/journal.pone.0199686.g002 tripeptide synthesis had orthogonal protecting groups. For α-modification, the protecting groups were Boc at the ε-position and Fmoc at the α-position. The protecting groups were switched for the ε-modification (Fig 4). tripeptide synthesis had orthogonal protecting groups. For α-modification, the protecting groups were Boc at the ε-position and Fmoc at the α-position. The protecting groups were switched for the ε-modification (Fig 4). Three derivatives of H-KPV-NH2 were prepared to test for activity against S. aureus and stability toward proteases: αG-KPV-NH2 (12a), H-K(εG)PV-NH2 (12b) and Ac-KPV-NH2 (12c). The abbreviation G represents the dihydroxylated piperidine in place of the α-NH2 or ε-NH2 group in compounds 12a and 12b respectively. The end-capped tripeptide Ac- KPV-NH2 (12c) was intended as a positive control. For both the α- and ε-modification, Fmoc deprotection of the tripeptide (10a or 10b) led to the free amine at the α- or ε-position, respec- tively. Each free amine was condensed with aldehyde 4 by reductive alkylation to afford tripep- tides 11a and 11b (Fig 5, with the sugar being designated as G’ in the furan form). The 1,2-acetonide functionality in compounds 11a and 11b was cleaved in TFA-water, liberating 8 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV Fig 3. Structure of compound 2. (a) ORTEP of the hydrate of compound 2 as determined by X-ray crystallography; (b) Piperidine ring of compound 2 showing the three pairs of equivalent hydrogens Hx, Hy, and Hz. of the hydrate of compound 2 as determined by X-ray crystallography; (b) Piperidine ring of compound 2 showing the three Fig 3. Structure of compound 2. (a) ORTEP of the hydrate of compound 2 as determined by X-ray crystallography; (b) Piperidine ring of compound 2 showing the three pairs of equivalent hydrogens Hx, Hy, and Hz. Fig 3. Structure of compound 2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Biological assays The sensitivity of various bacterial strains was tested using the agar diffusion method [33–36] with the compounds 12a-c that we had synthesized. Details are provided in S1 File. Whilst the positive control, ampicillin, showed inhibition of bacterial growth, no inhibition zones were observed for the negative control, water, and compounds 12a-c. Fig 4. Reaction scheme 2. Synthesis of H-KPV-NH2 derivatives 10a and 10b. https://doi.org/10.1371/journal.pone.0199686.g004 Fig 4. Reaction scheme 2. Synthesis of H-KPV-NH2 derivatives 10a and 10b. Fig 4. Reaction scheme 2. Synthesis of H-KPV-NH2 derivatives 10a and 10b. https://doi.org/10.1371/journal.pone.0199686.g004 Fig 4. Reaction scheme 2. Synthesis of H-KPV-NH2 derivatives 10a and 10b. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 9 / 14 Reductive "glycoalkylation" of KPV Fig 5. Reaction scheme 3. Synthesis of compounds 12a, 12b, and 12c. htt //d i /10 1371/j l 0199686 005 Fig 5. Reaction scheme 3. Synthesis of compounds 12a, 12b, and 12c. Fig 5. Reaction scheme 3. Synthesis of compounds 12a, 12b, and 12c. https://doi.org/10.1371/journal.pone.0199686.g005 To verify the activity of Ac-KPV-NH2 (12c), the peptide was purchased from Bachem (Bubendorf, Switzerland), the same supplier as was used by Charnley et al. [19], following pro- tocols similar to those reported by Cutuli et al. [12] and Charnley et al. [19]. Details are pro- vided in S1 File. Again, no inhibition of bacterial growth was observed. These results were surprising and disappointing because Ac-KPV-NH2 (12c) has been reported as an anti-microbial agent [12,19,37]. The original report by Catania and cowork- ers in 2000 described activity against both Staphylococcus aureus and Candida albicans, with effects over a broad range of concentrations, including “the physiological (picomolar) range [12].” In 2009, there was debate over the original report of antifungal activity [38,39]. Singh and Mukhopadhyay independently described the 90% staphylocidal activity of Ac- KPV-NH2 (12c) at micromolar concentrations and 50% activity in the nanomolar concen- tration range [37]. Charnley et al. reported broad range activity against both Gram-positive and Gram-negative bacteria [19]. On the other hand, without further discussion, Grieco et al. stated that “these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use [40].” Lau et al. recently performed direct compari- sons of 30 ultra-short antimicrobial peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans [41]. Their study included five tripeptides, Ac-KPV-NH2 (12c) amongst them; none of the tripeptides were active against the panel of skin pathogens, indicating MICs greater than 100 μM. PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Biological assays These results were surprising and disappointing because Ac-KPV-NH2 (12c) has been reported as an anti-microbial agent [12,19,37]. The original report by Catania and cowork- ers in 2000 described activity against both Staphylococcus aureus and Candida albicans, with effects over a broad range of concentrations, including “the physiological (picomolar) range [12].” In 2009, there was debate over the original report of antifungal activity [38,39]. Singh and Mukhopadhyay independently described the 90% staphylocidal activity of Ac- KPV-NH2 (12c) at micromolar concentrations and 50% activity in the nanomolar concen- tration range [37]. Charnley et al. reported broad range activity against both Gram-positive and Gram-negative bacteria [19]. On the other hand, without further discussion, Grieco et al. stated that “these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use [40].” Lau et al. recently performed direct compari- sons of 30 ultra-short antimicrobial peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans [41]. Their study included five tripeptides, Ac-KPV-NH2 (12c) amongst them; none of the tripeptides were active against the panel of skin pathogens, indicating MICs greater than 100 μM. 10 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV While the compounds did not show any antimicrobial activity under the variety of condi- tions tested, the impact of glycoalkylation could be assessed vis-à-vis improved stability to pro- teolytic enzymes. Pronase is a commercially-available cocktail of enzymes used routinely to digest proteins to their constituent amino acids [42]. Each of the three peptides (12a-c) was treated with pronase, and the composition of the mixture monitored by 1H NMR spectroscopy (see S1 File). The “parent” peptide, Ac-KPV-NH2 (12c) was degraded to its three constituent amino acids within 24 hours. The signal attributable to Hα of the proline (P) residue shifted upfield by about 0.2 ppm, with a concomitant change from an apparent triplet (in the tripep- tide) to a doublet of doublets in the free amino acid, consistent with a change in conformation of the pyrrolidine ring. The signal attributable to Hα of the valine (V) residue shifted upfield by nearly 0.5 ppm. These upfield shifts are in accordance with removal of the electron-with- drawing N-acyl group in each case. The α-glycoalkylated tripeptide (12a) was completely sta- ble under the conditions of the pronase experiment. Less clear-cut was the behavior of the ε- glycoalkylated tripeptide (12b). Biological assays The peptide appears to be stable, with Hα signals of both P and V remaining well-defined and with the same chemical shift and the molecular ion was still evident in the mass spectrum. The broad signals assigned to Hε and the protons of the piperi- dine ring reflect the dynamic nature of the Lys side chain. Upon prolonged incubation with the mixture of proteolytic enzymes, perhaps undergoing autoproteolysis, these signals gener- ally moved upfield and became broader. Conclusions We have developed the reaction chemistry to produce regioselectively glycoalkylated peptides. Specifically, reductive amination of D-glucose-derived aldehyde 4 with either the α- or ε- amino group of lysine residues gave a secondary amine. Upon liberation of the aldehyde derived from the anomeric carbon of glucose, an intramolecular reductive amination could be induced to afford a dihydroxylated piperidine moiety. Acknowledging that the impact of such a modification on biological activity is unlikely to be generalizable to peptides of assorted clas- ses, we sought to study the effect glycoalkylation on the antibacterial activity of Ac-KPV-NH2 (12c). Unfortunately, during the course of our work, controversy arose in the literature sur- rounding its alleged antimicrobial activity. Like others, we were unable to reproduce the results under a number of assay conditions. Nevertheless, we have shown that the internal peptide bonds of the glycoalkylated tripeptides, 12a and 12b, are stable over several days to pronase. Future work will involve application of the glycoalkylation concept to other sequences and we trust that this approach will appeal to others interested in improving the bioavailability, solu- bility and half-life of lysine-containing peptides. Acknowledgments The authors thank Dr. Frank Fronczek, Dr. Thomas Weldeghiorghis and Dr. Fengli Zhang, and Ms. Connie David for their help with crystal structure, NMR, and MS analysis, respec- tively. ACS thanks the Department of Chemistry, Louisiana State University, for her graduate research and teaching assistantships. Supporting information S1 File. Procedures and NMR spectra. Experimental procedures for the synthesis of aldehyde 4, 1H and 13C NMR spectra for the compounds involved in the synthesis of aldehyde 4, and 1H and 13C NMR spectra for the compounds in reaction schemes 1 (Fig 2), 2 (Fig 4), and 3 (Fig 5), computing details, atom coordinates, bond lengths and angles from the X-ray structure deter- mination of compound 2; NMR spectra over the timecourse of the pronase-stability experi- ments. S2 File. Crystallographic information file. Crystallographic information file for the hydrate of compound 2 as determined by X-ray crystallography. Data has been deposited at the CCDC 11 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0199686 June 28, 2018 Reductive "glycoalkylation" of KPV with deposition number 1825648. (CIF) Conceptualization: Megan A. Macnaughtan. Conceptualization: Megan A. Macnaughtan. Data curation: Abigael C. Songok, Pradip Panta. Formal analysis: Abigael C. Songok, Pradip Panta, William T. Doerrler, Megan A. Macnaugh- tan, Carol M. Taylor. Investigation: Abigael C. Songok, Pradip Panta, William T. Doerrler. Investigation: Abigael C. Songok, Pradip Panta, William T. Doerrler. Methodology: William T. Doerrler, Carol M. Taylor. Methodology: William T. Doerrler, Carol M. Taylor. Project administration: William T. Doerrler, Megan A. Macnaughtan, Carol M. Taylor. Resources: William T. Doerrler, Megan A. Macnaughtan, Carol M. Taylor. Supervision: William T. Doerrler, Megan A. Macnaughtan, Carol M. Taylor. Validation: Abigael C. Songok. Visualization: Abigael C. Songok, Pradip Panta. 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https://openalex.org/W2110283976	https://academicjournals.org/journal/AJPS/article-full-text-pdf/A1D698142813.pdf	English	null	Variability, heritability and genetic advance in upland cotton (Gossypium hirsutum L.)	African journal of plant science	2,014	cc-by	4,581	INTRODUCTION advance on the genetic architecture of 16 yield, yield components and fibre quality traits. Cotton is an important fibre crop of global importance which is grown in tropical and subtropical regions of more than 60 countries of the world. Despite threat from synthetic fibre or manmade fibre, cotton retains its reputation as “queen of the fibre plants‟‟. For multiple uses of lint and by- products, cotton is also referred to as “white gold”. In any crop improvement programme, knowledge on nature of gene action and inheritance of traits is essential so as to choose a suitable breeding methodology in crop improve- ment (Vineela et al., 2013). Development of an effective breeding programme depends on the existence of genetic variability for various economic characters in the gene pool. Selection is effective only when there is enough magnitude of variability in the breeding population. An understanding of precise magnitude of variability present in a population is important in formulating the most appro- priate breeding technique for improvement of various characters. The present investigation was carried out with 54 Gossypium hirsutum lines of diverse origin to estimate their per se performance, variability, heritability and genetic Full Length Research Paper Variability, heritability and genetic advance in upland cotton (Gossypium hirsutum L.) R. Dhivya, P. Amalabalu, R. Pushpa* and D. Kavithamani Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore-641 003, India. Accepted 23 September, 2013 The analysis of variance study indicated the presence of significant difference among all the traits in Gossypium hirsutum accessions. The highest phenotypic coefficient of variation (GCV) and genotypic coefficient of variation (GCV) were recorded by seed index, plant height, lint index and boll weight. Genotypic co-efficient of variation had a similar trend as PCV. High heritability along with high genetic advance was observed in traits viz., number of sympodia per plant, single plant yields, seed index and micronaire value. The combinations of high heritability with high genetic advance will provide a clear base on the reliability of that particular character in selection of variable entries. Based on per se performance, the accessions MCU5, TCH1715, TCH1716 and G cot 16 were identified as potential donors for single plant yield (g), number of bolls per plant, 2.5% span length (mm) and bundle strength (g/tex). So these accessions may be utilized for crossing programme to improve a particular character in crop improvement. Key words: Genetic variability, heritability, genetic advance, upland cotton. African Journal of Plant Science African Journal of Plant Science MATERIALS AND METHODS The study was conducted in the Department of Cotton, Tamil Nadu Agricultural University, Coimbatore during winter 2010. Fifty four G. hirsutum cotton genotypes were planted in randomized block design with two replications. Uniform spacing of 90 x 45 cm and all the recommended field operations were carried out. In each replication five competitive plants were randomly selected and observations were recorded for 16 characters viz., days to 50% flowering, plant height (cm), internode length (cm), number of sympodia/plant, number of ovules/plant, number of bolls/plant, boll weight (g), number of seeds/plant, seed setting percentage, seed cotton yield/plant, lint index, seed index, ginning outturn (%), 2.5% span length (mm), bundle strength (g/tex) and fibre fineness. Analysis of variance was carried out statistically utilizing the mean values (Panse and Sukhtame, 1995). The phenotypic and genotypic coefficient of variation was estimated using the formula suggested by Burton (1952) and expressed in percentage. The phenotypic and genotypic variances were calculated by utilizing the Corresponding author. E-mail: pushpa.saravanan@gmail.com. Afr. J. Plant Sci. 2 Table 1. Mean and range performance for different characters among G. hirsutum accessions. Characters Mean Minimum Maximum Days to 50% flowering 56.6 50.0 65.0 Plant height (cm) 108.8 78.0 147.0 Inter node length(cm) 5.4 4.0 6.5 No of Sympodia per plant 19.6 14.0 27.0 No of bolls per plant 23.6 19.5 38.5 Boll weight (g) 4.3 12.4 5.5 No of seeds per boll 27.4 23.0 35.5 No of ovules per flower 30.7 24.0 38.0 Seed setting percentage 89.6 71.5 100.0 Single plant yield (g / plant) 57.6 31.7 91.6 Lint index 5.7 4.0 7.9 Seed index 9.6 6.4 16.3 Ginning outturn (%) 37.3 30.8 46.1 2.5 % span length (mm) 28.4 24.3 34.0 Bundle strength (g / tex) 20.4 16.2 23.7 Micronaire value 4.3 3.5 5.2 (g/tex). These accessions may be exploited for further improvement of the above traits by breeding programmes. Regarding the identifica- tion of donor for specific trait, the highest performer of that particular trait can be considered. Among the accessions, TCH 1715 (5.5 g of boll weight), RB 488 (46.1% of GOT) were expressed the highest per se for that particular traits. The culture TCH 1710 (34 mm, 23.1g/tex) recorded the highest 2.5% span length. So these acces- sions may be utilized for crossing programme to improve that particular character in crop improve- ment. MATERIALS AND METHODS Anjali was found to be compact type which can be utilized in development of genotype suitable for high density planting. characters studied (Table 2) and infers existence of considerable genetic diversity among genotypes. Phenotypic variance, genotypic variance, phenotypic coefficient of variation, genotypic coefficient of variation, heritability in broad sense and genetic advance as percentage of mean which were estimated for 16 characters are shown in Table 3. The knowledge of nature and magnitude of variability available in the genotypes for different characters is an important prerequisite for making simultaneous selection over more number of characters to bring remarkable improvement in cotton. The analysis of variance study indicated the presence of significant difference among all the traits in the accessions. The heritable (genotypic) variation is usually masked by non-heritable variation creating respective mean sum of square from variance table (Lush, 1940). Heritability, expected genetic advance and genetic gain in the broad sense was calculated according to the formula suggested by Johanson et al. (1955). RESULTS AND DISCUSSION hirsutum accessions PCV was higher than the GCV for all characters. From this, we can understand that the environment plays a major role on expression of all these traits leading to increase in the PCV more than GCV. of sympodia per plant (12.1), lint index (11.2) and number of bolls per plant (10.9), plant height (10.2). The lowest PCV (4.5%) and GCV (3.5) values was observed in days to 50% flowering. In the present study, there was a close correspond- dence between phenotypic and genotypic variance for days to 50% flowering, inter node length, boll weight, single plant yield, lint index, micronaire value and 2.5% span length indicating less environmental influence. But plant height, number of sympodia per plant, number of bolls per plant, number of seeds per boll, seed setting percentage and ginning outturn showed higher variation indicating the influence of environment on these characters. Since the variations are influenced by the magnitude of the units of measurements of different traits, a measure of coefficient of variation which is independent of the unit of measurement is more useful in comparing between populations. In G. hirsutum accessions PCV was higher than the GCV for all characters. From this, we can understand that the environment plays a major role on expression of all these traits leading to increase in the PCV more than GCV. The highest PCV and GCV estimates were recor- ded for single plant yield indicating the presence of significant genetic variability in this character. Selection pressure can be applied on this character to isolate promising genotypes. Similar observations in cotton was reported by Dheva and Potdukhe (2002) and Preetha and Raveendran (2007). difficulty in exercising selection. Hence it becomes necessary to partition overall variability into heritable and non-heritable components to enable the breeders to plan for proper breeding programme. The plant height recorded the highest value for phenotypic variance (342.6) and the single plant yield recorded highest genotypic variance (219.9). The micronaire value exhibited the lowest phenol-typic variance (0.2) and in case of genotypic variance, the traits viz., inter node length, boll weight and micronaire value had recorded the lowest value (0.1). The co-efficient of phenotypic and genotypic variance were calculated for all the characters under study. The PCV ranged from 4.5 (days to 50% flowering) to 25.9% (single plant yield). RESULTS AND DISCUSSION The per se performance of yield and fibre quality characters were recorded on 54 germplasm accessions and the range of variations observed in respect of all the 16 traits studied are presented in the Table 1. Based on per se performance, the accessions such as MCU 5, TCH1715, TCH1716 and G cot 16 in G. hirsutum were identified as potential donors which recorded highest mean values for single plant yield, number of bolls per plant, 2.5% span length (mm) and bundle strength The analysis of variance showed highly signi- ficant differences among genotypes for all the Dhivya et al. 3 able 2. Analysis of variance for the different characters among the germplasm accessions of G. hirsutum. Source of variation Degrees of freedom Days to 50% flowering Plant height (cm) nter node length (cm) Number of sympodia / plant Number of bolls / plant Boll weight (g) Number of sees / boll Number of ovule / flower Seed setting percentage Single plant yield (g) Lint index Seed index Ginning out turn (%) 2.5 % span length (mm) Bundle strength(g /tex) Micronaire value Genotypes 53.0 10.4* 466.6 0.7 14.1 18.3 0.6* 10.8 17.1 90.4 441.9 1.3 4.6 21.1 10.2 6.1 0.3 Error 53.0 2.7 218.6 0.4 2.8 5.0 0.3 5.50 6.5 40.1 2.1 0.5 1.2 10.9 0.2 0.3 0.1 ,* Significant at 5 and 1% levels, respectively. difficulty in exercising selection. Hence it becomes necessary to partition overall variability into heritable and non-heritable components to enable the breeders to plan for proper breeding programme. The plant height recorded the highest value for phenotypic variance (342.6) and the single plant yield recorded highest genotypic variance (219.9). The micronaire value exhibited the lowest phenol-typic variance (0.2) and in case of genotypic variance, the traits viz., inter node length, boll weight and micronaire value had recorded the lowest value (0.1). The co-efficient of phenotypic and genotypic variance were calculated for all the characters under study. The PCV ranged from 4.5 (days to 50% flowering) to 25.9% (single plant yield). The highest PCV was followed by seed index (17.7%), plant height (17.0%), lint index (17.0%) and boll weight (15.4%). Genotypic co-efficient of variation had a similar trend as PCV. The range varied from 3.5 (Days to fifty percent flowering) to 25.8 (Single plant yield). RESULTS AND DISCUSSION Since the variations are influenced by the magnitude of the units of measurements of different traits, a measure of coefficient of variation which is independent of the unit of measurement is more useful in comparing between populations. In G. hirsutum accessions PCV was higher than the GCV for all characters. From this, we can understand that the environment plays a major role on expression of all these traits leading to increase in the PCV more than GCV. The highest PCV and GCV estimates were reco ded for single plant yield indicating the presenc of significant genetic variability in this characte Selection pressure can be applied on th character to isolate promising genotypes. Simil observations in cotton was reported by Dheva an Potdukhe (2002) and Preetha and Raveendra (2007). Moderate PCV and GCV estimates we noticed for the characters such as plant heigh number of sympodia per plant, number of bo per plant, lint index and seed index. Girase an Mehetne (2002) and Harshal (2010) also reporte the moderate PCV and GCV in various traits wi the suggestion that these characters can b improved though rigorous selection. The characte such as days to 50% flowering, 2.5% span leng and micronaire value exhibited low PCV and GC which indicated that the breeds should go f source of high variability for these characters make improvement. Similar suggestion we given by Kowsalya and Raveendran (1996), D Thi Ha An et al. ((2006) in their conclusion. In population, the observed variability is of sympodia per plant (12.1), lint index (11.2) and number of bolls per plant (10.9), plant height (10.2). The lowest PCV (4.5%) and GCV (3.5) values was observed in days to 50% flowering. In the present study, there was a close correspond- dence between phenotypic and genotypic variance for days to 50% flowering, inter node length, boll weight, single plant yield, lint index, micronaire value and 2.5% span length indicating less environmental influence. But plant height, number of sympodia per plant, number of bolls per plant, number of seeds per boll, seed setting percentage and ginning outturn showed higher variation indicating the influence of environment on these characters. Since the variations are influenced by the magnitude of the units of measurements of different traits, a measure of coefficient of variation which is independent of the unit of measurement is more useful in comparing between populations. In G. RESULTS AND DISCUSSION The maximum genotypic coefficient of variation (GCV) was observed for single plant of sympodia per plant (12.1), lint index (11.2) and number of bolls per plant (10.9), plant height (10.2). The lowest PCV (4.5%) and GCV (3.5) values was observed in days to 50% flowering. In the present study, there was a close correspond- dence between phenotypic and genotypic variance for days to 50% flowering, inter node length, boll weight, single plant yield, lint index, micronaire value and 2.5% span length indicating less environmental influence. But plant height, number of sympodia per plant, number of bolls per plant, number of seeds per boll, seed setting percentage and ginning outturn showed higher variation indicating the influence of environment on these characters. Since the variations are influenced by the magnitude of the units of measurements of different traits, a measure of coefficient of variation which is independent of the unit of measurement is more useful in comparing between populations. In G. hirsutum accessions PCV was higher than the GCV for all characters. From this, we can understand that the environment plays a major role on expression of all these traits The highest PCV and GCV estimates were recor- ded for single plant yield indicating the presence of significant genetic variability in this character. Selection pressure can be applied on this character to isolate promising genotypes. Similar observations in cotton was reported by Dheva and Potdukhe (2002) and Preetha and Raveendran (2007). Moderate PCV and GCV estimates were noticed for the characters such as plant height, number of sympodia per plant, number of bolls per plant, lint index and seed index. Girase and Mehetne (2002) and Harshal (2010) also reported the moderate PCV and GCV in various traits with the suggestion that these characters can be improved though rigorous selection. The characters such as days to 50% flowering, 2.5% span length and micronaire value exhibited low PCV and GCV which indicated that the breeds should go for source of high variability for these characters to make improvement. Similar suggestion were given by Kowsalya and Raveendran (1996), Do Thi Ha An et al. ((2006) in their conclusion. RESULTS AND DISCUSSION In a Source of variation Degrees of freedom Days to 50% flowering Plant height (cm) nter node length (cm) Number of sympodia / plant Number of bolls / plant Boll weight (g) Number of sees / boll Number of ovule / flower Seed setting percentage Single plant yield (g) Lint index Seed index Ginning out turn (%) 2.5 % span length (mm) Bundle strength(g /tex) Micronaire value Genotypes 53.0 10.4 466.6 0.7 14.1 18.3** 0.6* 10.8 17.1 90.4 441.9 1.3 4.6 21.1 10.2 6.1 0.3 Error 53.0 2.7 218.6 0.4 2.8 5.0 0.3 5.50 6.5 40.1 2.1 0.5 1.2 10.9 0.2 0.3 0.1 ,* Significant at 5 and 1% levels, respectively. culty in exercising selection. Hence it becomes essary to partition overall variability into able and non-heritable components to enable breeders to plan for proper breeding ramme. The plant height recorded the highest e for phenotypic variance (342.6) and the e plant yield recorded highest genotypic ance (219.9). The micronaire value exhibited owest phenol-typic variance (0.2) and in case enotypic variance, the traits viz., inter node th, boll weight and micronaire value had rded the lowest value (0.1). The co-efficient of notypic and genotypic variance were ulated for all the characters under study. The ranged from 4.5 (days to 50% flowering) to % (single plant yield). The highest PCV was wed by seed index (17.7%), plant height 0%), lint index (17.0%) and boll weight 4%). Genotypic co-efficient of variation had a ar trend as PCV. The range varied from 3.5 ys to fifty percent flowering) to 25.8 (Single t yield). The maximum genotypic coefficient of ation (GCV) was observed for single plant (25.8) followed by seed index (13.5), number of sympodia per plant (12.1), lint index (11.2) and number of bolls per plant (10.9), plant height (10.2). The lowest PCV (4.5%) and GCV (3.5) values was observed in days to 50% flowering. In the present study, there was a close correspond- dence between phenotypic and genotypic variance for days to 50% flowering, inter node length, boll weight, single plant yield, lint index, micronaire value and 2.5% span length indicating less environmental influence. But plant height, number of sympodia per plant, number of bolls per plant, number of seeds per boll, seed setting percentage and ginning outturn showed higher variation indicating the influence of environment on these characters. RESULTS AND DISCUSSION The highest PCV was followed by seed index (17.7%), plant height (17.0%), lint index (17.0%) and boll weight (15.4%). Genotypic co-efficient of variation had a similar trend as PCV. The range varied from 3.5 (Days to fifty percent flowering) to 25.8 (Single plant yield). The maximum genotypic coefficient of variation (GCV) was observed for single plant yield (25.8) followed by seed index (13.5), number Moderate PCV and GCV estimates were noticed for the characters such as plant height, number of sympodia per plant, number of bolls per plant, lint index and seed index. Girase and Mehetne (2002) and Harshal (2010) also reported the moderate PCV and GCV in various traits with the suggestion that these characters can be improved though rigorous selection. The characters such as days to 50% flowering, 2.5% span length and micronaire value exhibited low PCV and GCV which indicated that the breeds should go for source of high variability for these characters to make improvement. Similar suggestion were given by Kowsalya and Raveendran (1996), Do Thi Ha An et al. ((2006) in their conclusion. In a population, the observed variability is a Afr. J. Plant Sci. 4 4 Table 3. Components of variance for yield and fibre quality characters of G. hirsutum accessions. Table 3. Components of variance for yield and fibre quality characters of G. hirsutum accessions. RESULTS AND DISCUSSION High genetic advance, genetic gain and heritability were recorded for number of sympodia per plant, single plant yield, seed index, micronaire value and 2.5% span length indicated that selection can be resorted for the improvement of these characters in the future crop improvement programmes. %), number of sympodia per plant (26.2 %), number of bolls per plant (21.8 %). The lowest value of 7.0% was observed in the trait days to 50% flowering. High heritability along with high genetic advance was observed in traits viz., number of sympodia per plant, single plant yields, seed index and micronaire value in G. hirsutum (Do Thi Ha An et al., 2008). These traits are highly reliable during selection. High heritability combined with moderate genetic advance was found in the 2.5% span length. It was in accordance with of Muhammad et al. (2004). Among the study materials some of the accessions were identified as potential donors for the improvement of different characters (Table 4). The accessions with high mean performance are generally preferred for all the traits except days to 50% flowering, since earliness is the preferred attribute and early flowering was taken into consideration. From the results of the present study, it can be concluded that direct selection can be done for most of the yield attributing traits since it exhibited high genetic variability and high range of variation. A high PCV over GCV for the characters studied indicated that environment influences the expression of these characters under study. High genetic advance, genetic gain and heritability were recorded for number of sympodia per plant, single plant yield, seed index, micronaire value and 2.5% span length indicated that selection can be resorted for the improvement of these characters in the future crop improvement programmes. combined measure of genetic and environment causes, where as the genetic variability is the only estimate heritable from generation to generation. However, a measure of heritability alone does not give an idea about the expected gain in the next generation but it has to be considered in conjunction with genetic advance. The traits which expressed high heritability and high genetic advance as percentage of mean could be used as a powerful tool in selection process. According to Panes and Sukhatme (1995) such characters were found to be governed by additive genes and had minimum environment influence. The heritabi-lity estimates ranged from 23.5 (internode length) to 89.1% (single plant yield). RESULTS AND DISCUSSION Characters Phenotypic variance Genotypic variance PCV (%) GCV (%) h2 (%) A GA as percentage of mean Days to 50% flowering 6.6 3.9 4.5 3.5 58.5 4.0 7.0 Plant height (cm) 342.6 124.0 17.0 10.2 36.2 17.7 16.3 Inter node length(cm) 0.5 0.1 13.5 6.6 23.5 0.5 8.4 No of sympodia per plant 8.5 5.7 14.8 12.1 66.9 5.1 26.2 No of bolls per plant 11.7 6.7 14.4 10.9 57.2 5.2 21.8 Days to 50% flowering 6.6 3.9 4.5 3.5 58.5 4.0 7.0 Plant height (cm) 342.6 124.0 17.0 10.2 36.2 17.7 16.3 Boll weight (g) 0.4 0.1 15.4 8.4 29.8 0.5 12.1 No of seeds per boll 8.2 2.6 10.4 6.0 32.5 2.5 9.0 No of ovules per flower 11.8 5.3 11.2 7.5 44.5 4.0 13.1 Seed setting percentage 65.3 25.1 9.0 5.6 38.5 8.2 9.2 Single plant yield (g / plant) 222.0 219.9 25.9 25.8 89.1 39.0 27.3 Lint index 0.9 0.4 17.0 11.2 43.9 1.1 19.6 Seed index 2.9 1.7 17.7 13.5 58.0 2.6 27.1 Ginning outturn (%) 16.0 5.1 10.7 6.0 31.6 3.3 9.0 2.5 % span length (mm) 5.2 5.0 8.0 7.9 96.2 5.8 20.4 %), number of sympodia per plant (26.2 %), number of bolls per plant (21.8 %). The lowest value of 7.0% was observed in the trait days to 50% flowering. High heritability along with high genetic advance was observed in traits viz., number of sympodia per plant, single plant yields, seed index and micronaire value in G. hirsutum (Do Thi Ha An et al., 2008). These traits are highly reliable during selection. High heritability combined with moderate genetic advance was found in the 2.5% span length. It was in accordance with of Muhammad et al. (2004). Among the study materials some of the accessions were identified as potential donors for the improvement of different characters (Table 4). The accessions with high mean performance are generally preferred for all the traits except days to 50% flowering, since earliness is the preferred attribute and early flowering was taken into consideration. From the results of the present study, it can be concluded that direct selection can be done for most of the yield attributing traits since it exhibited high genetic variability and high range of variation. A high PCV over GCV for the characters studied indicated that environment influences the expression of these characters under study. RESULTS AND DISCUSSION The high heritability estimates of 89.1% were recorded by single plant yield followed by 2.5% span length (96.2 %) and bundle strength (91.8%) where as the low heritability was observed in inter node length (23.5%). The high heritability was registered in the traits viz., number of sympodia per plant, single plant yield, seed index, 2.5% span length and bundle strength. The inter node length exhibited low heritability in this investigation. For efficient selection, we cannot completely depend on heritability alone. The combinations of high heritability with high genetic advance will provide a clear base on the reliability of that particular character in selection of variable entries. The genotypic advance as percent of mean for 16 traits ranged from 7.0 to 27.3%. The higher genetic advance as percent of mean was recorded by single plant yield (27.3%) followed by seed index (27.1 Dhivya et al. 5 Table 4. Potential donors for yield and fibre quality traits. S/N Characters Potential accessions 1 Days to 50% flowering MCU-7, SVPR-3, TCH-1716, SVPR-2 2 Plant Height SCS 102, T CH 1715, Okra narrow 3 Inter node length MCU 5, RAC9740, SCS 102, F-1946 4 No. of Sympodia PSCL VII, Okra narrow, RHC 1694, SCS 102 5 No. of ovules NDLH 1588, TCH 1608 6 No. of Bolls Okra Narrow, G cot-16, F-1946 7 Boll weight(g) TCH 1715, MCU 13, LRA 5166 8 No. of seeds per boll NDLH 1588, SVPR 4 9 Seed setting % MCU 12, MCU 13, KC 3, TCH 1710 10 Single plant yield MCU 5, MCU 12, Surabhi, SVPR 2, SVPR 4, KC 2, TCH 1715, TCH 1716, TCH 1732, TCH 1734, TCH 1744, G. COT-16, CCH 2117, Pusa 953, ARB 2001, RHC 1694, LH 1961, RB 488, GISV201, MHIS-5, MHIS-7, Sara 2,Giza 1461, Sumangala 11 Lint index GISV 201, RAC 9544 12 Seed index SVPR 4, L-752, TCH 1715 13 Ginning Out Turn RB 488, TCH 1705 14 2.5 % span length TCH 1608, CNH 152, TCH 1705, RAC 9740, TCH 1732, MCU 5, MCU 13, TCH 1710, TCH 1715, TCH 1716 15 Bundle strength TCH 1710, TCH 1715, MCU 5, TCH 1732, TCH 1734, TCH 1744 16 Micronaire value MHIS-7, MCU 7, CCH 2117, NDLH 1588 Table 4. Potential donors for yield and fibre quality traits. 16 REFERENCES REFERENCES Kowsalya R, Raveendran TS (1996). Genetic variability and D2 analysis in upland cotton. Crop Res. 12(1):36-42. Burton GW (1952). Quantitative inheritance in grasses. Proceedings of the Sixth international Congress, pp.277-283. Lush JL (1940). Intra-sire correlation and regression of offspring on dams as a method of estimating heritability of characters in Proc. Am. Soc. Anim. Prod. 33:293-301. Dheva NG, Potdukhe NR (2002). Studies on variability and correlations in upland cotton for yield and its components. J. Indian Soc. Cotton Improv, pp.148-152. Muhammad I, Muhammad AC, Abdul J, Muhammad ZI, Muhammad-ul- Hassan and Noor-ul-Islam (2004). Inheritance of Earliness and other Characters in Upland Cotton. J. Biol. Sci. 3(6):585-590. p pp Do Thi Ha An, Ravikesavan R (2006). Genetic diversity in cotton (Gossypium sp): In: national conference on plant sciences research and development, APSI scientist meet, PSG CAS, Coimbatore, pp. 11-13. Panes VG, Sukhatme PV (1995). Statistical methods for agricultural workers, 3rd Ed., ICAR, New Delhi, p.58. Preetha S, Raveendran TS (2007). Genetic variability and association studies in three different morphological groups of cotton (Gossypium hirsutum L.) Asian J. Plant Sci. 6(1):122-128. Do Thi Ha An, Ravikesavan R, Iyanar K (2008). Genetic advance and heritability as a selection index for improvement of yield and quality in cotton. J. Cotton Res. Dev. 22(1):14-18. Vineela N, Samba Murthy JSV, Ramakumar PV, Ratna KS (2013). Variability Studies for Physio Morphological and Yield Components Traits in American Cotton (Gossypium hirsutum L). J. Agric. Vet. Sci. 4(3):07-10. ( ) Girase VS, Mehetre SS (2002). Correlation and Path analysis in cotton. J. Cotton Res. Dev. 16(1):1-7. ( ) Harshal EP (2010). Variability and correlation analysis by using various quantitative traits in released Bt cotton hybrids. J. Cotton Res. Dev. 24(2):141-144. Johanson AW, Robinson HF, Comstock RF (1955). Estimates of genetic and environmental variability in soybean. Agric. J. 47:314- 318.
https://openalex.org/W2905230218	https://www.matec-conferences.org/articles/matecconf/pdf/2018/105/matecconf_iswso2018_01031.pdf	English	null	Analysis of the Ecological Effects of Decadal Large Scale Intermittent Annual Water Allocation using Satellite Data in Baiyangdian Wetland, Northern China	MATEC web of conferences	2,018	cc-by	7,014	, 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 https://doi.org/10.1051/matecconf/201824601031 Analysis of the Ecological Effects of Decadal Large Scale Intermittent Annual Water Allocation using Satellite Data in Baiyangdian Wetland, Northern China Fei WANG 1,2,a, Ying ZHAO 3 1School of Life Science, Shanxi University, Taiyuan Shanxi 030006, China 030006, China 2School of Physical Education, Shanxi University, Taiyuan 030006, China 3Shanxi Academy of Environmental Research, Taiyuan 030027, China Abstract. In this study, the ecological effects of intermittent water allocation with emphasis on spatio- temporal responses of the corresponding vegetation were analyzed using remote sensing data and GIS-based buffer technology considering the period from 1st July 2000 to 31st December 2009. Three sampling sites (Angzh, Wangk, and Xidayang) with different water flow paths and three buffer distances were distinguished in the research. The Seasonal-Trend decomposition procedure based on Regression (STR) trend extraction and its corresponding linear regression and anomaly detection were executed to determine temporal variations of vegetation under the effects of water allocation. ANOVA and PCA methods were employed to identify the spatial responses of vegetation to different water flow paths and buffer distances. The results were as follows: (1) NDVI except NDVImin displayed higher values during the period without water allocation; (2) extremely significant decline trends (p<0.001) of all NDVI categories were observed in all sites at all buffer distance levels, except for NDVImin at buffer distances of 2 km and 4 km in Angzh, showing stronger fluctuations of frequency after 2008 as well as the decline gradient with the extent of buffer distance to river. The anomaly detection results provided similar evidence of stronger NDVI fluctuations after 2008; (3) water allocation had extremely significant effects on regional vegetation coverage (p<0.01) with a decline gradient of statistical p values along enlarged buffer distances. Our results provide evidence of spatial and temporal differences in vegetation response to water availability due to the intermittent frequency water allocation implemented via different river channels. The findings of this study will deepen our understanding of the effects of water division on regional vegetation restoration and can be used to develop a practical strategy for effective implementation of water allocation. © 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/). a Corresponding author: nemochina2008@hotmail.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/) P Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 es/by/4 0/) 1 Introduction contrast between high water usage in midstream areas (i.e. irrigation for agriculture) and water crisis in natural ecosystems along downstream areas has become increasingly prominent [6,7]. The higher consumption of water in the midstream area implies lower water availability for the downstream area, especially in areas with originally scarce water resources. The responses of wetland ecosystems to river flow regulation associated with water diversion involve many complex eco- hydrological processes [8,9]. Therefore, it is crucial to determine an appropriate level of water diversion considering both human needs and ecological restoration. Understanding changes in vegetation before and after water diversion and adjusting the eco-hydrological linkages between midstream and downstream areas are essential for water resource planning in water scarce areas. Vegetation cover is a critical component of wetland ecosystems in arid and semi-arid regions; however, water scarcity has remained a serious issue for a long time [1]. Vegetation dynamics are considered the primary ecological indicator of ecosystem response to water availability in wetland ecosystems. The dynamics of vegetation cover are dominated by the availability of water to a large extent in its temporal and spatial distribution [2]. Therefore, the relationship between vegetation and water availability is becoming increasingly important in the fields of global environmental change and ecological restoration. Managing water for the sustainable development of ecosystems is both a technical and a governance challenge in which balancing water loss and vegetation restoration plays a central role [3-5]. Implementation of water allocation aimed at maintaining ecosystem sustainability is a mechanism that may be used in basin-scale water management [3]. In arid and semiarid watersheds, the Water allocation has become a necessary measure to improve the reallocation of water resources for wetland ecosystem restoration in the Central Asia [10,11] and Northwestern China [12,13]. Remotely sensed data, such as normalized difference vegetation index (NDVI), serve , 0 (2018) MATEC Web of Conferences 246 1031 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 https://doi.org/10.1051/matecconf/201824601031 PCA could enhance our understanding of water allocation strategy and the response of ecological processes to intermittent water division as well as spatial distance. This study used 10-year NDVI time series with 10-day syntheses datasets (1st July 2000 to 31st December 2009) of Baiyangdian wetland to examine the temporal and spatial response of NDVI to water resources. 1 Introduction GIS-based three buffer distances (2 km, 4 km and 6 km distance to river channel center, respectively) were employed to assess the spatial response of NDVI to water resources. The NDVI series data were extracted to three distance gradient categories based on buffer levels at each site. The categories of each buffer distance level and each site included: the minimum value of NDVI of the all grid cells at the respective buffer distance level (NDVImin); the mean value of NDVI (NDVImean); the maximum pixel value of NDVI, meaning the value of the largest number of all grid cells at the respective buffer distance level (NDVImost); and the maximum value of NDVI (NDVImax). ANOVA analysis was performed using the data for trends and anomaly changes in each NDVI category and then temporal and spatial vegetation responses to water allocation for periods before water allocation, during water allocation and after water allocation. Our main objectives were: (1) to elaborate decadal trends and abrupt patterns caused by intermittent water division in each NDVI category; (2) to identify temporal and spatial responses of vegetation to intermittent water allocation as well as the relative importance of NDVI categories; and (3) to propose a new perspective for implementation of water allocation at different time scales via different division paths. as key indicators of vegetation status and are useful parameters in studies of terrestrial vegetation cover. The time series of NDVI data provide important information for evaluating vegetation change before and after water allocation to improve strategies of large-scale water division. The regional ecosystem of arid and semi-arid areas, including vegetation cover, is sensitive to fluctuations of local water resource availability [14-16]. The vegetation cover of wetland watersheds coupled with its eco-environmental quality and stability are crucial for maintaining a healthy ecosystem [17,18]. Understanding vegetation responses to combined water resource availability and the corresponding management planning would facilitate regional ecosystem management. Generally, ecosystem management strategies have been shown to have greater influence on vegetation distribution [19,20]. In the restoration of large-scale vegetation distributions, the effects of regional sustainable ecosystems could be reflected by NDVI [21,22]. Therefore, understanding gradients of NDVI variation and factors influencing NDVI response to water availability is important for sustainable ecosystem management. Omute et al. [23]revealed the strong interactions between NDVI and water levels especially in low precipitation years in Lake Victoria. Cao et al. 1 Introduction [24] compared the water balance in potential natural vegetation restoration to guide future ecological restoration planning. Zhang et al. (2016a) emphasized on the importance of water allocation to vegetation restoration rather than conservation of natural vegetation in China. Previous research focused more on water allocation models considering the evaluation of ecosystem services [25], gaining public acceptance for larger water projects and policy changes [26], affecting the responses of vegetation dynamics to hydro-climatic factors [11], fulfilling water reclamation management [27], changing operation rules with hydrologic state- dependent multi-reservoirs [3], as well as the relationships with soil, hydrology, vegetation and climate change [5,28,29]. These studies are mostly concentrated on the overall effects for a time period of water division or a specific effect during one water division, and on the final effects on the ecology and human society. Studies on the ecological effects of processes of intermittent water allocation, as well as the corresponding the strategy and spatial responses are lacking. 2.1 Study area The Baiyangdian watershed (38°10'–40°00' N and 113°40'–116°20' E), located 130 km south of Beijing (48°43'–39°02' N and 115°38'–116°07' E), is the largest natural wetland in the North China Plain (Fig. 1). In April 2017, Xiongan New Area as China's third state-level new area after Shenzhen Special Economic Zone and Shanghai Pudong New Area, achieved profound significance in economic and social development. Accordingly, the restoration of Baiyangdian wetland has become the top priority under the New District Construction. The wetland belongs to parts of the Haihe River basin, which is one of ten large basins in China. The total area of the Baiyangdian watershed is 31,200 km2 and includes eight rivers: the Caohe, Fuhe, Juma, Pinghe, Puhe, Tanghe, Xiaoyi and Zhulong rivers. There are three reservoirs upstream of the Lake: Angezhuan reservoir (Angzh), Wangkuai reservoir (Wangk), and Xidayang reservoir (Xidayang). The topography does not vary greatly in such a flood plain. The regional climate is dominated by continental monsoons of humid or sub-humid region with an average rainfall of 530.4 mm, and precipitation mostly occurs in July and August. There is a distinct seasonality in the annual patterns of precipitation, evaporation, and air temperature. p p g This study selected three sampling sites along water flow paths in the Baiyangdian watershed as examples of areas where constant water replenishment will be implemented in the future. These areas are the top priority for ecological conservation and restoration because the Xiongan New Area has been constructed as China's third state-level new area in April 2017. The sampling sites correspond to three water allocation paths with three upstream reservoirs; and they were selected considering less interference of river branches and ample distance from downstream lakes. With the anticipated population boom and the rapid economic development in the processes of regional construction, the consumption of water will increase dramatically and water availability for ecological processes will diminish. Determining the relative importance of NDVI categories at each site using 2 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 https://doi.org/10.1051/matecconf/201824601031 Figure 1. Geographical location of study area and sampling sites In the recent decades, the natural hydrologic regime of the upstream of the watershed reservoir, which is aimed at benefiting economic and social water users, has been profoundly changed. 2.1 Study area The annual natural outflow of the upstream reservoir is now inadequate to meet the environmental flow requirements of the downstream wetlands and their ecosystem health restoration. Water scarcity has become a crucial problem for the wetland restoration. The corresponding vegetation and agriculture have been greatly influenced. To alleviate water resource scarcity, water allocations from upstream reservoirs have been implemented since the 1990s (Table 1), resulting in significant impacts on regional hydro-ecological variations. sites Table 1. Recent implementations of water allocations from upstream three reserviors (2000-2009) Time period of water allocation Reservoir Discharge out of the reservoir (106m3) Discharge flow into the lake (106m3) Jul. 2000 Angezhuang 31.1 18.0 Dec. 2000−Jan. 2001 Wangkuai 79.0 40.6 Feb. 2001−Mar. 2001 Angezhuang 3.9 21.6 Jun. 2001−Jul. 2001 Wangkuai 90.8 45.1 Feb. 2002−Mar. 2002 Xidayang 50.2 35.0 Apr. 2002−May 2002 Xidayang 38.7 19.7 Jul. 2002−Aug. 2002 Wangkuai 61.1 31.0 Jan. 2003−Ma 2003 Wangkuai 200.0 116.3 Mar. 2005−Apr. 2005 Angezhuang 58.6 42.5 Mar. 2006 Angezhuang 32.0 8.3 Mar. 2006−Apr. 2006 Wangkuai 90.0 48.4 Jun. 2009−Jul. 2009 Angezhuang 69.7 17.3 Table 1. Recent implementations of water allocations from upstream three reserviors (2000-2009) 2.4.1 STR and Trend component extraction The seasonal variation in time series data is the most important characteristic and the basis of all seasonal adjustment procedures when building models to fit observation data. Various methods are available for handling seasonal components, such as Seasonal ARIMA (SARIMA), X-11-ARIMA [30] and X-12-ARIMA [31] and X-13ARIMA-SEATS [32], as well as STL (Seasonal- Trend decomposition using Loess), which has been widely used because of its availability in R [33]. However, only few of them afford the clarity, simplicity, and generality required for handling several problems that arise with seasonal data decomposition [34]. The STR (a Seasonal- Trend decomposition procedure based on Regression) could solves such problems by performing re-cast in the framework of ordinary least squares or quantile regression. In particular, the robust version of STR assumes a double exponential distribution for the residuals, and trend, seasonal and predictor coefficient changes compared to the normal distribution assumed in the STR model, thus providing detailed changes of the trend component. Anomaly detection of the extracted trend components was implemented using the Seasonal Hybrid Extreme Studentized deviate (S-H-ESD) method, which was built upon the Generalized ESD test for detecting anomalies. Machine learning was completed using the “AnomalyDetection” packages in R developed by Twitter (https://github.com/twitter/AnomalyDetection) [35]. With the packages, anomaly detection was achieved by employing time series decomposition and using robust statistical metrics, viz., median with ESD as well as piecewise approximation for high frequency time series data. The R package can detect both global and local anomaly values by setting the maximum anomaly probability among the all results. In this study, we assumed that the maximum anomaly month number may be induced during the implementation of water allocation. Thus, considering both positive and negative anomaly values, the maximum anomaly probability was set to 0.12 conservatively. The significant level was 95%. In the simplest STR model, time series data may be considered the sum of three components: one high- frequency seasonal component, one low-frequency long- term component (or trend), and a residual component (variation not explained by time), which are expressed as: Yt = Tt + Ssn(t), t + Rt (1) (1) where Tt is the trend, Ssn(t), t is the additive seasonal component, which is a k×n matrix (k is number of seasons and n is length of the time series), and Rt is the “remainder” component. the spatial distribution of NDVI series were all analyzed using ArcGIS (ESRI Arcgis Desktop, version 10.3). The extracted trend components were measured as continuous time series variables, and the linear regression analysis was performed to explore possible statistical trends of the magnitude of NDVI changes over time. Specifically, the simple linear regression analysis was employed to explain changes in the different NDVI categories over time at each buffer distance level at the three sites. The partial least squares (PLS) regression was applied to capture the overall sensitivity of temporal variables in different years. The overall goodness-of-fit of the regression model was evaluated by the regression coefficients and the p-value, and the proportion of the observed variations in the response variables could be explained by the PLS model. The linear regression analyses were performed in R (http://cran.R-project.org/). 2.4.3 ANOVA analysis To evaluate the effects of water allocations, three groups were set: before water allocation, during water allocation, and after water allocation. Due to the different lengths of duration of water transformation, we selected monthly time series data groups to represent the before (Ahead group), during (Current group), and after water allocation groups (After group). Significant variations of NDVI categories under different groups at each buffer distance level at the three sites were systematically analyzed by one-way ANOVA, and multiple comparisons were also conducted among groups under the t-test. The analyses were performed in R (http://cran.R-project.org/). p The STR model uses the maximum likelihood to estimate cross-validated residuals of the seasonal components, and cross validation to estimate the residuals of minimizing SSE (the sum of squares due to error) by finding optimal parameters using R core “stats” package. Considering that the Robust STR can tolerate outliers well using the quantile regression approach (only 0.5 quantile is used), which could provide details of the trend component, we selected Robust STR to extract the trend component in the research. All STR analyses were performed using the “stR” package in R language (http://cran.R-project.org/). Because the data have 10-day granularity, the yearly seasonality included a period of 36 observations. Thus, gaps of 36 observations were used for cross validation, and 95% confidence intervals were calculated in the STR analysis. A more complete description of this methodology can be found in Dokumentov and Hyndman [34]. 2.2 Data source Series records of water allocation data for Baiyangdian watershed were obtained from the Anxin Environmental Bureau. The sources for the corresponding 10-year NDVI time series datasets (1st July 2000 to 31st December 2009) with a spatial resolution of 1 × 1 km were the PROBA- VGT S10 TOC NDVI products obtained from European Space Agency (http://www.vito-eodata.be). The VGT S10 products (10-day syntheses) were compiled by merging segments (data strips) acquired in a 10-day period. All segments of this period were compared pixel-by-pixel to select the 'best' surface reflectance values. These products provided data from all spectral bands, the NDVI, and auxiliary data on image acquisition parameters. The NDVI product ensured data quality with a minimum effect of cloud covers, and the pixel brightness count was the ground area's reflectance (corrected for atmospheric effects). All NDVI images were reprojected in UTM coordinates as metadata for the research. The source of the corresponding water allocation records for decadal NDVI time series dataset (10-day syntheses with a spatial resolution of 1 km2) were employed to determine the ecological effects of water allocation on vegetation. The corresponding flow paths of water divisions from upstream reservoirs was distinguished, as shown in Figure 1. Detailed information of water transformation from three upstream reservoirs is summarized in Table 1. To evaluate the magnitudes of ecological improvement attributable to the frequency of water allocation, three buffer distance levels (distances of 2 km, 4 km and 6 km from the river center, abbreviated as bf2, bf4, and bf6 respectively) were considered in the research. Areas in the basin with relatively few river branches and weak anthropogenic activities, as well as relatively gentle slope were considered during the selection of sampling sites (Angzh, Wangk, and Xidayang). Grid numbers with 1 km2 spatial resolution of each grid at different buffer distances in the three sampling sites were summarized. The spatial buffer distance of each site and 3 https://doi.org/10.1051/matecconf/201824601031 https://doi.org/10.1051/matecconf/201824601031 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 , 0 (2018) MATEC Web of Conferences 246 1031 the spatial distribution of NDVI series were all analyzed using ArcGIS (ESRI Arcgis Desktop, version 10.3). 3.1. Descriptive Statistics Table 2 shows descriptive statistics of NDVI categories for each gird at three buffer distance levels at three sampling sites during the recorded periods. The results showed similar grid numbers at each buffer distance level in all Table 2. Descriptive summary of NDVI with spatial resolution 1km for each gird in 3 buffer distence levels at three sampling sties during 1st July 2000 to 31st December 2009 Site Buffer Distance Grid number NDVI within Allocation duration NDVI Without Allocation duration Max Min Most- pixes Mean SD Max Min Most- pixes Mean SD Angzh 2 km 92 0.728 0.036 0.266 0.268 0.102 0.848 -0.004 0.339 0.349 0.147 4 km 89 0.756 0.076 0.291 0.300 0.093 0.872 -0.008 0.388 0.391 0.172 6 km 50 0.752 0.1 0.309 0.311 0.103 0.872 -0.012 0.407 0.404 0.172 Wangk 2 km 91 0.8 0.004 0.306 0.301 0.180 0.868 -0.012 0.376 0.372 0.150 4 km 88 0.82 0.004 0.323 0.326 0.194 0.892 0.004 0.412 0.413 0.179 6 km 53 0.828 0 0.309 0.322 0.194 0.88 0.016 0.403 0.408 0.173 Xidayang 2 km 90 0.728 0.116 0.309 0.364 0.112 0.88 0.004 0.374 0.388 0.177 4 km 87 0.772 0.116 0.294 0.375 0.111 0.876 0 0.374 0.393 0.168 6 km 59 0.72 0.128 0.279 0.328 0.097 0.836 0 0.368 0.381 0.161 Table 2. Descriptive summary of NDVI with spatial resolution 1km for each gird in 3 buffer distence level during 1st July 2000 to 31st December 2009 criptive summary of NDVI with spatial resolution 1km for each gird in 3 buffer distence levels at three sampling sties during 1st July 2000 to 31st December 2009 red segments in the figures; no obvious NDVI trend was observed in response to water allocation. 2.4.4 PCA analysis PCA (Principal Component Analysis) was performed to investigate relatively important variables of all NDVI categories to identify the major NDVI variables responding to water allocation. The “pca3d” package in R was used for the PCA. In addition, the suitability of PCA for our data was tested and confirmed by performing the matrix of cumulative variance contribution. In the research, only four key variables for both PC1 axis and PC2 axis were obtained deliberately, and 95% individuals 2.4.2 Linear regression and anomaly detection 4 https://doi.org/10.1051/matecconf/201824601031 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 delineated with ellipse. The PCA analyses were conducted using R (http://cran.R-project.org/). three sites. During the period without water allocation, the NDVI values, except NDVImin, were clearly larger than those during the water allocation period. Some negatives NDVImin values were observed during the period of without water allocation, which implies the threat of water scarcity to vegetation. In addition, the relatively smaller NDVI values along the water flow from the Angzh reservoir were compared with that of the other two sites, showing the natural spatial differences of water resources in the watershed, as well as the assisted proof of negative NDVI values at the Angzh site. 3.2 NDVI Trends and Anomaly Detection The simple linear regression of NDVI trend components indicated extremely significant decline trends (p<0.001) for all NDVI categories at all buffer distance levels in all sites, except NDVImin on bf2 and bf4 at Angzh (Fig.2c). The significant decline trends of all NDVI categories show the insufficient water resource in the watershed. The Robust STR could tolerate outliers well and provide details of NDVI trend components (marked as blue circles) at the Angzh (Fig.2), Wangk (Fig.3), and Xidayang (Fig.4) sites. A common was the stronger fluctuation during the last years of the recorded duration, especially towards the end of 2009. The different buffer distances resulted in different NDVI responses, with a huge fluctuation gradient from bf2 to bf6, especially in sites Angzh (Fig.2) and Xidayang (Fig.4). The marginal box plots show the trend data distribution and indicate differences of both spatial effects and distance effects. The box plots of all sites show relatively centralized data distribution for NDVImean, NDVImax and NDVImost, and relatively higher values with increasing buffer distance, especially in NDVImax. In addition, a relatively narrower range of NDVImax and NDVImost was observed at the three buffer distance levels for all three sites, compared with the other two NDVI categories. The effects of water allocation are shown with The anomaly detection for the trend components from STR showed consistent variations in NDVImax at the end of 2009 for all three buffer distance levels (Fig.5). In the anomaly detection, we assumed the maximum anomaly probability as 0.12, which means that the anomalies are 12% at most. The anomaly results showed global anomaly values of NDVImax, whereas local anomaly values appeared only at bf2 around 2000 in site Angzh (Fig.5A- a). Some scattered anomaly values also appeared in other NDVI categories, such as in NDVImean at bf2 in site Xidayang (Fig.5C-a), NDVImean at bf4 and bf6 in site Angzh (Figs.5A-bc), NDVImost at bf4 in site Angzh (Fig.5A-b), and so on. 5 5 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 , 0 (2018) MATEC Web of Conferences 246 1031 https://doi.org/10.1051/matecconf/201824601031 Figure 2. Linear fitness for the trend extracted from STR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Angzh: the color background represents the water allocation occupancy. Figure 3. 3.2 NDVI Trends and Anomaly Detection 6 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 , 0 (2018) MATEC Web of Conferences 246 1031 https://doi.org/10.1051/matecconf/201824601031 Figure 5. Anomaly Detections for the trend from STR in NDVI categories in bf2(a), bf4(b) and bf6(c) in site Angezh(A), siteWangk(B) and Xidayang(C), respectively: the color background represents the water allocation occupancy. Figure 5. Anomaly Detections for the trend from STR in NDVI categories in bf2(a), bf4(b) and bf6(c) in site Angezh(A), siteWangk(B) and Xidayang(C), respectively: the color background represents the water allocation occupancy. Figure 6. ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Angzh: water allocation periods of A for Jul. 2000, B for Feb. 2001 - Mar. 2001, C for Mar. 2005 - Apr. 2005, D for Mar. 2006 and E for Jun. 2009 -Jul. 2009. 3.2 NDVI Trends and Anomaly Detection Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Wangk: the color background represents the water allocation occupancy. Figure 2. Linear fitness for the trend extracted from STR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Angzh: the color background represents the water allocation occupancy. Figure 2. Linear fitness for the trend extracted from STR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Angzh: the color background represents the water allocation occupancy. Fig re 3 Li fit f th t d f TR i NDVI ( ) NDVI (b) NDVI ( ) d NDVI (d) i diff t b ff di t Figure 3. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Wangk: the color background represents the water allocation occupancy. Figure 4. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Xidayang: the color background represents the water allocation occupancy. Figure 3. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Wangk: the color background represents the water allocation occupancy. Figure 4. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Xidayang: the color background represents the water allocation occupancy ar fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distanc in site Wangk: the color background represents the water allocation occupancy. Figure 3. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in in site Wangk: the color background represents the water allocation occupancy. in site Wangk: the color background represents the water allocation occupancy. Figure 4. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Xidayang: the color background represents the water allocation occupancy. Figure 4. Linear fitness for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in differen in site Xidayang: the color background represents the water allocation occupancy. for the trend from sTR in NDVImean(a), NDVImax(b), NDVImin(c) and NDVImost(d) in different buffer distances in site Xidayang: the color background represents the water allocation occupancy. 3.3 Vegetation Ecological Responses and Buffer Distance Effects The ecological responses of vegetation to water allocation in the three groups at different water transformation time in each site were analyzed using one-way ANOVA, comparisons were made between groups. As shown in Figures 6-8, extremely significant differences were observed between the groups at all buffer distance levels in all sites, except for that between the Ahead group and After group. At the beginning of the recorded water allocations (before July 2001) in sites Angzh and Wangk, NDVI values were relatively higher when receiving the transformed water than that in the same period before the water allocation (Figs. 6A, 6B, 7A). Moreover, NDVI values increased with the extension of buffer distance. However, at the end periods of the recorded water allocations (starting from 2009) opposite changes were detected in the comparisons between the Ahead group and Current group in Angzh site (Fig.6E). Among other comparisons of the three groups, the results showed no- significant differences: Ahead group and Current group from June to July 2001 in Wangk site (p>0.05) (Fig.7B), Ahead group and After group from March to April 2005 in Angzh site (p>0.05) (Fig.6C), and Ahead group and After group from February to May 2005 in Angzh site (p>0.05) (Fig.8). The remaining comparatives groups showed extremely significant differences (p<0.01). Moreover, with the increase of buffer distance, the p value of t-test became relatively higher, which indicates the effects of distance on vegetation acquiring water. The results imply the benefit of vegetation restoration with water resource availability after the implementation of water transformation. In addition, we should emphasize on the importance of water allocation time. In the research, the response of vegetation to water acquisition was weak in spring (Figs. 6C,7E) and especially in summer, which is the water transformation time (Figs 6E,7B). Figure 6. ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Angzh: water allocation periods of A for Jul. 2000, B for Feb. 2001 - Mar. 2001, C for Mar. 2005 - Apr. 2005, D for Mar. 2006 and E for Jun. 2009 -Jul. 2009. 7 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 , 0 (2018) MATEC Web of Conferences 246 1031 https://doi.org/10.1051/matecconf/201824601031 Figure 7. 3.3 Vegetation Ecological Responses and Buffer Distance Effects ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Wangk: water allocation periods of A for Dec. 2000 -Jan. 2001, B for Jun. 2001 - Jul. 2001, C for Jul. 2002 -Aug. 2002, D for Jan. 2003 - Ma 2003 and E for Mar. 2006 - Apr. 2006. water resources in Xidayang site (Fig.9C). The results reflect the effects of water transformation strategies aimed at vegetation restoration. When water allocation was implemented via the Wangk channel, riparian vegetation close to rivers was sensitive. While water transformation was accomplished by means of the Angzh channel, non- riparian vegetation far from rivers was sensitive to water resource. Figure 9. PCA analysis showing the four relative primary variables for each axis in sites Angzh(A), Wangk(B) and Xidayang(C) Figure 9. PCA analysis showing the four relative primary Figure 7. ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Wangk: water allocation periods of A for Dec. 2000 -Jan. 2001, B for Jun. 2001 - Jul. 2001, C for Jul. 2002 -Aug. 2002, D for Jan. 2003 - Ma 2003 and E for Mar. 2006 - Apr. 2006. Figure 8. ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Xidayang during Feb. 2002 - May 2002 Figure 8. ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Xidayang during Feb. 2002 - May 2002 Figure 8. ANOVA analysis of month-fold NDVI response to water allocation among ahead water allocation, within water allocation and after water allocation in site Xidayang during Feb. 2002 - May 2002 Figure 9. PCA analysis showing the four relative primary variables for each axis in sites Angzh(A), Wangk(B) and Xidayang(C) 3.4 Relative important variables for ecological effects by PCA The analysis of descriptive statistics showed that all NDVI categories (except NDVImin) had relatively high values in all sites during the period without water allocation. Omute et al. [23] revealed that NDVI could be a robust predictor for water level variations during low precipitation years around Lake Victoria. This result indicates the strong correlation of NDVI with water availability when natural water resources are not sufficient. Wang et al [36] revealed peak values of NDVI during the growing season when NDVI was highly correlated with precipitation on a monthly scale. Our results were similar only for NDVImin. In fact, water allocation is mostply implemented during the A total of 12 variables in each site (combinations of four NDVI categories with three buffer distance levels) showed the ecological responses to water resource availability. The PCA could identify the relative important variables for their ecological sensitivity to water resource availability. As shown in Figure 9, NDVImin at bf4 and NDVImax at both bf4 and bf6 in Angzh are the most influential variables (Fig.9A). In Wangk, NDVImin at bf2 appears to be relatively more sensitive to water resources (Fig.9B). The variables of NDVImax at all buffer distance levels and NDVImin at bf4 display relatively higher sensitivity to 8 , 0 (2018) MATEC Web of Conferences 246 10 ISWSO 2018 31 https://doi.org/10.1051/matecconf/201824601031 https://doi.org/10.1051/matecconf/201824601031 of the remote sensing data. The former data are intermittent records at an approximately month scale. The later data are representative of 10-day periods and we acknowledge that the temporal resolution contributes to uncertainty in the estimated significant changes in vegetation. Nevertheless, the extractions of NDVI should be robust because we used an unbiased method. In terms of spatial resolution, each 1-km2 spatial resolution generally consisted of several different types of vegetation. It is important to acknowledge that the extracted NDVI values are representative of vegetation communities rather than individual species. non-growing season when vegetation has less greenness representing the corresponding lower NDVI values. The negative NDVI value in the period without water allocation implies the threat of water scarcity to vegetation because of the relatively moistened conditions during the water allocation period. In addition, the negative NDVI value may represent more bare land rather than water bodies. The NDVI trend by Robust STR detection revealed stronger fluctuation during the last years of the recorded duration, especially in 2009. 5 Conclusions We analyzed the ecological effects of intermittent water allocation emphasizing on both temporal and spatial responses of the corresponding vegetation using remote sensing data and GIS-based buffer technology. The STR trend extraction and its corresponding linear regression and anomaly detection methods were used to determine temporal variations of vegetation under the effects of water allocation. ANOVA and PCA methods were used to identify the spatial responses of vegetation in different water flow paths and buffer distances. Our conclusions are as follows: (1) NDVI, except NDVImin, displayed higher values during the period without water allocation. The results imply implied water scarcity in the watershed and suggest an urgency for ecological restoration. (2) The extremely significant decline trends (p<0.001) of all NDVI categories (except NDVImin) at all buffer distance levels in all three sites at bf2 and bf4 in Angzh showed stronger fluctuations after 2008, as well as the decline gradient with buffer distance from rivers. The anomaly detection provided similar evidence of stronger NDVI fluctuations after 2008. (3) The ecological response of vegetation to water allocation was significant, with regional vegetation coverage (p<0.01) showing a decline gradient of statistical p values along wide buffer distances. Our results provide evidences for the spatial and temporal differences of vegetation responses to water availability due to the intermittent frequency water allocation implemented via different river channels. Regional water resources have undergone substantial spatial and temporal variations since the implementation of water allocation. The intermittent frequency with different flow paths caused the spatial and temporal differences of vegetation distribution. Our results provide evidence for the spatial and temporal differences of vegetation responses to water availability. For example, NDVI values were relatively higher during the allocation period in Angzh beginning from 2000, while opposite changes occurred after 2008. Similar outcomes were also observed at different buffer distances. The above conclusions are also supported by the relative importance detection using PCA. For example, NDVImin at bf4 and NDVImax at both bf4 and bf6 in Angzh were the most prominent variables (Fig.9A), whereas NDVImin at bf2 had relatively higher sensitivity to water resource in Wangk (Fig. 9B). The PCA results also implied the effects of water allocation on vegetation at different buffer distances, which should be considered when the exact water flow path must be determined from multiple pathways with emphasis on large-scale vegetation restoration. 3.4 Relative important variables for ecological effects by PCA Similar results were also reported by Wang et al [36]; RDA (redundancy analysis) results showed that hydrological conditions, especially water availability, had important effects on NDVI dynamics. As the economic and agriculture demand for water increased, water crisis occurred in all watersheds in the last years of the recorded data. Supporting evidence showed that relative small discharge flowed into the lake Baiyangdian in 2009, and evidence from the anomaly detection showed that global consistent variations of NDVImax occurred at the end of 2009. Our results also showed the effects of distance to water availability on vegetation with an obvious decline gradient from bf2 to bf6. Ling et al [28] reported significantly positive correlation of ecological water requirement with distance from rivers on both banks. The ecological water requirement of riparian vegetation can be satisfied over a stretch between 4.9 km and 10.0 km from the river [28]. Our results were similar with this result at least within the range of 6 km from rivers. The ANOVA results also indicated the effects of distance on vegetation response to water availability. Moreover, the p value of the t-test became relatively higher as the distance from the river was decreased, indicating the effects of distance on water acquisition by vegetation. Vegetation close to rivers could consume more water than that stored in soil during dry conditions compared with vegetation farther away from rivers. References 29. W. Zhang, G. Hu, Y. Dang, D.C. Weindorf, and J. Sheng, J. Arid Environ. 130, Supplement C(2016) 1. X. Feng, B. Fu, S. Piao, S. Wang, P. Ciais, Z. Zeng, Y. Lü, Y. Zeng, Y. Li, and X. Jiang, Nat. Clim. Change 6, 11(2016) 30. G. Cao and L. Wu, Energy 115, (2016) 31. R.A. Wildman, J. Stat. Educ. 25, 1(2017) 2. X. Chen and J. Niu, Environ. Nat. Resour. Res. 6, 4(2016) 32. A. Phinikarides, G. Makrides, B. Zinsser, M. Schubert, and G.E. Georghiou, Renew. Energ. 77, (2015) 33. F. Wang, X. Wang, Y. Zhao, and Z. Yang, Int. J. Environ. Sci. Te. 11, 2(2014) 3. S. Nabinejad, S. Jamshid Mousavi, and J.H. Kim, Water Resour. Manage. 31, 11(2017) 34. A. 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https://openalex.org/W3037167856	https://www.aclweb.org/anthology/2020.sigmorphon-1.12.pdf	English	null	Low-Resource G2P and P2G Conversion with Synthetic Training Data	null	2,020	cc-by	3,869	1 Introduction In this system paper, we discuss the participation of the University of Alberta team in the SIGMOR- PHON 2020 Task 1: Multilingual Grapheme-to- Phoneme Conversion (Gorman et al., 2020). This is a sequence-to-sequence transduction task, in which a word, represented by a sequence of graphemes, must be converted into the sequence of phonemes representing its pronunciation. For example, given the French word connaissent the correct output is the phoneme sequence [k O n E s]. Following previous SIGMORPHON shared tasks, in addition to the standard setting with 3600 training examples for each language (which we refer to as the high-resource setting), we deﬁne a low-resource setting in which training data is lim- ited to 100 examples. This emulates a plausible scenario of working with a low-resource language for which only a small quantity of reliable phono- logical data is available. For example, a typical IPA description of the phonological inventory of a single language contains about a hundred phonetic transcriptions of individual words (IPA, 1999). We analyze the relative performance of different sys- tems depending on the size of the training data. The principal contributions of this paper include a novel G2P data augmentation method that lever- ages multiple systems and text corpora, as well as a thorough comparison of several G2P and P2G systems in both low-resource and high-resource settings. Abstract version (G2P), in which the goal is to predict the spelling of a word given its phonetic transcription (Rentzepopoulos and Kokkinakis, 1996). While G2P reﬂects the difﬁculty of reading, P2G may indicate the complexity of writing in a given lan- guage. Training instances for one of the two tasks can easily be applied to the other one by simply re- versing the input and output. We use the shared task datasets to investigate how systems designed for G2P perform on P2G. We also leverage raw text corpora to improve the accuracy on P2G, which indirectly leads to improvements on G2P as well. This paper presents the University of Al- berta systems and results in the SIGMOR- PHON 2020 Task 1: Multilingual Grapheme- to-Phoneme Conversion. Following previous SIGMORPHON shared tasks, we deﬁne a low- resource setting with 100 training instances. We experiment with three transduction ap- proaches in both standard and low-resource settings, as well as on the related task of phoneme-to-grapheme conversion. We pro- pose a method for synthesizing training data using a combination of diverse models. We develop a novel method of mitigating re- source limitations by synthesizing additional train- ing data using a combination of multiple G2P and P2G models. The underlying intuition is that a P2G model should be the inverse of the correspond- ing G2P model. Since models trained on a small number of instances tend to have limited accuracy, we attempt to distinguish between the correct and incorrect predictions by ensuring that P2G model output matches the corresponding G2P model input. The precision of this approach is further improved by comparing predictions of different systems. Fig- ure 1 illustrates this idea. Low-Resource G2P and P2G Conversion with Synthetic Training Data Bradley Hauer, Amir Ahmad Habibi, Yixing Luan, Arnob Mallik, Grzegorz Kondrak Department of Computing Science University of Alberta, Edmonton, Canada {bmhauer,amirahmad,yixing1,amallik,gkondrak}@ualberta.ca 3 Methods In this section, we ﬁrst describe DTLM, a multi- purpose string discriminative transduction system which we apply to both G2P and P2G tasks. We then introduce our approach to synthesizing addi- tional training data from unannotated texts. 3.2 Data Augmentation Inspired by the data hallucination technique for neural model training (Silfverberg et al., 2017; Anastasopoulos and Neubig, 2019), we introduce a method to synthesize additional training instances from unannotated texts. For each language under consideration, we train base transduction models on the available training data, and extract a list of words from a text corpus. A naive self-training ap- proach would be to simply apply a base G2P model to the words in the list to produce new training instances. However, without some mechanism to ﬁlter out incorrect predictions, a model trained on the augmented data would learn to replicate many of the errors made by the base model. Instead, we 2 Prior Work Our methods build upon the prior work of the Uni- versity of Alberta teams on string transduction. Di- recTL, a feature-based discriminative transducer, was originally designed for the G2P task (Jiampo- jamarn et al., 2008). In DirecTL+ (Jiampojamarn et al., 2010), the feature set was augmented with The task of phoneme-to-grapheme (P2G) conver- sion is the inverse of grapheme-to-phoneme Con- 117 Proceedings of the Seventeenth SIGMORPHON Workshop on Computational Research in Phonetics, Phonology, and Morphology, pages 117–122 Online, July 10, 2020. c⃝2020 Association for Computational Linguistics https://doi.org/10.18653/v1/P17 Figure 1: Our approach to synthesizing additional G2P training data. Figure 1: Our approach to synthesizing additional G2P training data. joint n-grams deﬁned on both source and target substrings. The system was applied to related tasks such as transliteration (Jiampojamarn et al., 2009), morphological inﬂection (Nicolai et al., 2015), stemming (Nicolai and Kondrak, 2016), and cog- nate projection (Hauer et al., 2019), proving to be particularly competitive in low-resource settings. DTLM (Nicolai et al., 2018), our principal tool in this work, is a successor of DirecTL+, which in- corporates target-side language models and a high- precision alignment. DTLM achieved state-of-the- art results on several tasks in which plain word types constitute the transduction target strings. Fi- nally, our data augmentation approach is inspired by the self-training approach of Hauer et al. (2017). et al., 2007) in a two-step process. In the ﬁrst step, M2M+ induces a one-to-one alignment in which null symbols may be inserted on either side. In the second step, the null links on the source side are removed by merging adjacent target symbols. The accuracy of DTLM can be enhanced by leveraging target character and word language mod- els. A 4-gram character languages model, which is induced from a set of word types extracted from a text corpus, encourages the prediction of high-probability letter sequences. A unigram word language model (which we also refer to as word counts) biases DTLM toward the production of known word-forms, with more common words and preﬁxes being preferred. Thus, DTLM is able to take advantage of existing multi-lingual text cor- pora, such as Wikipedia, to improve its accuracy on P2G. Since we have no access to any corpora of phonetic transcriptions, the language model com- ponent is not used for G2P. 3.1 Discriminative String Transduction The core of DTLM, adapted from DirecTL+, is a dynamic programming algorithm which uses a set of feature templates to transduce multiple charac- ters in a single operation. The feature set includes context features (n-grams on the source side), tran- sition features (target side bigrams), linear-chain features (conjunction of context and transition fea- tures), and joint n-gram features (on both source and target). The transduction quality of DTLM depends on a high precision one-to-many alignment, which is performed with M2M+ aligner (Jiampojamarn 118 Language DTLM -LM -WC -LM -WC Dutch 21.6 25.6 25.1 29.8 French 28.2 28.4 48.4 52.2 Greek 33.1 40.9 52.0 59.6 Table 1: WER for variants of DTLM on P2G develop- ment sets in the standard (high-resource) setting. reduce the noise by cross-checking the predictions of the independent base transduction systems ap- plied in both directions. Figure 1 illustrates the data augmentation pro- cess. For each word in the word list, we perform multiple sanity checks before accepting a new train- ing instance. First, both G2P models (in this case, DTLM and FST) must agree on their phoneme pre- dictions. Second, when applied to the common G2P prediction, the corresponding base P2G mod- els must not only agree, but also output the original orthographic word. If both G2P models predict the same phoneme sequence, and both P2G mod- els recover the original grapheme sequence, that grapheme–phoneme pair is added to the synthetic training data. The ﬁnal augmented model is trained on the combined original and synthetic data. Table 1: WER for variants of DTLM on P2G develop- ment sets in the standard (high-resource) setting. translation (Vaswani et al., 2017). Our choice of TRANSFORMER over LSTM was based on initial development experiments.1 The system is imple- mented using the Fairseq toolkit (Ott et al., 2019). Unlike FST, which only needs to be tuned on the size of n-grams, TRANSFORMER requires exten- sive tuning which may take several days to com- plete. We attempted to follow the tuning guidelines as they became available. We kept the hyperparam- eters as speciﬁed in the source code, with the max- imum number of training epochs set to 400. The tuning was performed separately for each language in terms of word error rate (WER). We trained the models on two Nvidia Titan RTX GPUs, using Adam optimizer. We varied dropout probability be- tween 0.1, 0.2, and 0.3. 4 Development In this section, we describe our development ex- periments on both G2P and P2G with three differ- ent transduction systems and the synthetic training data. 4.1 Datasets We created low-resource datasets of 100 instances from each standard (high-resource) training set of 3600 instances (Lee et al., 2020). We extracted every 36th instance, starting from the ﬁrst instance, in a deterministic manner, to ensure replicability. The P2G datasets were created by swapping the grapheme and phoneme strings in the task datasets. The ofﬁcial development sets of 450 instances were used for model tuning only. Unfortunately, we were ultimately unsuccessful in replicating the ofﬁcial results of TRANSFORMER. The implementation used for producing the ofﬁcial results was not available at the system submission time, and used different hyperparameter settings.2 3.1 Discriminative String Transduction and batch size between 256, 512, and 1024 in the high-resource setting, and 64 in the low-resource setting. Due to the underspec- iﬁcation in the guidelines, instead of tuning the number of epochs, we took the model checkpoint of the last epoch. 1However, the ofﬁcial baseline results, show LSTM as more accurate than TRANSFORMER on most languages. The model results and predictions were not available at the system submission time. 2Unlike the earlier implementation that we used, it tuned the number of training epochs without a ﬁxed maximum. 1However, the ofﬁcial baseline results, show LSTM as more accurate than TRANSFORMER on most languages. The model results and predictions were not available at the system submission time. 2Unlike the earlier implementation that we used, it tuned the number of training epochs without a ﬁxed maximum. 4.2 Task Baselines DTLM was our principal system for both G2P and P2G. The models were tuned on the ofﬁcial de- velopment sets separately for each task (G2P and P2G), language, and setting (high-resource and low-resource). The context size was varied from 1 to 3 in low-resource, and from 2 to 7 in high- resource settings. We also varied joint n-gram fea- tures from 1 to 6, and Markov order from 0 to 2, with and without linear chain features. The task organizers provided implementations of three baseline systems, which are referred to as FST, LSTM, and TRANSFORMER. These are not baselines in the traditional sense of “the simplest possible algorithm” (Manning and Schutze, 2001, page 234), but rather sophisticated systems capable of achieving state-of-the-art results on related tasks. Rather than develop a novel competitive approach, our goal was to combine the unmodiﬁed baselines and DTLM to achieve a relative improvement with respect to the individual systems. For P2G models, we extracted word frequency lists for each language from the ﬁrst one million As our neural base system, we selected TRANS- FORMER, an encoder-decoder architecture with fully-connected layers and self-attention mecha- nism, which was originally developed for machine 119 High Resource Low Resource Language DTLM FST TF DTLM FST TF Adyghe 18.2 16.7 21.3 53.1 56.0 87.8 Armenian 4.9 5.1 8.0 14.0 27.3 80.7 Bulgarian 6.0 6.4 8.4 20.9 28.7 83.8 Dutch 23.8 27.3 21.1 34.0 66.7 90.4 French 28.7 50.4 51.3 51.6 72.4 94.0 Georgian 1.1 0.7 1.1 4.4 6.4 74.7 Greek 32.9 59.6 56.9 41.3 89.1 97.6 Hindi 3.8 12.0 15.1 18.0 45.8 86.9 Hungarian 4.0 6.9 8.0 14.9 28.7 81.8 Icelandic 13.6 12.0 15.6 28.0 45.6 82.4 Japanese 4.4 9.8 3.6 61.1 59.3 97.8 Korean 39.1 50.0 32.7 96.7 97.3 100 Lithuanian 4.0 3.6 3.3 15.1 25.8 75.1 Romanian 1.8 1.3 2.9 17.8 15.6 57.3 Vietnamese 16.2 18.4 16.2 71.8 85.6 96.9 Average 13.5 18.7 17.7 36.2 50.0 85.8 Table 2: WER on P2G test sets. lines of Wikipedia3, excluding words with fre- quency less than 10, shorter than 4 characters, or containing non-alphabetic characters. From the word lists, we generated 4-gram character language models using the CMU Toolkit4. Target language models are not used for the G2P task because of the lack of phonetic transcription corpora. Table 1 demonstrates the impact of word counts (WC) and character language models (LM) on P2G accuracy. 7 FST, which is not included in Table 3, obtains 22.0% WER average in the standard setting according to the ofﬁcial results, and 58.1% WER average in the low-resource setting, as our submission with RunID=5. 6We note that the P2G accuracy is particularly high on Georgian, which, unlike French, seems to be easier to write than to read. 3https://dumps.wikimedia.org 4http://www.speech.cs.cmu.edu/SLM 5Only 36% of the graphemes in the Korean test set are observed in the low-resource train set. The corresponding number in Japanese is 90%. 4.2 Task Baselines The results on three challenging lan- guages suggest that most of the DTLM advantage comes from leveraging monolingual text corpora. Furthermore, word counts help more than charac- ter LMs. Without those two components, DTLM results on P2G in the standard (high-resource) set- ting were in the same range as FST and TRANS- FORMER. Table 2: WER on P2G test sets. 5 Test Results Table 2 shows the P2G results on the test sets. All models are trained on the same training sets, with- out any synthesized instances. TRANSFORMER (TF) completely fails with only 100 training in- stances (low resource), but outperforms FST with 3600 training instances (high resource).6 DTLM is substantially more accurate on average than the other two systems in both settings. Although DTLM beneﬁts from information extracted from freely-available unannotated text corpora, the re- sults of the three systems are directly comparable because they all use the same annotated training material. This further conﬁrms the claim of Nicolai et al. (2018) that DTLM achieves state-of-the-art results on the task of phoneme-to-grapheme con- version. 3https://dumps.wikimedia.org 4http://www.speech.cs.cmu.edu/SLM Acknowledgements We thank Garrett Nicolai for the assistance with DTLM. We thank the organizers of the shared task for their effort. In particular, we thank Kyle Gor- man for promptly answering our questions during the pandemic lockout. This research was supported by the Natural Sciences and Engineering Research Council of Canada, Alberta Innovates, and Alberta Advanced Education. IPA, 1999. Handbook of the International Phonetic As- sociation. Cambridge University Press. Sittichai Jiampojamarn, Aditya Bhargava, Qing Dou, Kenneth Dwyer, and Grzegorz Kondrak. 2009. Di- recTL: a language independent approach to translit- eration. In Proceedings of the 2009 Named Entities Workshop: Shared Task on Transliteration (NEWS 2009), pages 28–31, Suntec, Singapore. Association for Computational Linguistics. 4.4 Synthetic Training Data Th T d l i d h d The data augmentation approach was successful in our development experiments on the standard high-resource datasets, reducing the average WER with respect to base TRANSFORMER from 17.0% to 16.0%, We obtained improvements on 13 out of 15 languages, with the exception of Bulgarian and Korean.5 The TRANSFORMER models trained on the data The TRANSFORMER models trained on the data 120 High Resource Low Resource Language DTLM TF TF+ DTLM TF TF+ RunID 1 2 3 4 6 - Adyghe 29.8 28.9 28.2 54.4 92.9 58.4 Armenian 16.9 13.1 16.0 36.4 82.9 36.2 Bulgarian 35.8 30.0 36.7 67.6 93.3 66.4 Dutch 19.6 19.3 16.9 58.7 93.6 57.6 French 7.6 6.4 6.4 53.3 94.9 44.9 Georgian 28.2 25.8 27.1 39.6 84.4 42.2 Greek 15.8 17.1 17.3 39.1 88.0 44.0 Hindi 12.2 10.7 8.7 48.2 89.8 43.1 Hungarian 5.3 6.0 5.3 27.6 87.6 22.7 Icelandic 13.1 10.2 11.3 61.6 90.9 62.0 Japanese 8.7 6.7 6.7 57.8 98.0 53.1 Korean 45.3 45.1 45.1 95.1 100 100 Lithuanian 21.8 22.7 24.4 62.7 90.7 64.0 Romanian 11.3 12.7 10.7 30.2 69.3 28.9 Vietnamese 7.8 7.3 8.7 75.3 95.3 87.3 Average 18.6 17.5 18.0 53.8 90.1 54.1 Table 3: WER on G2P test sets. References Antonios Anastasopoulos and Graham Neubig. 2019. Pushing the limits of low-resource morphological in- ﬂection. In Proceedings of the 2019 Conference on Empirical Methods in Natural Language Processing and the 9th International Joint Conference on Natu- ral Language Processing (EMNLP-IJCNLP), pages 984–996, Hong Kong, China. Association for Com- putational Linguistics. Table 3: WER on G2P test sets. augmented with synthesized instances (labeled as TF+ in Table 3) achieved consistently higher results in our development experiments in the standard (high resource) setting (Section 4.4). Unfortunately, a corresponding improvement is not seen in the ofﬁcial test results. Possible explanations include the limit of 400 on the number of epochs made by the task organizers, as well as the suboptimal tuning procedure, which might have accidentally resulted in the overﬁtting of the augmented model. This is also suggested by the fact that the results of our TRANSFORMER models are often better than the ofﬁcial results on the test datasets. Kyle Gorman, Lucas F.E. Ashby, Aaron Goyzueta, Arya D. McCarthy, Shijie Wu, and Daniel You. 2020. The SIGMORPHON 2020 shared task on multilin- gual grapheme-to-phoneme conversion. In this vol- ume. Bradley Hauer, Amir Ahmad Habibi, Yixing Luan, Rashed Rubby Riyadh, and Grzegorz Kondrak. 2019. Cognate projection for low-resource in- ﬂection generation. In Proceedings of the 16th Workshop on Computational Research in Phonetics, Phonology, and Morphology, pages 6–11, Florence, Italy. Association for Computational Linguistics. On the other hand, the data augmentation ap- proach is remarkably successful in the low-resource setting, yielding an average WER improvement over 35% with respect to base TRANSFORMER. We interpret these results as a strong proof-of-concept of the validity of our data augmentation approach; when training data is limited, it can dramatically improve the accuracy of neural models, without any change to their architecture. Bradley Hauer, Garrett Nicolai, and Grzegorz Kondrak. 2017. Bootstrapping unsupervised bilingual lexicon induction. In Proceedings of the 15th Conference of the European Chapter of the Association for Compu- tational Linguistics: Volume 2, Short Papers, pages 619–624. IPA, 1999. Handbook of the International Phonetic As- sociation. Cambridge University Press. 4.4 Synthetic Training Data For our data augmentation approach outlined in Section 3.2, we required base G2P and P2G trans- duction systems. We preferred FST and DTLM over TRANSFORMER, as they performed better on small training datasets in terms of both accuracy and speed. Although data augmentation could also be applied to P2G, we used it exclusively for G2P, which is the primary focus of this shared task. The starting point for generating the synthetic training data were the word lists extracted from Wikipedia, as described in Section 4.3. We applied the base models to the lists, and ﬁltered out the instances on which the models disagreed or failed to recover the original spelling from their own pho- netic predictions. We further limited the number of synthetic training instances to 20,000 per language. This process failed to produce a substantial num- ber of new instances for Vietnamese and Korean, which we attribute to the unusual characteristics of the two scripts. Table 3 shows the G2P results on the test sets. The DTLM models were trained without any syn- thetic data or target language models. Although DTLM results are generally lower than on P2G, it outperforms FST in both settings.7 TRANS- FORMER again fails in the low resource setting, In the standard (high resource) setting, DTLM is about 6% worse on average than TRANSFORMER in terms of WER, but 10% better in terms of PER (3.9% vs 4.3% according to the ofﬁcial results). In addition, DTLM is much easier and faster to train. 6 Conclusion We have presented a novel data augmentation method that combines the strengths of multiple string transduction methods. We have also explored both G2P and P2G tasks in both the standard high- resource setting, and a low-resource setting of our own design. The results demonstrate that the weak- ness of neural systems in low-resource settings can be mitigated through the application of data aug- mentation. Sittichai Jiampojamarn, Colin Cherry, and Grzegorz Kondrak. 2008. Joint processing and discriminative training for letter-to-phoneme conversion. In Pro- ceedings of ACL-08: HLT, pages 905–913. Sittichai Jiampojamarn, Kenneth Dwyer, Shane Bergsma, Aditya Bhargava, Qing Dou, Mi-Young 121 CoNLL SIGMORPHON 2017 Shared Task: Univer- sal Morphological Reinﬂection, pages 90–99, Van- couver. Association for Computational Linguistics. Kim, and Grzegorz Kondrak. 2010. Translitera- tion generation and mining with limited training re- sources. In Proceedings of the 2010 Named Entities Workshop, pages 39–47, Uppsala, Sweden. Associa- tion for Computational Linguistics. Kim, and Grzegorz Kondrak. 2010. Translitera- tion generation and mining with limited training re- sources. In Proceedings of the 2010 Named Entities Workshop, pages 39–47, Uppsala, Sweden. Associa- tion for Computational Linguistics. Ashish Vaswani, Noam Shazeer, Niki Parmar, Jakob Uszkoreit, Llion Jones, Aidan N Gomez, Łukasz Kaiser, and Illia Polosukhin. 2017. Attention is all you need. In Advances in neural information pro- cessing systems, pages 5998–6008. Sittichai Jiampojamarn, Grzegorz Kondrak, and Tarek Sherif. 2007. Applying many-to-many alignments and hidden Markov models to letter-to-phoneme conversion. In Human Language Technologies 2007: The Conference of the North American Chap- ter of the Association for Computational Linguistics; Proceedings of the Main Conference, pages 372– 379, Rochester, New York. Association for Compu- tational Linguistics. Jackson L. Lee, Lucas F.E. Ashby, M. Elizabeth Garza, Yeonju Lee-Sikka, Sean Miller, Alan Wong, Arya D. McCarthy, and Kyle Gorman. 2020. Massively multilingual pronunciation mining with WikiPron. In Proceedings of the 12th Language Resources and Evaluation Conference, pages 4216–4221, Mar- seille. Christopher D. Manning and Hinrich Schutze. 2001. Foundations of Statistical Natural Language Pro- cessing. The MIT Press. Garrett Nicolai, Colin Cherry, and Grzegorz Kondrak. 2015. Inﬂection generation as discriminative string transduction. In Proceedings of the 2015 conference of the North American chapter of the association for computational linguistics: human language tech- nologies, pages 922–931. Garrett Nicolai and Grzegorz Kondrak. 2016. Lever- aging inﬂection tables for stemming and lemmatiza- tion. In Proceedings of the 54th Annual Meeting of the Association for Computational Linguistics (Vol- ume 1: Long Papers), pages 1138–1147. Ashish Vaswani, Noam Shazeer, Niki Parmar, Jakob Uszkoreit, Llion Jones, Aidan N Gomez, Łukasz Kaiser, and Illia Polosukhin. 2017. Attention is all you need. In Advances in neural information pro- cessing systems, pages 5998–6008. 6 Conclusion Garrett Nicolai, Saeed Najaﬁ, and Grzegorz Kondrak. 2018. String transduction with target language mod- els and insertion handling. In Proceedings of the Fifteenth Workshop on Computational Research in Phonetics, Phonology, and Morphology, pages 43– 53. Myle Ott, Sergey Edunov, Alexei Baevski, Angela Fan, Sam Gross, Nathan Ng, David Grangier, and Michael Auli. 2019. fairseq: A fast, extensible toolkit for sequence modeling. In Proceedings of the 2019 Conference of the North American Chap- ter of the Association for Computational Linguistics (Demonstrations), pages 48–53, Minneapolis, Min- nesota. Association for Computational Linguistics. Panagiotis A. Rentzepopoulos and George K. Kokki- nakis. 1996. Efﬁcient multilingual phoneme-to- grapheme conversion based on HMM. Computa- tional Linguistics, 22(3):351–376. Miikka Silfverberg, Adam Wiemerslage, Ling Liu, and Lingshuang Jack Mao. 2017. Data augmentation for morphological reinﬂection. In Proceedings of the 122

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